yeast fermentation experiment in different concentrations of sucrose

The fermentation of sugars using yeast: A discovery experiment

Charles Pepin (student) and Charles Marzzacco (retired), Melbourne, FL

Introduction

Enzyme catalysis 1  is an important topic which is often neglected in introductory chemistry courses. In this paper, we present a simple experiment involving the yeast-catalyzed fermentation of sugars. The experiment is easy to carry out, does not require expensive equipment and is suitable for introductory chemistry courses.

The sugars used in this study are sucrose and lactose (disaccharides), and glucose, fructose and galactose (monosaccharides). Lactose, glucose and fructose were obtained from a health food store and the galactose from Carolina Science Supply Company. The sucrose was obtained at the grocery store as white sugar. The question that we wanted to answer was “Do all sugars undergo yeast fermentation at the same rate?”

Sugar fermentation results in the production of ethanol and carbon dioxide. In the case of sucrose, the fermentation reaction is:

\[C_{12}H_{22}O_{11}(aq)+H_2 O\overset{Yeast\:Enzymes}{\longrightarrow}4C_{2}H_{5}OH(aq) + 4CO_{2}(g)\]

Lactose is also C 12 H 22 O 11  but the atoms are arranged differently. Before the disaccharides sucrose and lactose can undergo fermentation, they have to be broken down into monosaccharides by the hydrolysis reaction shown below:

\[C_{12}H_{22}O_{11} + H_{2}O \longrightarrow 2C_{6}H_{12}O_{6}\]

The hydrolysis of sucrose results in the formation of glucose and fructose, while lactose produces glucose and galactose.

sucrose + water \(\longrightarrow\) glucose + fructose

lactose + water \(\longrightarrow\) glucose + galactose

The enzymes sucrase and lactase are capable of catalyzing the hydrolysis of sucrose and lactose, respectively.

The monosaccharides glucose, fructose and galactose all have the molecular formula C 6 H 12 O 6  and ferment as follows:

\[C_{6}H_{12}O_{6}(aq)\overset{Yeast Enzymes}{\longrightarrow}2C_{2}H_{5}OH(aq) + 2CO_{2}(g)\]

In our experiments 20.0 g of the sugar was dissolved in 100 mL of tap water. Next 7.0 g of Red Star ®  Quick-Rise Yeast was added to the solution and the mixture was microwaved for 15 seconds at full power in order to fully activate the yeast. (The microwave power is 1.65 kW.) This resulted in a temperature of about 110  o F (43  o C) which is in the recommended temperature range for activation. The cap was loosened to allow the carbon dioxide to escape. The mass of the reaction mixture was measured as a function of time. The reaction mixture was kept at ambient temperature, and no attempt at temperature control was used. Each package of Red Star Quick-Rise Yeast has a mass of 7.0 g so this amount was selected for convenience. Other brands of baker’s yeast could have been used.

This method of studying chemical reactions has been reported by Lugemwa and Duffy et al. 2,3  We used a balance good to 0.1 g to do the measurements. Although fermentation is an anaerobic process, it is not necessary to exclude oxygen to do these experiments. Lactose and galactose dissolve slowly. Mild heat using a microwave greatly speeds up the process. When using these sugars, allow the sugar solutions to cool to room temperature before adding the yeast and microwaving for an additional 15 seconds.

Fermentation rate of sucrose, lactose alone, and lactose with lactase

Fig. 1 shows plots of mass loss vs time for sucrose, lactose alone and lactose with a dietary supplement lactase tablet added 1.5 hours before starting the experiment. All samples had 20.0 g of the respective sugar and 7.0 g of Red Star Quick-Rise Yeast. Initially the mass loss was recorded every 30 minutes. We continued taking readings until the mass leveled off which was about 600 minutes. If one wanted to speed up the reaction, a larger amount of yeast could be used. The results show that while sucrose readily undergoes mass loss and thus fermentation, lactose does not. Clearly the enzymes in the yeast are unable to cause the lactose to ferment. However, when lactase is present significant fermentation occurs. Lactase causes lactose to split into glucose and galactose. A comparison of the sucrose fermentation curve with the lactose containing lactase curve shows that initially they both ferment at the same rate.

Fig. 1. Comparison of the mass of CO 2 released vs time for the fermentation of sucrose, lactose alone, and lactose with a lactase tablet. Each 20.0 g sample was dissolved in 100 mL of tap water and then 7.0 g of Red Star Quick-Rise Yeast was added.

However, when the reactions go to completion, the lactose, lactase and yeast mixture gives off only about half as much CO 2  as the sucrose and yeast mixture. This suggests that one of the two sugars that result when lactose undergoes hydrolysis does not undergo yeast fermentation. In order to verify this, we compared the rates of fermentation of glucose and galactose using yeast and found that in the presence of yeast glucose readily undergoes fermentation while no fermentation occurs in galactose.

Fig. 2. Comparison of the mass of CO 2 released vs time for the fermentation of sucrose, glucose and fructose. Each 20 g sugar sample was dissolved in 100 mL of water and then 7.0 g of yeast was added.

Fermentation rate of sucrose, glucose and fructose

Next we decided to compare the rate of fermentation of sucrose with that glucose and fructose, the two compounds that make up sucrose. We hypothesized that the disaccharide would ferment more slowly because it would first have to undergo hydrolysis. In fact, though, Fig. 2 shows that the three sugars give off CO 2  at about the same rate. Our hypothesis was wrong. Although there is some divergence of the three curves at longer times, the sucrose curve is always as high as or higher than the glucose and fructose curves. The observation that the total amount of CO 2  released at the end is not the same for the three sugars may be due to the purity of the fructose and glucose samples not being as high as that of the sucrose.

Fermentation rate and sugar concentration

Next, we decided to investigate how the rate of fermentation depends on the concentration of the sugar. Fig. 3 shows the yeast fermentation curves for 10.0 g and 20.0 g of glucose. It can be seen that the initial rate of CO 2  mass loss is the same for the 10.0 and 20.0 g samples. Of course the total amount of CO 2  given off by the 20.0 g sample is twice as much as that for the 10.0 g sample as is expected. Later, we repeated this experiment using sucrose in place of glucose and obtained the same result.

Fig. 3. Comparison of the mass of CO 2  released vs time for the fermentation of 20.0 g of glucose and 10.0 g of glucose. Each sugar sample was dissolved in 100 mL of water and then 7.0 g of yeast was added.

Fermentation rate and yeast concentration

After seeing that the rate of yeast fermentation does not depend on the concentration of sugar under the conditions of our experiments, we decided to see if it depends on the concentration of the yeast. We took two 20.0 g samples of glucose and added 7.0 g of yeast to one and 3.5 g to the other. The results are shown in Fig. 4. It can clearly be seen that the rate of CO 2  release does depend on the concentration of the yeast. The slope of the sample with 7.0 g of yeast is about twice as large as that with 3.5 g of yeast. We repeated the experiment with sucrose and fructose in place of glucose and obtained similar results.

Fig. 4. Comparison of the mass of CO 2 released vs time for the fermentation of two 20.0 g samples of glucose dissolved in 100 mL of water. One had 7.0 g of yeast and the other had 3.5 g of yeast.

In hindsight, the observation that the rate of fermentation is dependent on the concentration of yeast but independent of the concentration of sugar is not surprising. Enzyme saturation can be explained to students in very simple terms. A molecule such as glucose is rather small compared to a typical enzyme. Enzymes are proteins with large molar masses that are typically greater than 100,000 g/mol. 1  Clearly, there are many more glucose molecules in the reaction mixture than enzyme molecules. The large molecular ratio of sugar to enzyme clearly means that every enzyme site is occupied by a sugar molecule. Thus, doubling or halving the sugar concentration cannot make a significant difference in the initial rate of the reaction. On the other hand, doubling the concentration of the enzyme should double the rate of reaction since you are doubling the number of enzyme sites.

The experiments described here are easy to perform and require only a balance good to 0.1 g and a timer. The results of these experiments can be discussed at various levels of sophistication and are consistent with enzyme kinetics as described by the Michaelis-Menten model. 1  The experiments can be extended to look at the effect of temperature on the rate of reaction. For enzyme reactions such as this, the reaction does not take place if the temperature is too high because the enzymes get denatured. The effect of pH and salt concentration can also be investigated.

• Jeremy M. Berg, John L. Tymoczko and Lubert Stryer,  Biochemistry , 6th edition, W.H. Freeman and Company, 2007, pages 205-237.
• Fugentius Lugemwa, Decomposition of Hydrogen Peroxide,  Chemical Educator , April 2013, pages 85-87.
• Daniel Q. Duffy, Stephanie A. Shaw, William D. Bare, Kenneth A. Goldsby, More Chemistry in a Soda Bottle, A Conservation of Mass Activity,  Journal of Chemical Education , August 1995, pages 734-736.
• Jessica L Epstein, Matthew Vieira, Binod Aryal, Nicolas Vera and Melissa Solis, Developing Biofuel in the Teaching Laboratory: Ethanol from Various Sources,  Journal of Chemical Education , April 2010, pages 708–710.

More about April 2015

Department of Chemistry 200 University Ave. W Waterloo, Ontario, Canada N2L 3G1

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Fermentation of glucose using yeast

• Four out of five

Carry out this practical and use the follow-up questions to explore an important fermentation reaction

Add some fresh context to this classic experiment with  Is fermented food and drink good for us?  in  Education in Chemistry . The article tucks into the science of fermentation and its everyday applications, from kombucha to kefir, and puts the supposed health benefits under the microscope. 

Beer and wine are produced by fermenting glucose with yeast. Yeast contains enzymes that catalyse the breakdown of glucose to ethanol and carbon dioxide. In this experiment, learners will set up a glucose solution to ferment and then test the products. You may also demonstrate distilling the fermentation mixture to separate the ethanol formed or set this as a learner activity. 

Download this

Carry out the fermentation of glucose using yeast with 14–16 learners. Observe and test the products, follow up with questions to consolidate learning and/or the distillation of ethanol.

The experiment is part of the  Nuffield practical collection , developed by the Nuffield Foundation and the Royal Society of Chemistry. Delve into a wide range of chemical concepts and processes with this collection of over 200 step-by-step practicals.

Learning objectives

• Carry out and observe a fermentation reaction.
• Test the products of a fermentation reaction.
• Explain the conditions needed for a fermentation reaction.

The experiment allows learners to cover the first two learning objectives. Use the questions to test their results and observations. Questions 4–6 cover the third learning objective and ask learners to explain the conditions required. Use question 7 to see if learners can connect this experiment to rates of reaction. To stretch learners, expand this question and ask them to write a full plan. Find the answers in the Teacher notes . 

How to use this resource

Set learners the first part of the experiment. It usually yields results within a lesson if the water is at the correct temperature and the reaction mixture is well mixed to begin with. It also depends on the freshness of the yeast. Dried yeast does work. If fermentation is not rapid because of the yeast used, then carry the whole experiment over to the next lesson.

For an alternative practical arrangement to part 1, use a bung and delivery tube to bubble the carbon dioxide through limewater. Or watch the Identifying ions practical video from 08:20 to see how to use a pipette to collect the gas when testing for carbonate ions. 

In the second part of the experiment, you can demonstrate distilling the reaction mixture. Watch the Fractional distillation  and Simple distillation videos and download the accompanying resources for setup, method and more learner-facing activities on simple distillation.

If you demonstrate distillation, pool the class results and filter the groups’ solutions into your distillation flask. Significant quantities of yeast will produce foaming and you can carry this over into the product if you do not filter the reaction mixture. Collect the fraction between 77–82°C. Ethanol boils at 78°C. This fraction should burn easily compared with the non-flammable original solution. Pour the ethanol away immediately and do not keep or reuse it.

Alternatively, set the distillation practical as a learner activity. Individuals or pairs may not produce enough ethanol to complete the distillation so learners may need to combine their solutions and work in groups.

More resources

• Use our  organic chemistry worksheet on alcohols  with 14–16 learners for practice in applying knowledge in context, including burning alcohols in cooking and as fuels.
• Link your lessons on fermentation and bioethanol to UN sustainable development goal 8 while developing learners’ literacy skills with this resource on  E10 petrol and climate change .
• Learn how a circular approach to manufacturing at British Sugar means there is virtually zero waste, including how they create coproducts such as bioethanol, by watching Paul’s  video job profile .
• Download the classroom activity and display the  fractional distillation  poster in your classroom to help 14–16 learners understand this important separating technique.

Technician notes

Read our  Standard health and safety guidance  and carry out a risk assessment before running any live practical.

Ensure learners wear safety glasses.

Be aware that if the fermentation is fast, the mixture may overflow from the flask.

Equipment (per group)

• 100 cm 3  conical flask
• 50 cm 3  measuring cylinder
• Boiling tube
• Boiling tube rack
• Access to a mass balance, correct to 1 decimal place
• Cotton wool – enough to plug the conical flask
• Safety glasses

Chemicals (per group)

• Glucose, 5 g – not currently classed as hazardous. See CLEAPSS Hazcard HC040c for more information.
• Yeast (as fast acting as possible), 1 g

Wear eye protection and measure 5 g of calcium hydroxide.

Add, while stirring, to 300 cm 3 of water in a large beaker.

Continue to stir the suspension, then pour it into a clean, labelled 2.5 dm 3 screw-top bottle using a funnel.

Fill the bottle with distilled water and tightly close the lid. Invert it to mix.

Leave the bottle overnight to allow the suspension to settle.

When required, slowly pour the limewater into small, labelled bottles.

Add more distilled water and/or calcium hydroxide to the stock bottle as required.

• 50 cm 3  of warm water 30–40°C
• Put 5 g of glucose in the conical flask and add 50 cm 3 of warm water. Swirl the flask to dissolve the glucose.
• Add 1 g of yeast to the solution and loosely plug the top of the flask with cotton wool.
• Wait while fermentation takes place. The time it takes will depend on the temperature, how well you mixed the reactants and the yeast’s freshness.
• Add 5 cm 3 of limewater to the boiling tube. Avoid contact with your skin as limewater is an irritant.
• Remove the cotton wool and pour the invisible gas into the boiling tube containing limewater. Take care not to pour in any liquid as well.
• Gently swirl the limewater in the boiling tube and note what happens.
• Replace the cotton wool in the top of the flask.

Source: © Royal Society of Chemistry

Set up the equipment as shown or use a pipette or bung and delivery tube instead of cotton wool to bubble the carbon dioxide through limewater

• Remove the cotton wool and note the smell of the solution.

If you are going to observe the distillation then you, or your teacher, will:

• Filter all the groups’ solutions into a distillation flask.
• Distil the mixture and collect the distillation fraction between 77–82°C.

The distillation fraction should easily burn.

Fermentation of glucose using yeast student sheet

Fermentation of glucose using yeast teacher notes, additional information.

This is a resource from the  Practical Chemistry project , developed by the Nuffield Foundation and the Royal Society of Chemistry. This collection of over 200 practical activities demonstrates a wide range of chemical concepts and processes. Each activity contains comprehensive information for teachers and technicians, including full technical notes and step-by-step procedures. Practical Chemistry activities accompany  Practical Physics  and  Practical Biology . Updated in 2024 with additional student questions by Neil Goalby.

© Nuffield Foundation and the Royal Society of Chemistry

More Neil Goalby

Carbon allotropes | Johnstone’s triangle worksheets | 14–16 years

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Metallic bonding | Johnstone’s triangle worksheets | 14–16 years

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Specification

• Ethanol is produced industrially by fermentation of glucose. The conditions for this process.
• Ethanol produced industrially by fermentation is separated by fractional distillation and can then be used as a biofuel.
• AT.3 Use of appropriate apparatus and techniques for conducting and monitoring chemical reactions, including appropriate reagents and/or techniques for the measurement of pH in different situations.
• Aqueous solutions of ethanol are produced when sugar solutions are fermented using yeast. Students should know the conditions used for fermentation of sugar using yeast.
• AT4 Safe use of a range of equipment to purify and/or separate chemical mixtures including evaporation, filtration, crystallisation, chromatography and distillation.
• 9.33C Describe the production of ethanol by fermentation of carbohydrates in aqueous solution, using yeast to provide enzymes
• 9.34C Explain how to obtain a concentrated solution of ethanol by fractional distillation of the fermentation mixture
• 3 Use of appropriate apparatus and techniques for conducting and monitoring chemical reactions, including appropriate reagents and/or techniques for the measurement of pH in different situations
• Use of appropriate apparatus and techniques for conducting and monitoring chemical reactions, including appropriate reagents and/or techniques for the measurement of pH in different situations
• (s) how ethanol (an alcohol) is made from sugars by fermentation using yeast

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• Fermentation of Food

Testing Substrate Specificity in Yeast Fermentation

As Buchner discovered at the turn of the 20th century, the process of fermentation is a multistep, enzyme-catalyzed reaction. Inherent to maximal enzyme action is a defined set of optimal conditions and substrates. Therefore, the enzymes responsible for glycolysis and subsequent fermentation reactions will exhibit optimal reaction rates in an environment that mimics physiological conditions. To demonstrate this point, we will use “active dry” yeast packets from the supermarket (such as Fleischmann’s or Red Star brands). This common product is a freeze dried collection of Saccharomyces cerevisiae , also known as “baker’s yeast.”

Naturally found on ripe fruits, like grapes, as well as on and in the human body, S. cerevisiae is a facultative anaerobe, and its biodiversity and carbon utilization is dictated by the carbon and energy sources available in its specific habitat. This demonstrates the incredible metabolic flexibility housed in these tiny eukaryotes, however, some substrates are more efficiently metabolized than others. In the absence of oxygen, S. cerevisiae will switch on its fermentation pathway as a mechanism to maintain a favorable cellular redox status (fermentation regenerates NAD + , which is essential for glycolysis), generating ethanol and carbon dioxide as byproducts. 

To understand how different sugar substrates are utilized by S. Cerevisiae , we can measure the amount of CO 2 produced. If you recall the stoichiometry for fermentation, for every mole of glucose, yeast cells will produce two moles of CO 2 , which makes a quantification of sugar metabolism fairly straightforward. While scientists have invented a number of devices to quantifiably measure the rate CO 2 production resulting from fermentation in yeast, these devices are not practical for classroom settings. Here we will use a basic 15ml conical tube (such as Falcon or Corning brands) with gradation markings as a device to measure CO 2 production in response to a variety of carbon sources.

Corner Store: Grocery Items

• Dextrose (glucose), Galactose, Lactose, Maltose, Sucrose (table sugar)
• Baker’s Yeast Packets or a jar

Common Items

Laboratory equipment.

• Water bath, hot plate, or pan & stove
• Beakers or flat-bottomed bowl/baking dish
• Micropipettes & tips, sterile transfer pipettes (or straws or medicine syringe)
• 15mL Conical Tubes and a needle OR test tubes and glass slides to cover

Preparation

• Prepare sugar stock solutions at 40% w/v (40g per 100ml of H 2 O)
• For a recommended starting concentration of 0.5%, v/v, you’ll need 0.5 ml stock in 10 ml final volume
• Heat water baths and/or hot plates to 4 0 ° C
• Warm all solutions to be used in the 40 °C bath
• Prepare a gas-collection setup (see below)
• Immediately before the experiment, prepare the 7% yeast solution in H 2 O

In the example at right, plastic tubes were used with a few holes poked into each plastic cap using a needle. The tubes were filled with yeast solution and food source (e.g. sucrose), covered, and inverted quickly into the water bath. The samples should be held inside the tubes and displaced into the water bath as gas is produced.

A similar setup can be used with test tubes and stoppers with holes (cover with your finger when inverting) or using a glass slide to invert each tube into the bath before sliding off the slide and allowing the fermentation gas produced to displace the yeast solution. You may also be able to use a typical gas collection apparatus from a chemistry laboratory at your school.

Finally, volumes of gas can be determined by emptying a marked tube and filling it with water exactly to the mark. Then that water can be poured into a graduated cylinder of appropriate size to determine the volume of gas produced.

• Fill 15ml conical tube with 8ml of a sugar solution
• Mix the 7% yeast solution to be a uniform suspension. Fill the remainder of the tube (~7ml) with yeast solution such that the meniscus rises above the lip of the tube. 
• NOTE: make sure that there are no sizeable air bubbles in the tube
• NOTE: you can also test the effect of temperature on fermentation by adjusting temperature of water bath or hot plate
• Immediately mark the bottom of the CO 2 bubble (if there is one). Mark this point at 5 minute intervals for 30 minutes.
• At the end of the experiment, record the level of CO 2 produced at each time interval by emptying the tube, filling with water to the mark, and pouring the water into a graduated cylinder or onto a balance for accurate measurement

What conclusions can you draw about the metabolism efficiency of different substrates by  S. cerevisiae ?

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Article Contents

Introduction, sucrose in nature and in human society, natural occurrence and importance of s. cerevisiae in human history, sucrose as an important industrial substrate for s. cerevisiae, molecular background of sucrose consumption in s. cerevisiae, physiology of s. cerevisiae during growth on sucrose, engineering sucrose utilization by s. cerevisiae.

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Sucrose and Saccharomyces cerevisiae : a relationship most sweet

• Article contents
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• Supplementary Data

Wesley Leoricy Marques, Vijayendran Raghavendran, Boris Ugarte Stambuk, Andreas Karoly Gombert, Sucrose and Saccharomyces cerevisiae : a relationship most sweet, FEMS Yeast Research , Volume 16, Issue 1, February 2016, fov107, https://doi.org/10.1093/femsyr/fov107

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Sucrose is an abundant, readily available and inexpensive substrate for industrial biotechnology processes and its use is demonstrated with much success in the production of fuel ethanol in Brazil. Saccharomyces cerevisiae , which naturally evolved to efficiently consume sugars such as sucrose, is one of the most important cell factories due to its robustness, stress tolerance, genetic accessibility, simple nutrient requirements and long history as an industrial workhorse. This minireview is focused on sucrose metabolism in S. cerevisiae , a rather unexplored subject in the scientific literature. An analysis of sucrose availability in nature and yeast sugar metabolism was performed, in order to understand the molecular background that makes S. cerevisiae consume this sugar efficiently. A historical overview on the use of sucrose and S. cerevisiae by humans is also presented considering sugarcane and sugarbeet as the main sources of this carbohydrate. Physiological aspects of sucrose consumption are compared with those concerning other economically relevant sugars. Also, metabolic engineering efforts to alter sucrose catabolism are presented in a chronological manner. In spite of its extensive use in yeast-based industries, a lot of basic and applied research on sucrose metabolism is imperative, mainly in fields such as genetics, physiology and metabolic engineering.

Yeasts are the major producers of biotechnology products worldwide, exceeding production by any other group of industrial microorganisms. In this scenario, Saccharomyces cerevisiae is the principal cell factory, which is mainly due to a long history of safe use, and consequently its Generally Regarded As Safe (FDA, USA) status; an extensive understanding of its physiology; and the availability of genetic systems for cloning and expression (Demain, Phaff and Kurtzman 2011 ). Saccharomyces cerevisiae was the first eukaryotic cell that had its complete genome sequenced (Goffeau et al . 1996 ) and also the first eukaryote for which an in silico genome-scale metabolic model was reconstructed (Förster et al . 2003 ).

Besides its use in the food and beverage markets, S. cerevisiae is also applied for the production of heterologous proteins, pharmaceuticals, bulk and fine chemicals (Hensing et al . 1995 ; Bekatorou, Psarianos and Koutinas 2006 ; Ro et al . 2006 ). A frequent bottleneck in these bioprocesses is substrate cost, which can overshadow product advantage, especially when petroleum-derived products are the competitors (Abbott et al . 2009 ).

In tropical countries, such as Brazil, sucrose obtained from sugarcane has been used as a substrate in biorefineries for several decades. The Brazilian fuel ethanol industry successfully demonstrates the cost effectiveness of cane sugar (Brazilian Sugarcane Industry Association 2015a). Despite recent progress in second-generation fuel ethanol, in which lignocellulosic hydrolysates are used as a substrate, sucrose still remains as a preferred and abundant carbon and energy source for yeast fermentations, in great part due to its low price, when compared to other substrates (Maiorella et al . 2009 ; Gombert and van Maris 2015 ). Nevertheless, there are still a number of scientific challenges in sucrose fermentation that remain to be addressed. These challenges and the recent scientific achievements in this field constitute the focus of this minireview, as well as a brief history of sucrose and yeast usage by humankind.

Natural occurrence of sucrose and its role in nature

Sucrose (α-D-glucopyranosyl-(1↔2)-β-D-fructofuranoside) is the most abundant free low molecular weight carbohydrate in the world (Peters, Rose and Moser 2010 ). It can be synthesized by a wide range of organisms including some prokaryotes (photosynthetic proteobacteria, cyanobacteria, planctomycetes and firmicutes) (Reed and Stewart 1985 ; Khmelenina et al . 2000 ; MacRae and Lunn 2012 ) and eukaryotes (single-celled photosynthetic protists and green plants) (Porchia and Salerno 1996 ). Two enzymes are essential for sucrose biosynthesis: sucrose phosphate synthase (SPS, EC 2.4.1.14) and sucrose phosphate phosphatase (SPP, EC 3.1.3.24) (Fig. 1A ). SPS synthesizes sucrose 6-phosphate from fructose 6-phosphate (an intermediate from the Calvin–Benson cycle) and a nucleoside-diphosphoglucose (usually UDP-glucose, which can be obtained from fructose 6-phosphate). Next, SPP hydrolyzes sucrose 6-phosphate into orthophosphate and sucrose (MacRae and Lunn 2012 ). Besides SPS and SPP, there is another enzyme that can synthesize sucrose called Sucrose synthase (SuSy; EC 2.4.1.13). SuSy catalyses the reversible synthesis of sucrose from NDP-glucose and fructose (Fig. 1B ). However, in general, this enzyme acts towards sucrose cleavage without major impacts for photosynthetic sucrose synthesis (Geigenberger and Stitt 1993 ; Ruan 2014 ).

Sucrose biosynthesis and cleavage. ( A) Biosynthesis: two enzymes are essential: SPS and SPP. These enzymes probably originated in bacteria and were transferred to plants through the cyanobacterial ancestor of chloroplasts (MacRae and Lunn 2012 ). ( B) Sucrose cleavage via sucrose synthase (SuSy) in green plants; via hydrolases in S. cerevisiae (e.g. Sucp; Malx2p and Imap) and via phosphorolysis in bacteria such as P. saccharophila (SPase: sucrose phosphorylase).

The main roles played by sucrose in biological systems are related to osmoregulation, tolerance to temperature and desiccation, cell signalling and carbon transport and storage (MacRae and Lunn 2008 ). Mutant cyanobacteria that are unable to synthesize sucrose are still viable. However, in green plants, sucrose biosynthesis is a prerequisite for life (Salerno and Curatti 2003 ). This is the reason why sucrose is widespread in Viridiplantae (green algae and the land plants which evolved from them). It can be found in green algae (e.g. Chlorophyceae and Ulvophyceae) (Winkenbach, Grant and Bidwell 1972 ; Salerno 1985a , b ; Kolman et al. 2015 ) and in Streptophyta (e.g. Charales and Embryophytes) (Macrae and Lunn 2012 ). In Bryophytes, for instance, this disaccharide protects the organism against desiccation (Smirnoff 1992 ). Among Tracheophyta, Monilioformopses (ferns and their allies) have genes related to sucrose synthesis (Hawker and Smith 1984 ). Also, sucrose metabolism in Gymnosperms is barely studied, a notable exception being the conifers where sucrose synthesis and degradation are tightly related to seasonal changes (Egger et al . 1996 ). On the other hand, studies in Angiosperms have revealed sucrose as the major form of carbon transport among plant tissues (Ayre 2011 ; Macrae and Lunn 2012 ). The physicochemical properties of sucrose could be the reason for this preference (Kühn et al . 1999 ). The viscosity of sucrose solutions is low even in highly concentrated solutions (e.g. phloem sap, 200 to 1600 mM), allowing high translocation rates (0.5–3 m h −1 ). Since sucrose is a non-reducing sugar, it can be accumulated in high amounts inside the cells, without reacting with proteins or other molecules, as do reducing sugars such as maltose, glucose or fructose. One possible disadvantage could be the size of the molecule, i.e. only a few carbon atoms are transported, since sucrose is a disaccharide and not a larger polymer. However, this is compensated by the high osmotic potential created at similar weight/volume ratios, thereby increasing phloem transport efficiency (Lang 1978 ; Kühn et al . 1999 ; van Bell 1999 ). A more detailed review on sucrose biosynthesis is well described by MacRae and Lunn ( 2008 , 2012 ).

Sucrose and human society

According to Shaffer ( 2001 ), sugar crystallization started around 350 AD in India. Originally from Southeast Asia, sugarcane ( Saccharum spp .) was the first sucrose source utilized by humans and its domestication started about 8000 BC in New Guinea (Roach and Daniels 1987 ). Recent sugarcane varieties can accumulate up to 12–20% (w/w) sucrose in the internods (Linglea et al . 2009 ). In addition to its applications in cooking, sucrose was also used as a medicine by Greeks (UCLA 2002 ). Later, during the Arab agricultural revolution in the seventh century, sugar production increased due to the advent of sugar mills and larger plantations (Watson 1974 ). During the crusades in the 11th century, sugar was brought to Europe, where it supplemented honey, the only sweetener available at that time. However, sucrose remained as a luxury product until its price decay was caused by the extensive and cheaper production in the New World in the 16th century (Mintz 1986 ).

In 1747, sucrose was first crystallized from sugar beet ( Beta vulgaris ) by the German scientist Andreas Marggraf ( 1747 ). Soon after, his student, Franz K. Achard, built the first sugar factory based on this temperate-climate crop (Achard 1799 ; Wolff 1953 ). The sucrose content of sugar beet is about 16–19% (w/w) and the world average yield of harvested sugar beet is around 60 metric tons per hectare. Each hectare produces approximately 10–12 tonnes of sugar (Hoffmann 2010 ; CEFS 2013 ; FAO 2015a ). The main sugar beet producer is the Russian Federation (39.3 million metric tons harvested in 2013). By region, the European Union is the main producer of this cultivar with approximately 167 million metric tons harvested in 2013 (67.9% of the world production) (FAO 2015a ). Besides the edible sugar market, about 10% of the aforementioned amount is destined for the production of ethanol (ARD 2012 ). Despite the high ethanol yield (7000 litres per hectare from sugar beet, compared to 5000 litres/hectare from sugarcane and 3000 litres/hectare from corn) (Nersesian 2010 ), sugar beet use in the ethanol industry remains ‘not promising’ due to its costly and energy intensive processing, when compared to other European alternatives such as wheat and other cereals (Nersesian 2010 ; ARD 2012 ).

Sugarcane and sugarbeet constitute the main sources of edible sugar currently produced, with sugarcane accounting for approximately 80% of the world sugar production (ARD 2015 ). Besides these sources, date palm ( Phoenixdactylifera ), sorghum ( Sorghum vulgare ) and the sugar maple ( Acer saccharum ) are other minor commercial sugar crops (van Putten, Dias and de Jong 2013 ).

Brazil is the world leader in sugar and sugarcane production with more than 653 million tons harvested in the crop year 2013/2014, twice the amount produced by India, the second largest producer (Brazilian Sugarcane Industry Association 2015a ; FAO 2015b ). For the crop season 2015/2016, an increment of 18 million tons is expected due to more favourable rainy conditions. In Brazil, around 50% of the harvested sugarcane is used for producing edible sugar, and the rest is employed for fuel ethanol production (Brazilian Sugarcane Industry Association 2015b ). Brazilian sugarcane plantations yield approximately 70–80 metric tons per hectare (Sugarcane Technology Center 2011 ). Concerns about the use of sugarcane to produce biofuels/biochemicals instead of food are still real and somewhat polemical. To assuage the critics, it is important to highlight that only 1.1% (≈9 million hectares) of the Brazilian territory is currently used for sugarcane plantation (Brazilian Sugarcane Industry Association 2015a ) and the latest national agro-ecological zoning reports the existence of additional 65 million hectares available for sugarcane culture, without making use of protected areas (e.g. Amazon forest) (Manzatto et al . 2009 ). Although Brazilian intellectual property regulations still require substantial improvements, Brazil has become a hotspot for biotech industries due to the low cost of feedstock (mainly sucrose from sugarcane) by the well-established sugarcane crushing industry (Nielsen 2012 ).

The Latin word ‘ Saccharomyces ’ literally means ‘sugar fungus’ and clarifies that this ascomycetous genus is preferentially found in sugar-rich environments (Gerke, Chen and Cohen 2006 ). Saccharomyces cerevisiae in particular is characterized by a long history of coexistence with Homo sapiens due to its role in the manufacture of bread, wine, sake and beer, among others (‘ cerevisiae ’ is a Latin word for ‘of beer’) (Schneiter 2004 ). Humans have gradually incorporated yeast in their diet, and Bacteroides thetaiotaomicron (and a limited number of other Bacteroidetes ) present in the human gut microbiota have evolved a complex machinery to metabolize the highly complex yeast cell-wall mannans. While most of the gut microbes target the components derived from the human diet, Bacteroides digests the human domesticated and ingested yeasts, thereby contributing to the overall activity of the human microbiota and, consequently, to human health (Cuskin et al . 2015 ).

The DNA of S. cerevisiae was found in wine jars from the tomb of the King Scorpion, in Abydos (3,150 BC) (Cavalieri et al . 2003 ) and the earliest evidence for winemaking dates back to 7000–5500 BC from pots found in China (McGovern et al . 2004 ). This long history of domestication led to the concept that natural isolates of S. cerevisiae would be ‘refugees from human-associated cultures’ instead of truly ‘wild’ exemplars (Mortimer 2000 ; Plech, De Visser and Korona 2014 ). However, recent genomics studies provide strong evidence for the presence of ‘wild’ S. cerevisiae in nature (Fay and Benavides 2005 ; Liti et al . 2009 ; Wang et al . 2012 ; Cromie et al . 2013 ; Leducq 2014 ; Plech, De Visser and Korona 2014 ). Wang et al . ( 2012 ) isolated S. cerevisiae from environments close and far from human activity, and added eight new lineages (named CHN I to CHN VIII) to the five previously known ‘wild’ strains (Liti et al . 2009 ). They show evidence that indicates primeval forests, situated in Far Eastern Asia, as the origin of the S. cerevisiae species. For instance, the oldest lineage CHN I and other basal ones (CHN II-V) were only found in China. In other words, these authors present evidence that any S. cerevisiae lineage associated to human activity worldwide was originated from wild lineages from China (Wang et al . 2012 ). In nature, S. cerevisiae species can be isolated from a vast range of habitats such as oak and beech bark, plant exudates, soil underneath trees (e.g. forest and orchard soil) (Bowles and Lachance 1983 ; Sniegowski, Dombrowski and Fingerman 2002 ; Fay and Benavides 2005 ; Sampaio and Gonçalves 2008 ) in fruits (e.g. fig, Lychee), in flower nectars (e.g. from Bertram palm; Liti et al . 2009 ), in rotten wood (Wang et al . 2012 ), in stromata from the obligate tree parasite ascomycetes (e.g. Cyttaria hariotii , since their fructifying body is rich in sugars; Libkind et al . 2011 ), in the intestines of insects (Stefanini et al . 2012 ), in human infections (Wheeler et al . 2003 ; Muller et al . 2011 ), etc. According to Goddard and Greig ( 2015 ), this vast range of habitats points towards a nomad model to understand yeast ecology. In support to this model is the highly diverse tolerance spectrum of yeasts, towards, for instance, pH, osmolarity and temperature (Petrovska, Winkelhausen and Kuzmanova 1999 ; Serrano et al . 2006 ; Salvado et al . 2011 ), as well as the low density of S. cerevisiae in habitats such as fruits and oak barks, which contradicts the idea that these might be the species' niche (Taylor et al . 2014 ; Kowallik, Miller and Greig 2015 ). Therefore, it is perfectly possible that S. cerevisiae is a ‘nomad, able to survive as a generalist at low abundance in a vast ranges of habitats’ (Goddard and Greig 2015 ).

Saccharomyces cerevisiae , as other strains in the same genus, is capable of consuming several different substrates as carbon sources (e.g. sucrose, maltose, glycerol, ethanol, etc.) (Samani et al . 2015 ). Opulente et al . ( 2013 ) compared patterns of sugar consumption and structure of metabolic pathways in 488 different Saccharomyces strains. Based on this, the authors were able to ‘partially predict’ the substrate specificity of a strain based on the environment from which it was isolated (Opulente et al . 2013 ). Because S. cerevisiae has the metabolic capacity for sucrose consumption (Grossmann and Zimmermann 1979 ), one of the main questions that arise is: Where, in nature, does S. cerevisiae feed on sucrose? Experiments with plants show accumulation of sucrose in wounded tissues, rather than other sugars such as glucose and fructose (van Dam and Oomen 2008 ; Schmidt, Schurr and Röse 2009 ). During certain periods, when glucose sources such as fruits and flower nectar are not available, yeasts could grow on sucrose present in plant exudates (e.g. as a consequence of insect damage), as speculated by Lemaire et al . ( 2004 ). Furthermore, it is also possible that S. cerevisiae spores remain dormant until the environmental conditions get favourable again. According to Neiman ( 2011 ), the ecological role of sporulation might be related to yeast dispersion via insects as vectors.

Besides its use as sweetener, sucrose has been explored by humans as an industrial substrate for the microbial production of different compounds/products or, in some cases, the yeast itself is the desired product. Around 400 million kilograms of yeast biomass are produced each year worldwide (Gómez-Pastor, Pérez-Torrado and Matallana 2011 ). Industrial production and commercialization of yeast started at the end of the 19th century, after being intensively studied by Louis Pasteur, who first demonstrated the role of yeast in alcoholic fermentation ( 1857 ). Today, yeast cells (in different formulations) are used as animal feed, in the bakery and fermentation industries (brewing, beverages, biofuels, pharmaceutical, enzymes and chemicals) (Swanson and Fahey 2004 ; Bekatorou, Psarianos and Koutinas 2006 ).

More than 4000 years ago, in ancient Egypt, yeast fermentation was already employed to leaven bread (Sugihara 1985 ). Today, S. cerevisiae is employed in the bakery industries all over the world. In many cases, sucrose is added to the dough up to 30% (w/w), causing a collateral osmotic stress (Sasano et al . 2012 ). Besides osmotolerance, other important traits of yeast in bread making processes have been the object of intensive research in the recent years, such as rapid fermentation rates, capacity to endure freeze-thawing stress and production of large amounts of CO 2 (Randez-Gil, Córcoles-Sáez and Prieto 2013 ).

The main advantages of S. cerevisiae as a host for the production of heterologous enzymes are correct protein folding, post-translational modifications and efficient protein secretion (Mattanovich et al . 2012 ; Nielsen 2013 ), as demonstrated in the production of insulin by Novo Nordisk. Despite the advantages mentioned above, the following disadvantages could limit its extensive use as protein factory: (i) high-mannose type N-glycosylation, which results in a reduced half-life of the glycoprotein in vivo , which prejudices its therapeutic use (Nielsen 2013 ); (ii) retention of the exported protein in the periplasmic space; (iii) S. cerevisiae metabolism is preferentially fermentative (Crabtree effect, discussed further below), which prejudices biomass propagation (Nevoigt 2008 ).

Besides its use in the production of recombinant proteins, S. cerevisiae is also an attractive industrial host for fine and bulk chemicals production. Compared to chemical synthesis or extraction from nature, industrial microbiology requires less energy input, has decreased generation of toxic wastes and, most importantly, is based on renewable feedstock utilization (Demain, Phaff and Kurtzman 2011 ). Lactic acid production, for instance, is carried out using fermentation with lactic acid bacteria. However, pH control represents a considerable manufacturing cost in these processes (Bozell and Petersen 2010 ). Due to its higher physiological activity in acidic conditions, S. cerevisiae is a great alternative for the production of lactic and other organic acids (van Maris et al . 2004 ; Abbott et al . 2009 ). Another example is succinic acid, which has a market size around US$ 7 billion and recently started to be produced with engineered S. cerevisiae to compete with petroleum counterparts (Jansen, van de Graaf and Verwaal 2012 ; Myriant 2012 ; Reverdia 2012 ).

Fuel ethanol production is, by far, the largest industrial activity that uses sucrose as a substrate for yeast fermentation (at least in Brazil). Sugarcane juice contains by weight 8–20% sucrose and 0.3-2.5% of reducing sugars, e.g. glucose and fructose (Basso, Basso and Rocha 2011 ; OECD 2011 ). Despite the high sugar concentration, sugarcane juice is deficient in phosphorous and nitrogen. The composition varies depending on the sugarcane variety and maturity, the soil composition and the climate, as well as juice processing conditions (OECD 2011 ). In Brazilian industrial mills, sugarcane juice is also used for edible sugar production, which generates a sugar rich by-product called ‘molasses’. Molasses is composed of 45–60% (w/w) sucrose, 5–20% (w/w) glucose and fructose, low levels of phosphorus and high levels of minerals such as potassium and calcium, and some yeast growth inhibitors (Basso, Basso and Rocha 2011 ; OECD 2011 ). Molasses is diluted in water to a final sugar concentration of about 14–18% and added to the fermentation reactor in addition to sugarcane juice (Amorim et al . 2011 ).

Another example of a sucrose-rich substrate already used in industry is sugar beet, which can be converted into ethanol (ARD 2012 ). According to Ogbonna, Mashima and Tanaka ( 2001 ), sugar beet juice (16.5% sucrose, w/w) is complete in nutrients required for S. cerevisiae growth and ethanol production, and inhibitory compounds are not present in detrimental levels.

One key step in sucrose metabolism in S. cerevisiae is its cleavage by invertase (β-fructofuranosidase, EC 3.2.1.26) into glucose and fructose (Fig. 1B and Table 1 ). Other organisms can cleave sucrose in different ways. Besides the reaction carried out by plant sucrose synthase (mentioned before, Fig. 1B ), some bacteria (e.g. Pseudomonas saccharophila ) express sucrose phosphorylase, an enzyme that converts sucrose and inorganic phosphate into fructose and glucose 1-phosphate (Fig. 1B ) (Weimberg and Doudoroff 1954 ; Goedl et al . 2010 ).

Saccharomyces cerevisiae enzymes that hydrolyse sucrose.

The authors do not specify which invertase(s), i.e. which gene-encoded proteins, were assayed. It may be SUC2 and/or its paralogues depending on the yeast strain used.

Mal12p and Mal32p have similar hydrolytic parameters because they are 99.7% identical at the amino acid level (Voordeckers et al . 2012 ).

IMA3 ORF is 100% identical to IMA4 at the nucleotide level (Teste, François and Parrou 2010 ).

Saccharomyces cerevisiae 's invertase was already studied more than 100 years ago and was the enzyme used by Michaelis and Menten for their classic paper ‘ Die Kinetik der Invertinwirkung ’ (Berthelot 1860 ; Brown 1902 ; Michaelis and Menten 1913 ; Johnson and Goody 2011 ). This enzyme is named invertase because the hydrolysis of sucrose causes an inversion of optical rotation in the sugar solution, from positive to negative. The easiness of optical rotation determination is the reason why invertase was already studied during the early 20th century. Besides sucrose, invertase can also hydrolyse raffinose (α-D-galactopyranosyl-(1→6)-α-D-glucopyranosyl-(1↔2)-β-D-fructofuranoside) producing fructose and melibiose (α-D-galactopyranosyl-(1→6)-α-D-glucopyranoside), and the polysaccharide inulin (linear chains of β-2,1-linked D-fructofuranose molecules terminated by a glucose residue) (Gascón and Lampen 1968 ; Wang and Li 2013 ; Yang et al . 2015 ). Yeast invertase has also a low transfructosylating activity, allowing the synthesis of fructo-oligosaccharides from sucrose (Lafraya et al . 2011 ).

In S. cerevisiae , sucrose consumption starts with its hydrolysis by invertase in the periplasmic space (outside of the cells, between the cell wall and the cytoplasmic membrane). Subsequently, the monosaccharides (glucose and fructose) enter the cells by facilitated diffusion and become available for their intracellular phosphorylation by gluco- and hexokinases, which corresponds to the first enzymatic step in the classical Embden-Meyerhof-Parnas glycolytic pathway.

Yeast invertase is encoded by the so-called SUC genes, which constitute a gene family originally identified by Winge and Roberts ( 1952 ) and later confirmed by Hawthorne ( 1955 ). Nine SUC genes ( SUC1-SUC5, SUC7-SUC10 ) have been already found in telomeric loci in different chromosomes and S. cerevisiae strains (Korshunova, Naumova and Naumov 2005 ; Naumov and Naumova 2010 ). SUC2 is the only one positioned in a subtelomeric region (left end of chromosome IX; however, this position can vary according to the strain, Naumov and Naumova 2011 ), and is postulated as the ancestral gene since it can be found in every S. cerevisiae strain, as well as in other Saccharomyces yeasts, such as S. paradoxus (Carlson and Botstein 1983 ; Naumov et al . 1996 ). Nevertheless, there is significant sequence variation in the SUC2 gene from these Saccharomyces yeasts, and this sequence variation has been proposed as a method to identify different yeast strains (Oda et al . 2010 ).

The molecular characterization of five SUC genes ( SUC1 - SUC5 ) present in different S. cerevisiae strains revealed that all these genes encode functional invertases (Grossmann and Zimmermann 1979 ; Hohmann and Zimmermann 1986 ). Regarding the other SUC genes ( SUC7 - SUC10 ), they have been only studied at the genetic level (chromosomal location and gene nucleotide sequence). The results published recently by Naumova et al . ( 2014 ) show that while the sequences of SUC2 from 17 different S. cerevisiae strains have 98.9-100% similarity, in the case of the other telomeric invertase genes the one closer to SUC2 is SUC1 (95.4-95.6% identity), while the other SUC genes ( SUC3 - SUC5 and SUC7 - SUC10 ) are 99.4–100% identical to each other and have a similarity of 92.3–95.6% to SUC2 . Their data also show that SUC3 and SUC5 have identical nucleotide sequences. All other Saccharomyces yeasts ( S. arboricola , S. bayanus , S. cariocanus , S. paradoxus , S. kudriavzevii and S. mikatae ) seem to have a single invertase gene with an overall 88.0–99.8% identity. All these SUC genes seem to encode functional invertases, since most nucleotide polymorphisms are silent (Naumova et al . 2014 ).

Baker's, brewer's and distiller's yeasts were found to contain multiple copies of SUC genes, and it was postulated that this reflects an adaptation to sucrose-rich broths (Codón, Benítez and Korhola 1998 ; Naumova et al . 2013 ). However, the Brazilian industrial fuel ethanol yeast strains (e.g. BG-1, CAT-1, PE-2, SA-1 and VR-1; Stambuk et al . 2009 ; Babrzadeh et al . 2012 ) and wine strains contain only one copy of SUC2 , such as the laboratory strains S288c and those from the CEN.PK family (Carlson and Botstein 1983 ). According to Stambuk et al . ( 2009 ), invertase activity in these sugarcane industrial strains is probably not a limiting step in sucrose catabolism.

Besides secreted invertases, S. cerevisiae also produces cytosolic forms of invertase. The SUC2 gene can be transcribed into two different mRNAs that differ in their 5’ ends, with lengths 1.9 and 1.8 Kb, respectively. The longer one includes the coding sequence for a signal peptide (20 amino acids) that directs the protein into the secretory pathway (Carlson and Botstein 1982 ; Perlman, Halvorson and Cannon 1982 ; Hohmann and Gozalbo 1988 ). Both invertase types behave similarly with respect to pH and temperature, with optima in the range of pH 4.6-5.0 and 35ºC –50ºC (Gascón and Lampen 1968 ). The intracellular form is a homodimer that weighs about 120–135 kDa. The extracellular form is also a homodimer, which aggregates into tetramers, hexamers and/or octamers. Glycosylation occurs only in the extracellular form and contributes to 50% of the protein mass, which is about 240–270 kDa for the homodimer (Gascón and Lampen 1968 ; Gascón, Neumann and Lampen 1968 ; Trimble and Maley 1977 ; Deryabin et al . 2014 ). Glycosylation renders invertase resistant to attack by proteases, allows proper protein oligomerization and traps this enzyme between the plasma membrane and the cell wall (Esmon et al . 1987 ; Tammi et al . 1987 ; Reddy et al . 1988 ).

Invertase belongs to family 32 of the glycoside hydrolases (GH32) that includes inulinases, levanases and transglycosylases with fructose transferase activity (Cantarel et al . 2009 ). GH32 enzymes have a characteristic N-terminal 5-fold β-propeller catalytic domain surrounding a central negatively charged active site cavity, and an additional β-sandwich domain appended to the catalytic domain. An aspartate located close to the N-terminus acts as the catalytic nucleophile and a glutamate acts as the general acid/base catalyst. Despite the long history of research on yeast invertase, the high degree of glycosylation of this enzyme challenged the determination of the crystal structure of the protein (Sainz-Polo et al . 2012 , 2013 ). The molecular mass of the purified intracellular invertase (expressed in Escherichia coli ) is 428 kDa, consistent with an octamer association which is best described as a tetramer of dimers that oligomerize by intersubunit extension of the two β-sheets that end in the β-sandwich domain within each subunit. The intracellular enzyme has two classes of dimers (‘open’ and ‘closed’) located at opposite vertices of the octameric rectangle. The ‘closed’ dimers form a more narrowed pocket at the active site (when compared to the ‘open’ domains), and are unable to accommodate oligosaccharides with more than three or four sugar units. Interestingly, the model for the extracellular invertase predicts an octameric aggregate of only ‘closed’ dimers, which may explain its predominant invertase (and not inulinase) character at the molecular level (Sainz-Polo et al . 2012 , 2013 ).

The utilization of sucrose by S. cerevisiae was also a nice model to unravel the complex regulation of glucose repressible genes in yeast. Mutants defective in sucrose utilization were isolated by Carlson, Osmond and Botstein ( 1981 ), and besides mutations in the SUC2 gene, these authors were able to isolate also several new snf − ( s ucrose n on- f ermenting) mutants (Neigeborn and Carlson 1984 ) that were shown to play key roles in glucose repression, including SNF1 , a protein kinase required for transcription of glucose-repressed genes and several other metabolic functions in yeast (Celenza and Carlson 1984 ); SNF2 , SNF5 and SNF6 that are part of the chromatin remodelling complex involved in transcriptional regulation (Laurent, Treitel and Carlson 1991 ); SNF3 , which encodes for a low-affinity glucose sensor, with homology to sugar transporters, that regulates HXT gene expression (Özcan et al . 1996 ); and SNF4 , part of the Snf1p kinase complex (Celenza, Eng and Carlson 1989 ).

Indeed, transcriptional regulation of SUC2 is complex. Intracellular invertase is expressed constitutively at low levels, while extracellular invertase is subjected to glucose repression (Carlson and Botstein 1982 ). The repressors that have been shown to bind to the SUC2 promoter are Rgt1 (inactivated through phosphorylation by Snf3/Rgt2 in the presence of glucose), Mig1/Mig2 (inactivated through phosphorylation by Snf1 under low glucose concentration), Sfl1 (inactivated through phosphorylation by Tpk2 under low glucose concentration) and, less important, there is Sko1, which weakly binds to the SUC2 promoter. Sko1 is inactivated through phosphorylation, at the end of the HOG pathway, only under high glucose concentration. Sko1 represses the glucose transporter gene HXT1 in the absence or at low glucose. The mentioned repressors have to bind Cyc8-Tup1 to be active, besides extensive chromatin remodelling carried out by the SWI/SNF complex (Gancedo 1992 ; Trumbly 1992 ; 1998 ; Geng and Laurent 2004 ; Belinchón and Gancedo 2007 ; 2008 ; Bendrioua et al . 2014 ; Weinhandl et al . 2014 ). The role played by low amounts of glucose in the inactivation of some of these repressors is in agreement with data reported by Özcan et al . ( 1997 ). Their experiments show that SUC2 expression is about 5- to 10-fold higher in the presence of low glucose or fructose concentration (0.1% w/v) than in the absence of these sugars. Although an activator of SUC genes is predicted in all models of gene regulation, up to now the identity of such transcriptional activator is still unknown (Belinchón and Gancedo 2007 ). Dynamic regulation of gene expression using sucrose is much desired in industries where sugarcane is the feedstock. To address this issue, Williams et al . ( 2015 ) identified four genes which are differentially regulated by sucrose. Employing a heterologous RNA interference module, overexpression/repression of promoter-GFP fusion was achieved using sucrose as an inducer (Williams et al . 2015 ).

From an ecological point of view, SUC2 regulation is a classic example of an optimized strategy for the efficient consumption of mixed substrates. When glucose is not abundant or even absent in nature, SUC2 expression is probably at a basal level (Fig. 2 ). In an environment rich in sucrose, this basal invertase activity could be sufficient to create a low concentration of glucose/fructose around the cells which may cause maximum expression of SUC2 . Besides the absolute concentration of glucose/fructose, temporal changes in concentration are also connected to SUC2 expression. In other words, S. cerevisiae only maximizes the induction of genes related to glucose/fructose consumption if it is able to utilize them (Özcan et al . 1997 ; Bendrioua et al . 2014 ). On the contrary, when glucose/fructose accumulates above a certain threshold (2.5–3.2 g/L), SUC2 is repressed leading to the consumption of the hexoses already available (Meijer et al . 1998 ; Elbing et al . 2004 ). These opposite effects exerted by glucose balance invertase levels and optimize sugar consumption in S. cerevisiae (Özcan et al . 1997 ).

Dynamics of invertase expression ( A ) according to the amount of glucose/fructose released extracellularly ( B ) in a sucrose-rich environment. Initially, when glucose is zero, extracellular SUC2 is expressed at a basal level causing glucose/fructose release. SUC2 achieves its maximum expression when extracellular hexoses are around 0.001 g/L. At an extracellular glucose/fructose concentration higher than 2.5-3.2 g/L, SUC2 is repressed. When the concentration of hexoses decreases again, SUC2 repression is relieved and may return to its maximum expression levels, if hexose concentration decreases to very low levels (<0.001 g/L). Based on values reported by Özcan et al . ( 1997 ) and Meijer et al . ( 1998 ).

The extracellular hydrolysis of sucrose has been extensively studied as an interesting model for social microbial behaviour, its dynamics and evolution. The secretion of a public good (invertase) by cooperators (e.g. a SUC2 yeast strain) allows the hydrolysis of sucrose, producing glucose and fructose that diffuses away from the cooperator cell and can be consumed by other cells of the population, including cheaters (e.g. a suc2 Δ yeast strain) that will not have the metabolic cost of synthesizing the enzyme (Greig and Travisano 2004 ; Gore, Youk and van Oudenaarden 2009 ). Consequently, in well mixed batch cultures, cheaters can exploit the public good and invade populations of cooperators, depending on factors such as cell density and frequency, spatial population expansion, presence of other species of cheaters (e.g. E. coli bacteria), presence of environmental stresses and sucrose concentration (Greig and Travisano 2004 ; MacLean and Brandon 2008 ; Gore, Youk and van Oudenaarden 2009 ; MaClean et al . 2010 ; Koschwanez, Foster and Murray 2011 ; Celiker and Gore 2012 ; Dai et al . 2012 ; Damore and Gore 2012 ; Dai, Korolev and Gore 2013 ; Sanchez and Gore 2013 ; Van Dyken et al . 2013 ). However, the real significance of this ‘social trait’ in natural populations of Saccharomyces has been recently challenged since a survey of over 100 wild yeast isolates (80 strains of S. paradoxus and 30 strains of S. cerevisiae ) revealed no cheater strains (Bozdag and Greig 2014 ). All the strains had significantly high levels of invertase activity; all S. paradoxus strains had only the SUC2 gene in chromosome IX, while from the S. cerevisiae wild yeasts only three strains (isolated from sucrose-rich palm nectars) had additional SUC genes ( SUC3 , SUC8 and SUC9 ), besides SUC2 , which was present in all strains. Therefore, there is no evidence to support the idea that non-producing cheaters may occur among wild Saccharomyces yeasts (Bozdag and Greig 2014 ).

Nevertheless, S. cerevisiae strains lacking invertase activity ( suc2 mutants) are still able to consume sucrose (Badotti, Batista and Stambuk 2006 ; Badotti et al . 2008 ), suggesting the existence of alternative genes allowing sucrose consumption. Known native S. cerevisiae hydrolases that act on sucrose are listed in Table 1 . Since sucrose is also an α-glucoside, maltases (Mal12p and Mal31p) are as active on sucrose as they are on maltose (Zimmermann, Khan and Eaton 1973 ), but their catalytic efficiency (K cat /K m ) is not as high as that of invertase (Reddy and Maley 1996 ). Isomaltases (Ima1-Ima5p), which share the same ancestry with maltases, can also hydrolyze sucrose (Brown, Murray and Verstrepen 2010 ; Voordeckers et al . 2012 ). Interestingly, Ima proteins are inhibited by high isomaltose concentrations, a phenomenon that does not occur when sucrose is the substrate (Deng et al . 2014 ).

Since maltases and isomaltases are intracellular proteins, sucrose must be transported into the cytoplasm to be hydrolyzed by these enzymes. Stambuk et al . ( 1999 ) first demonstrated that a maltose proton symporter (encoded by the AGT1 gene) can also transport sucrose, besides other α-glucosides (Han et al . 1995 ). Later, Stambuk, Batista and De Araujo ( 2000 ) determined the kinetics of active sucrose transport in S. cerevisiae , revealing the presence of a high-affinity (K m = 7.9 ± 0.8 mM) sucrose transport activity mediated by Agt1p, and a low-affinity (K m = 120 ± 20 mM) transport activity by the maltose transporters encoded by MALx1 genes ( x refers to the locus number). Each MALx1 is located at a different telomere-associated MAL locus in the S. cerevisiae genome (Chow, Sollitti and Marmur 1989 ; Cheng and Michels 1991 ; Needleman 1991 ; Naumov, Naumova and Michels 1994 ; Duval et al . 2010 ). According to the known regulation of MAL genes, no sucrose transport can be observed without addition of the inducer maltose to the medium, or the strain needs to be MAL constitutive to express the transporters and enzymes that will allow sucrose utilization (Badotti, Batista and Stambuk 2006 ; Badotti et al . 2008 ).

Growth of S. cerevisiae on sucrose compared to other carbon sources

In industrial processes, S. cerevisiae is exposed to a variety of sugars other than sucrose. Grape must, for instance, is mainly composed of glucose and fructose (Fleet and Heard 1993 ). In the case of beer wort, maltose and maltotriose are also present, besides sucrose (Bamforth 2003 ). Even in sugarcane juice, which is predominantly composed of sucrose, glucose and fructose are also present (Wheals et al . 1999 ). Due to glucose repression, sugars other than glucose are only consumed after the depletion of this monosaccharide. Glucose activates signalling cascades that repress the transcription of genes necessary for the metabolism of other carbon sources (e.g. sucrose, maltose, galactose, ethanol, glycerol) (Trumbly 1992 ; Verstrepen et al . 2004 ; Kim et al . 2013 ). The presence of glucose in the medium is responsible for the so-called Crabtree effect in S. cerevisiae (De Deken 1966 ), meaning that under such conditions, even when the oxygen supply is abundant, cells perform fermentation instead of (or together with) respiration, which is a consequence of both glucose repression (described above) and insufficient respiratory capacity, also termed overflow metabolism at the level of pyruvate (Fiechter and Gmünder 1989 ). In spite of the lower amount of ATP obtained per mole of substrate consumed, the ‘Crabtree’ effect offers, at least, the following potential advantages to S. cerevisiae : (i) consumption of glucose at higher rates, meaning that the sugar becomes less available for competing organisms in the same niche; (ii) accumulation of ethanol to toxic levels, meaning that competing organism may be killed ( S. cerevisiae 's tolerance to ethanol is one of its hallmarks) and that the accumulated ethanol may be later used by S. cerevisiae as a carbon and energy source, as long as oxygen is available for respiration (Pfeiffer, Schuster and Bonhoeffer 2001 ; Verstrepen et al . 2004 ; Piškur et al . 2006 ; Hagman et al . 2013 ).

Depending on the carbon source and strain, yeast physiology can vary. The specific growth rate of strain CEN.PK122 (diploid) on glucose (μ glucose ≈ 0.38 h −1 ) is slightly lower than on sucrose (μ sucrose ≈ 0.41 h −1 ) or maltose (μ maltose ≈ 0.40 h −1 ), determined using shake-flask cultures (van Dijken et al . 2000 ). Accordingly, anaerobic batch cultures of the CEN.PK113-7D strain (haploid) also reveal faster growth on sucrose (μ sucrose = 0.35 ± 0.00 h −1 ; Basso 2011 ) than on glucose (μ glucose = 0.30 ± 0.01 h −1 ; van Hoek, van Dijken and Pronk 2000 ). The major difference between sucrose and glucose metabolism relies on the extracellular hydrolysis of sucrose by invertase, but since the CEN.PK strains are MAL constitutive, the active sucrose transport described above might be responsible for the increased growth rate on sucrose, when compared to glucose.

Another probable and maybe more accurate explanation can be related to the G protein coupled receptor GPR1 , which activates the cAMP signalling pathway, thereby increasing the glycolytic flux (Tamaki 2007 ). Lemaire at al . ( 2004 ) demonstrated that this receptor has a higher affinity for sucrose than for glucose. In accordance with the above explanation, Badotti et al . ( 2008 ) also suggest the influence of GPR1 on the faster growth of yeast on sucrose, as compared to glucose. From an ecological point of view, GPR1 can be associated to S. cerevisiae's feast/famine cycles in nature. This receptor can be activated by low sucrose concentration in famine periods, and serve for the detection of high glucose concentration during periods of feast, when fruits and flower nectar are available (Lemaire et al . 2004 ).

Growth of S. cerevisiae in a medium containing both glucose and sucrose can be divided into four phases. In the first phase, glucose is fermented and no sucrose is consumed, the respiratory quotient (RQ = moles of CO 2 produced/moles of O 2 consumed) is high (RQ ≈ 9) because there is no significant consumption of O 2 (Raamsdonk et al . 2001 ). The second phase starts after glucose depletion and is characterized by sucrose fermentation, which slightly decreases the RQ value (RQ ≈ 6) because the glucose repression effect becomes less intense. Next, after sucrose depletion, the ethanol produced in the previous phases is consumed by respiration. In this stage, no fermentation takes place and the RQ drastically drops to 0.6, in agreement to the stoichiometry of ethanol respiration. At last, when ethanol is exhausted, the acetate previously produced is consumed increasing RQ to 1 accordingly to acetate respiration stoichiometry (Dynesen et al. 1998 ; Raamsdonk et al . 2001 ).

In agreement with the classical Embden–Meyerhof–Parnas pathway coupled to ethanolic fermentation, two ATPs are produced from each glucose converted into ethanol and CO 2 by S. cerevisiae . This would lead to an ATP yield of four ATPs per sucrose consumed. However, in the case of sugars actively transported into the cells, the real yield is only three ATPs, since one ATP is consumed by H + -ATPase pumps to extrude the proton imported together with the disaccharide (Weusthuis et al . 1993 ). This difference in one ATP (25% less than the four ATP/sucrose yield) can be detected in anaerobic cultures through the biomass yield on substrate, a parameter proportional to cell free-energy yield (Verduyn et al . 1990 ; De Kok et al . 2011 ). For instance, when the CEN.PK113-7D strain is cultivated in maltose-limited anaerobic chemostats, a 25% smaller biomass yield (Y x/s (MALTOSE) = 0.072 ± 0.000 g g gluc eq −1 ) is observed, when compared to glucose- (Y x/s (GLUCOSE) = 0.095 ± 0.002 g g gluc eq −1 ) (De Kok et al . 2012 ) or sucrose-limited chemostats (Y x/s (SUCROSE) = 0.09 ± 0.01 g g gluc eq −1 ) (Basso et al . 2011 ).

Compared to growth rate values observed when S. cerevisiae is grown on glucose, sucrose or maltose as the sole carbon and energy source, growth on galactose is much slower. Saccharomyces cerevisiae strain CEN.PK122 grows with μ galactose ≈ 0.28 h −1 on galactose as the sole carbon source in aerobic shake flasks (van Dijken et al . 2000 ). This is industrially relevant since galactose is present in cheese whey (Siso 1996 ) and in lignocellulosic hydrolysates (De Bari et al . 2014 ). The reasons for this slower growth of yeast on galactose can be related to the galactose uptake rate, which is around three times slower than glucose uptake (Ostergaard et al . 2000 ). Bro et al . ( 2005 ) showed that PGM2 (phosphoglucomutase) expression limits fluxes through the Leloir pathway, which is one of the first steps in galactose metabolism (Frey 1996 ).

Saccharomyces cerevisiae 's physiology in sucrose-limited chemostats

Surprisingly, only very limited data are available describing the growth of S. cerevisiae in sucrose-limited chemostat cultivations, either under aerobiosis or under anaerobiosis. Results reported by Diderich et al . ( 1999 ), Abbott et al . ( 2008 ) and Basso et al . ( 2010 , 2011 ) can be directly compared, since the yeast strain, medium composition and chemostat parameters employed were identical.

Under anaerobiosis, the specific sucrose consumption rate is higher than during aerobiosis (Table 2 ). Because the energetic yield is lower under fermentative metabolism (when compared to respiratory metabolism), the glycolytic flux is higher, in order to guarantee enough ATP supply for cell growth and maintenance. A similar behaviour is observed for the situation in which glucose is the limiting substrate (Table 2 ).

Physiology of S. cerevisiae in aerobic and anaerobic carbon-limited chemostat cultures at a dilution rate of 0.1 h −1 * .

Cultivation conditions: 1 L working volume; 30ºC; pH 5.0; dissolved oxygen above 60% for aerobic cultures; synthetic medium according to Verduyn et al . ( 1992 ). Averages and mean deviations were obtained from duplicate experiments.

N. D.: Not determined.

Conversion of values presented in the cited references was carried out in order to establish unit uniformity.

Reactor stirrer speed is slightly different, 700 rpm for Basso et al . ( 2010 ) and 800 rpm for the other authors.

Data extracted from Abbott et al . ( 2008 ). The authors used S. cerevisiae CEN.PK113-7D and give averages ± standard deviations for three independent cultures.

Data extracted from Basso et al . ( 2011 ). The authors used S. cerevisiae CEN.PK113-7D.

Data extracted from Diderich et al . ( 1999 ). The authors used S. cerevisiae CEN.PK113-7D.

Data extracted from Basso et al . ( 2010 ). The authors used auxotrophic S. cerevisiae CEN.PK113-5D and then supplemented with uracil in the medium.

The referred ‘residual substrate’ is glucose or sucrose depending on the ‘carbon source in the medium vessel’ used.

Another parameter drastically influenced by oxygen availability in sugar-limited chemostats of S. cerevisiae is the biomass yield (Y x/s ). Y x/s with sucrose or glucose as the sole carbon source is about 5-fold higher under aerobiosis compared to anaerobiosis (Table 2 ). As mentioned before, biomass yield is directly proportional to cell ATP yield, which is higher under respiratory metabolism than in fermentative assimilation (Verduyn et al . 1990 ). Besides this, it is not possible to detect differences in the Y x/s values, when sucrose-limited chemostat cultures are compared to glucose-limited cultivations. This can easily be explained by the fact that the metabolism of both sugars results in the same amount of ATP per hexose equivalent consumed. Besides the biomass yield, the ethanol yield (Y e/s ) is also similar, when the two situations are compared.

Under aerobic conditions, at a dilution rate of 0.1 h −1 , no ethanol is produced (Table 2 ). Under these low growth rate conditions, the so-called Crabtree effect (De Deken 1966 ) is not observed and in connection with the very low residual substrate concentration, every substrate molecule is oxidized by respiration. Above dilution rates of around 0.3 h −1 (the exact value depends on the strain), the ‘Crabtree’ effect sets in, leading to respirofermentative metabolism even in fully aerated glucose-limited chemostats (Diderich et al . 1999 ; de Kock, du Preez and Kilian 2000 ).

Similar to the behaviour of ethanol, in sucrose-limited chemostats at 0.1 h −1 , glycerol is only produced by S. cerevisiae under anaerobiosis (Table 2 ). Glycerol is known as a ‘redox valve’, the role of which is the regeneration of NAD + . Unlike sugar conversion to ethanol (and accompanying CO 2 ), which is a redox neutral process, sugar conversion into biomass results in a net generation of NADH, which mainly takes place during the oxidative decarboxylation reactions related to amino acid and lipid biosyntheses. In this context, the NADH-dependent reduction of dihydroxyacetone phosphate to glycerol-3-phosphate (G3P), which is subsequently dephosphorylated to glycerol, is crucial to maintain yeast redox balance (van Dijken and Scheffers 1986 ; Bakker et al . 2001 ).

The last parameter shown on Table 2 , residual sugar substrate, is low for all the conditions presented, which shows that high-affinity hexose transporters (e.g. HXT ) are involved in the uptake of the residual sugars (Diderich et al . 1999 ; Abbott et al . 2008 ; Basso et al . 2010 , 2011 ).

To conclude, the physiology of S. cerevisiae during sucrose-limited chemostats at 0.1 h −1 seems to be highly similar to that observed on glucose, at least when the scarce available data are inspected. More quantitative data are required, in order to verify whether this behaviour holds for different strains and for different dilution rates. The use of more precise/sensitive analytical methods could aid in making these comparisons more solid. It will be interesting to see whether the critical dilution rate (which corresponds to the dilution rate value at which alcoholic fermentation sets in in aerobic sugar-limited chemostat cultivations carried out at increasing dilution rates) for sucrose-limited chemostat cultivations will or not be the same as the corresponding value observed for glucose-limited chemostat cultivations, when a particular strain is evaluated. Also, the physiology of yeast under other nutrient limitations (e.g. nitrogen) using sucrose-based media and chemostat cultivations is an unexplored area that has the potential to deliver results different from those obtained using glucose- or maltose-based media, due to the different degrees of glucose repression that is expected to take place when these different sugars are used.

Prior to the advent of metabolic engineering, many studies achieved tremendous success in elucidating the mechanisms of sucrose consumption by yeasts (Zimmermann, Khan and Eaton 1973 ; Santos et al . 1982 ; Oda and Ouchi 1991a , b ). A breakthrough study was carried out by Batista, Miletti and Stambuk ( 2004 ), who investigated the uptake of sucrose by an S. cerevisiae strain which is devoid of hexose transport. In this unique background, the authors could determine the contribution of active sugar uptake for sucrose metabolism. When the high-affinity sucrose-H + symporter gene AGT1 was deleted in the hxt -null background, the resulting strain could no longer grow on sucrose, confirming the role of AGT1 in active sucrose uptake (Table 3 ) (Batista, Miletti and Stambuk 2004 ). In another study, the AGT1 permease gene was deleted from a laboratory strain that lacks invertase activity, but can still cleave sucrose intracellularly through cytoplasmic α-glucosidases and also transport sucrose through low-affinity (for sucrose) MALx1 maltose permeases; a decreased sugar uptake was observed, with increased respiratory metabolism, leading to 1.5- to 2-fold more biomass as compared to the reference strain, with a concomitant decrease in ethanol production. The phenotype achieved (Table  3 ) is economically relevant for biomass-related applications in which ethanol is an undesired by-product (Badotti et al . 2008 ).

Previous works on the metabolic engineering of sucrose consumption in S. cerevisiae .

Compared to CEN.PK113-7D, which is the S. cerevisiae reference strain. Decreased accumulation of extracellular fructose, glucose and glycerol was also observed.

This strain was obtained by evolution in prolonged anaerobic sucrose-limited chemostats.

Later, the same research group engineered a laboratory strain of S. cerevisiae , with the aim of increasing the ethanol yield on sugar. The signal peptide encoding sequence was deleted from the SUC2 gene, in order to abolish extracellular invertase activity. In this engineered strain, sucrose has to be internalized by proton symporters, which leads to the indirect expenditure of one ATP per sucrose molecule taken up, because of the energetic cost involved in proton extrusion by Pma1p, necessary to keep intracellular pH homeostasis. In such a strain, the carbon flux towards ethanol increases, resulting in a higher ethanol yield compared to the reference strain (Table 3 ) (Basso et al . 2011 ; Stambuk et al . 2011 ). Moreover, using an evolutionary engineering approach, Basso et al . ( 2011 ) submitted the ‘i SUC2 strain’ from Stambuk et al . ( 2011 ) to a long-term sucrose-limited anaerobic chemostat cultivation. The evolved lineage could produce approximately 0.42 g ethanol g glucose eq −1 , which is around 11% higher than the yield obtained with the reference strain CEN.PK113-7D. These authors also observed that a duplication of the AGT1 gene was involved in the observed phenotype (Table 3 ), which is in agreement with transport assays that confirmed that the limiting step for efficient sucrose metabolism was sucrose transport, namely transport capacity (Basso et al . 2011 ).

An evolutionary engineering approach was also used by Koschwanez et al ., to select evolved yeast strains in low (1 mM) sucrose media. The analysis of more than 12 evolved populations (which grew better and outcompeted the parental strain in low sucrose concentrations) revealed that none of them increased sucrose transport activity, which unfortunately may reflect the genetic MAL negative phenotype of the W303 yeast, which they used in their experiments. Nevertheless, when AGT1 was overexpressed through a strong promoter, the cells could grow in 1 mM sucrose (Koschwanez, Foster and Murray 2013 ). A total of 10 evolved clonal populations had increased (3–21 fold) invertase expression, and in most cases the expression of HXT sugar transporters was also increased to facilitate hexose (from sucrose hydrolysis) uptake by the cells. However, the most predominant phenotype found in almost all the evolved strains was the ability to form multicellular clumps due to a failure in cell separation (Koschwanez, Foster and Murray 2013 ). In such clumps of cells, the hexoses produced by one cell hydrolyzing sucrose can be efficiently taken up by the adjacent cell, which will not occur if the two cells were separate and distant one from the other (Koschwanez, Foster and Murray 2011 , 2013 ). Indeed, sucrose has also been described as a potent inducer of yeast filamentation and/or pseudohyphal growth (Van de Velde and Thevelein 2008 ), which may explain why flocculant yeasts are the predominant type of yeasts isolated from the industrial production of fuel ethanol from sugarcane in Brazil (Basso et al .  2008 ).

Sucrose has been with humans since time immemorial. To eat for the sheer pleasure is a human trait and food with sugar (desserts) is a delectable treat. For the biotechnology industry, sucrose is an abundant, readily available and inexpensive substrate, mainly in tropical areas, such as in Brazil. S. cerevisiae , which naturally evolved to efficiently consume sugars such as sucrose, is currently one of the most important cell factories due to its robustness, stress tolerance, inexpensive nutrient requirements and genetic accessibility. For these reasons, this review focused on sucrose metabolism by S. cerevisiae , a surprisingly unexplored subject in the scientific literature, when compared to the knowledge accumulated on the metabolism of sugars that occur more frequently in temperate climate crops, such as maltose. Thus, it can be concluded that sucrose has been a ‘neglected’ sugar or carbon source by the research community. As described in this review, sucrose transporters and hydrolases are vast in yeast, which makes the construction of sucrose knockout strains still a challenge. Not much information is available on the physiology of S. cerevisiae grown on sucrose-based media in chemostat cultivations (only a few datasets from sucrose-limited chemostats at a dilution rate of 0.1 h −1 are available, for instance). The number of published works exploring the engineering of sucrose utilization in S. cerevisiae is rather low. Some of the key issues to be addressed in the coming years are as follows: (1) What are the similarities and differences in the physiology and regulation of metabolism of S. cerevisiae , when growth on sucrose is compared to growth on glucose or maltose? The following aspects can be considered of particular importance: the identification of all genes that should be eliminated to render a yeast strain incapable of thriving on sucrose and the degree of glucose repression to which cells are exposed during the release of glucose provoked by sucrose hydrolysis via invertase and/or via other enzymes or even chemical hydrolysis. (2) How can this knowledge be employed to improve sucrose-based industrial processes? (3) How can this knowledge lead us to a better understanding of the original habitat of S. cerevisae , before it started being in close contact with human societies?

This work was financially supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, São Paulo, Brazil) (grant number 2012/05548-1), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brasília, Brazil), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brasília, Brazil) through a PNPD grant to VR.

Conflict of interest. None declared.

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Determining the Effects of Cellular Respiration on Different Sucrose Concentration during Ethanol (Yeast) Fermentation1

To know if there is a difference between rate of cellular respiration in the yeast if different sucrose solution was added by measuring the depth or height of the bubbles (CO_2 ) produced and computing its volume by using the formula of cylindrical area was the main purpose of the study. To find the rate of cellular respiration, an experiment has been conducted. The set-ups produced different amount of bubbles was showed by the experiment, however there was a significant amount of bubbles produced by the set-ups and the rate of CO_2 production with sucrose solution compared to the small amount produced by the controlled set-up which is the tap water or the set-up with 0% of sucrose solution. Test tube 1, the controlled set-up, produced the least amount of CO2 at 883.13 mm3 at a rate of 44.16 mm3/min, followed by test tube 2, and 3. Test tube 4 with greatest sucrose concentration produced the greatest amount of CO2 at 6711.75 mm3 and at the fastest rate of 335.59 mm3/min. There is a difference on the rate of different sucrose concentrations on the cellular respiration in the yeast and the greater sucrose solution will produce more carbon dioxide was concluded by the researcher. To shake the set-ups properly and wash and dry the equipment used for measuring the sucrose solution after the solution has been transferred to avoid the mixing of the different sucrose solution for each set-up was recommended.

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Muriel Nuñez

Chiara Sanmartin , F. Venturi , Angela Zinnai , G. Andrich

Over the last two decades, most of Italian vines have produced grapes with higher sugar to total acid ratios, greater concentrations of phenols and aromatic compounds and greater potential wine quality. As a consequence, the musts obtained by these grapes are more difficult to process because of the risk of slowing or stuck of fermentation. With the aim of describing the time evolution of the sugars bioconversion during alcoholic fermentation, the kinetics of the D-glucose and D-fructose degradations, promoted by two yeast strains (Saccharomyces cerevisiae (strain C) e Saccharomyces bayanus (strain B)), was investigated using synthetic media, added or not with ethanol. The concentrations of both the substrates and the products of the sugars conversions, as well as the number of viable cells of yeasts, were determined as a function of the alcoholic fermentation time and the related kinetics constants determined. If the reaction medium contained high concentrations of both glucose and fructose, the strains showed significant different fermentatory ability. In these conditions a stuck of fermentation occurred and the remaining sugar was only fructose (strain C) or prevailing fructose (strain B). If the reaction medium contained only glucose as substrate, the strain C seemed more efficient while the kinetics behavior changed completely in presence of only fructose. On the basis of the information collected using this kinetic approach, it would be possible to develop technical data sheets, specific for each yeast strain, useful to choose the optimal microbial strain as a function of the different operative conditions. Moreover the kinetic constant of hexose conversion could be adopted as bio-markers in selection and breeding of wine yeast strains having a lower tendency for sluggish fructose fermentation.

Proceeding of the 2nd International Seminar on Chemistry

Safri Ishmayana , Robert P Learmonth

High sugar concentration is more preferred in industrial bioethanol production, as it can increase the amount of ethanol produced by the end of fermentation. However, when high sugar concentration is used in the media, yeast cells are exposed to high osmotic stress, which can affect the fermentation performance. The present experiment aimed to investigate the fermentation performance of the yeast S. cerevisiae at high sugar concentration, in poor (Yeast nitrogen base/YNB) and rich (yeast extract peptone/YEP) media. Growth parameters and sugar utilization were monitored during the fermentations. The results indicate that all three strains used had stuck fermentations, leaving 50 to 60% residual sugar. Therefore, a follow up experiment was conducted by using media of different nutritional value, including YNB with nitrogen supplementation and YEP, as well as different sugars (sucrose or glucose). Yeast cell grown in YEP had better fermentation performance indicated by higher sugar utilization. Addition of ammonium sulphate to YNB media did not change fermentation performance of the yeast cells. In YEP media, cells grown with glucose tended to maintain better viability than sucrose. Our study confirmed that nutrient availability is very important for fermentation performance. Comparison of YNB and YEP media indicates that nutritionally insufficient media are not suitable for high concentration sugar fermentation.

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Sucrose Concentration and Fermentation Temperature Impact the Sensory Characteristics and Liking of Kombucha

1 Department of Food Science, University of Massachusetts Amherst, Amherst, MA 01003, USA

David A. Sela

2 Department of Nutrition, University of Massachusetts Amherst, Amherst, MA 01003, USA

3 Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, MA 01655, USA

Alissa A. Nolden

Associated data.

Data will be available upon request from the corresponding author.

Kombucha is a fermented tea beverage consumed for its probiotics and functional properties. It has a unique sensory profile driven by the properties of tea polyphenols and fermentation products, including organic acids. Fermentation temperature and sucrose content affect the fermentation process and the production of organic acids; yet less is known about their impacts on the sensory profile and consumer acceptance. Thus, we aimed to examine the impact of sucrose concentration and fermentation temperature on sensory attributes and liking. For this study, kombucha tea was fermented at three different concentrations of sucrose and fermented at two temperatures for 11 days. Fermentation was monitored by pH, brix, and titratable acidity, and consumers ( n = 111) evaluated the kombucha for sensory attributes and overall liking. The fermentation temperature resulted in significant differences in titratable acidity, with higher temperatures producing more organic acids, resulting in higher astringency, and suppressed sweetness. The lower fermentation was reported as significantly more liked, with no difference in liking between the 7.5% and 10% sucrose kombucha samples. Fermentation temperature had the greatest impact on the sensory profile rather than sucrose concentration, which had a greater effect on the fermentation rate and production organic acids.

1. Introduction

Kombucha is a fermented tea beverage that has been consumed for centuries, dating back to 220 BCE, and was introduced from China to Eastern Europe [ 1 , 2 ]. While this beverage has been consumed for many years, it has recently garnered renewed commercial success around the world for its probiotic benefits [ 2 , 3 , 4 ]. The kombucha market is growing, with the current U.S. market valued at approximately 2.64 billion $USD as of 2021, and it is expected to reach 9.7 billion $USD by 2030 [ 3 ]. This is driven, in part, by its perceived nutraceutical properties such as its antioxidant activity, aiding digestion, and lowering cholesterol [ 4 ].

The beverage is made by fermenting black or other types of tea with sucrose and using previously fermented tea as the starter culture. It is generally prepared using sweetened black tea ( Camellia sinensis ); however, other types of tea, such as green or oolong, can be used. The starter tea is referred to as the “mother” symbiotic culture of bacteria and yeast (SCOBY), which initiates fermentation [ 5 , 6 , 7 ]. According to the prior literature, most kombuchas are made with 10% w / v sucrose concentration and fermentation is held at temperatures ranging from 18 to 30 °C [ 2 , 8 , 9 , 10 ]. Fermentation is carried out for 7 to 14 days [ 11 ]. However, some studies have exceeded 14 days to observe the bacterial and chemical dynamics [ 8 , 12 ]. The microorganisms that dominate this probiotic tea are mostly acetic acid bacteria, yeasts, and lactic acid bacteria. The dominant yeast and bacterial genera are Zygosaccharomyces and Komagataeibacter , respectively [ 13 ]. The SCOBY metabolizes sucrose and ethanol to produce organic acids such as acetic, glucuronic, and gluconic acids that provide the beverage’s unique sensorial attributes, with acetic acid providing the dominant acid [ 11 , 14 , 15 ]. The pH and titratable acidity also play a role in the kombucha’s sensory attributes. The tartness and sourness of kombucha have been associated with the pH, titratable acidity [ 14 ], and volatile organic compounds [ 16 ].

While kombucha has become an alternative to soft drinks, it can contain between 1 and 24 g of sugar per serving of kombucha [ 5 ]. Serving sizes vary across products from 8–16 oz. (250–480 mL); therefore, the present study considers 250 mL a standard serving size. In comparison, soft drinks can contain 26 g or more of sugar per serving (250 mL). While kombucha may provide less sucrose per serving than soft drinks, drinking a serving of kombucha with 24 g of added sugar is roughly half the recommended daily value for added sugar intake (based on a 2000-calorie diet). Therefore, it is important to identify how different concentrations of sucrose impact the sensory attributes and determine the lowest concertation of sucrose that can be used without compromising consumer liking.

There is extensive prior research on the production of kombucha and fermentation characteristics such as substrate concentration, type of substrate, tea type, and fermentation temperatures [ 1 , 7 , 10 , 12 , 17 ]. Much of this work has provided valuable information on the impact of fermentation temperature on bacterial, physical, and fermentation characteristics. There is a scientific gap, however, in understanding the impact that these conditions have on the sensorial properties of kombucha [ 5 ]. One study discussed the resulting microbial loads following fermentation at 20 °C and 30 °C, noting a difference in the production of gluconic and glucuronic acids [ 12 ]. However, the most abundant acid in kombucha, acetic acid, was not quantified and did not undergo sensory analysis. One of the most comprehensive studies examining the sensory profile of kombucha examined products fermented at two concentrations of sucrose (63 g/L and 94 g/L; equivalent to roughly 16 and 24 g per serving (250 mL), respectfully) and fermented at two temperatures (21 °C and 25.5 °C) [ 18 ]. The study concluded that fermentation temperature and sucrose concentration significantly impacted the sensory profile, with lower temperature and higher sucrose concentrations producing a higher sweetness intensity [ 18 ]. The quantitative descriptive sensory analysis results provide a comprehensive assessment of the sensory profile, but it does not directly assess consumer liking. In the study by Phetxumphou and colleagues (2023), the lowest sucrose concentration was 6.3% ( w / v ) or roughly 16 g of added sugar per serving (250 mL). The study reported herein expands on this finding by investigating a wider range of sucrose concentrations (5%, 7.5% and 10%), which would equate to 12.5 to 25 g of sucrose per serving, helping to determine if lower amounts of sucrose can result in a well-liked kombucha beverage.

Due to recommendations to reduce added sugar intake, it is important to determine if kombucha can be fermented at lower concentrations of sucrose and how this impacts flavor attributes and consumer liking. Therefore, the aim of the present study is to examine the effect of sucrose concentration and fermentation temperatures on the perceived intensity of sensory attributes and overall liking. This will inform the judicious selection of the lowest sucrose concentrations that will not compromise the liking or sensory profile of kombucha tea. Through sensory testing, the effects of sucrose concentration and fermentation temperature on the flavor intensities and liking of kombucha were observed. Chemical and analytical sampling was conducted to follow fermentation patterns and verify the safety of the product. In addition, titratable acidity and pH are crucial factors in the acidity and sourness of a food product [ 19 ], thus the effect of titratable acidity and pH on the products were evaluated over the period of fermentation.

2. Materials and Methods

2.1. kombucha preparation.

Kombucha tea was prepared in a food-grade facility following good manufacturing practices. Ingredients were sourced from local grocery retailers. Starter culture tea was prepared by fermenting 1 L of black tea, using 8 g/L of loose-leaf black tea (Lipton, India) [ 11 ], 100 g of white granulated sugar (Stop and Shop, Quincy, MA, USA), and 10% starter tea from a commercial raw kombucha brand (GT’s Living Foods, Vernon, CA, USA) [ 4 ]. The tea was steeped in boiling water for 10 min and then cooled to room temperature before adding the raw kombucha [ 6 ]. Once the raw kombucha was mixed in, the starter tea was placed in a 30 °C incubator to ferment for two weeks prior to experimentation. A 40 L batch of black tea was distributed into 12 1-gallon food-grade glass vessels (ULINE, Pleasant Prairie, WI, USA). Glassware was sterilized in boiling water for 10 min before use. After steeping the loose-leaf tea for 15 min [ 12 ], the tea leaves were strained using a cheesecloth and dispensed into glass jars. Each vessel of tea contained 3.2 L. Based on previous studies, this experiment fermented kombucha at three concentrations of sucrose 5.0% (50 g/L), 7.5% (75 g/L), and 10% (100 g/L) w / v % and at two fermentation temperatures, 20 °C (+/− 1.5 °C) and 30 °C (+/− 1.5 °C) [ 10 , 12 ]. Preliminary experiments resulted in variations in small-scale fermentation. To minimize the variability between experimental conditions, the experiment was replicated (i.e., carried out in duplicate) within a single experiment. Duplicate batches for each experimental condition were combined at the end of the fermentation period for sensory testing. After the tea and sucrose mixtures reached room temperature, 224 mL (7% v / v ) of the prepared liquid starter culture tea was mixed into each batch [ 13 ]. Fermentation was halted based on pH and titratable acidity measurements on day 11.

2.2. Chemical Analysis: pH, Titratable Acidity, Brix

The pH, brix, and titratable acidity measurements were taken daily throughout the fermentation period. Triplicate measurements were taken for each fermentation vessel, resulting in six measures for each experimental condition. Each sample (30 mL) was drawn using a sterile wine thief (E.C. Kraus, Independence, MO, USA) and held in 50 mL falcon tubes (Fisher Scientific, Newington, NH, USA) for analysis. The pH meter (Oakton pH 6+, Vernon Hills, IL, USA) was calibrated daily using pH buffers 4.0, 7.0, and 10.0. The brix refractometer (Milwaukee Instruments MA871, Rocky Mount, NC, USA) was calibrated using distilled water. Sucrose concentrations were measured in units of sucrose (°Brix). Brix measurements from day 0 and day 11 were converted to specific gravity and used to estimate the amount of ethanol in the final product using a simple equation [ 20 ]. Manual titrations were performed using 0.1 N NaOH (titrant) and phenolphthalein (color indicator). Titratable acidity was expressed in units of acetic acid. Once fermentation was completed, the cellulosic biofilm was removed from the experimental batches, and teas were stored in a 4 °C food-safe refrigerator. Duplicate batches were combined immediately prior to the sensory evaluation. Alcohol by volume percentages were calculated (following methods described elsewhere [ 20 ]). For commercial kombucha, U.S. regulations mandate that the alcohol content be under 1.2%. All batches prepared contained 0.5% or less ethanol, thus considered non-alcoholic. Participants were aware that samples contained trace alcohol in the consent form and the pre-screener questionnaire.

2.3. Sensory Analysis

Participants were recruited from the University of Massachusetts and the surrounding area. Individuals were eligible to participate based on the following inclusion criteria: 18 years or older, no tongue, lip, or cheek piercings, not currently pregnant or breastfeeding, no loss of taste or smell function due to COVID-19, had not smoked in the last 30 days, and were willing to consume fermented products with trace amounts of ethanol. In total, 148 participants completed the screener, and 111 people participated in the sensory study. Seven participants were removed from the dataset based on their performance during the training session (described below), resulting in 104 participants (age 27.5 ± 8.7 and 54 female). All protocols were reviewed and approved by the University of Massachusetts Amherst Institutional Review Board for Human Research (IRB #4169).

Participants were invited to complete an in-person sensory test (~20 min). For this study, participants rated the intensity of the samples and overall liking of the kombuchas. General questions about kombucha were asked to obtain information on the regularity of consumption, why one chooses to drink kombucha or consume fermented foods, types of flavors or brands they are familiar with, and their concern with added sugar in food products.

Prior to tasting samples, participants received instructions on the use of the generalized Visual Analog Scale (gVAS) and practiced rating 9 remembered or imagined sensations [ 21 ]. The gVAS is a scale used to make intensity ratings, with attributes placed at the ends of the scale: 0 (no sensation of any kind) and 100 (strongest imaginable sensation of any kind). This orientation helps participants practice identifying specific intensities in the context of all sensations, not just taste. Additionally, this practice ensures that participants understand how to use the scale appropriately. For this study, participants rated the brightness of a dimly lit room below the brightness of the sun, with a total of 7 participants removed.

Each participant was served six samples in reusable cups containing 30 mL of kombucha. Samples were presented at room temperature and were blinded with 3-digit codes. The order of samples presented was counterbalanced via a Willams Design. Participants were instructed to taste each sample and drink as much or little as they wanted. Participants first rated eight attributes, selected from a larger set of attributes from [ 18 ]. The intensities were sweetness, sourness, astringency, vinegar flavor, fizzy or carbonation, apple juice/cider flavor, lemony/citrus flavor, and yeast flavor. While no formal training was provided, descriptions of each attribute were provided. For example, the sweetness was described as the taste of cotton candy, and astringency was described as a puckering or drying sensation often felt after mouthwash or drinking wine. After reporting the intensity, participants reported their overall liking on a 9-point hedonic scale [ 14 , 22 ]. Participants were instructed to rinse between samples with water (filtered via reverse osmosis). Finally, after tasting all samples, participants were asked to rank most liked to least liked for all six samples. All sensory data were collected using Compusense ® (Compusense Inc, Guelph, ON, Canada).

2.4. Statistical Analysis

Statistical analysis was conducted to assess the relationship between temperature and sucrose concentrations on reported liking via analysis of variance (ANOVA) and a T-test, respectively. Significant ANOVA models were followed with Tukey’s honest significant difference (HSD) to examine differences between pairs. Separate ANOVA models were conducted to determine the impact of sucrose concentration and fermentation temperature and their interaction effect on each sensory attribute and overall liking. A stepwise regression was conducted to identify the attributes associated with reported overall liking. The relationship between titratable acidity and sensory ratings (astringency and sourness) was examined using linear regression with an adjusted R 2 value reported. Differences in titratable acidity across fermentation temperatures were examined via Student’s t -test. All analyses were conducted using RStudio (version 2023.03.1).

3.1. Determination of pH, Brix, and Titratable Acidity

pH, brix, and titratable acidity measurements were recorded daily throughout fermentation ( Figure 1 ). During the fermentation period, all samples decreased in pH and increased in titratable acidity as expected. The pH of kombucha on day 0 (day of preparation) was below 4.6, the pH level at which spoilage organisms are less likely to grow [ 23 ]. The final pH of 30 °C batches ranged between 2.25 and 2.38, while 20 °C batches ranged between 1.95 and 2.21 ( Figure 1 a). Fermentation was halted on day 11, with a final range of pH 1.95–2.38. Sucrose, measured in °Brix, varied over the course of fermentation ( Figure 1 b). All samples fermented at 30 °C had a lower pH and higher titratable acidity (expressed in g/L of acetic acid). The titratable acidity of the samples fermented at 20 °C ranged from 3.26 to 3.96 g/L, while 30 °C ranged from 12.83 to 18.24 g/L on the final day of fermentation ( Figure 1 c). Titratable acidity was significantly associated with fermentation temperature ( t -test; p < 0.05).

Measurements (mean ± SD) for ( a ) pH, ( b ) Brix, and ( c ) titratable acidity throughout the 11-day fermentation period.

Batches were refrigerated following the fermentation and combined prior to sensory analysis and the titratable acidity of the combined batches was evaluated. The final titratable acidity for 20 °C was (3.52 ± 0.6, 4.24 ± 0.06, 4.36 ± 0.03) and for 30 °C was (14.57 ± 0.0, 13.81 ± 0.06, 21.14 ± 0.06), for the 5%, 7.5% and 10% sucrose concentrations, respectfully.

3.2. Sensory Evaluation

3.2.1. summary of participant characteristics.

A total of 111 participants took part in the study. After removing seven participants that did not complete the training properly (see Section 2.3 ), the final data set included 104 participants, with 50 identifying as male and 54 identifying as female. The pool of participants reported that 41% did not consume fermented tea. However, 90% answered that they consume fermented foods regularly. Participants that reported that they did not eat fermented foods were prompted by another question asking, “For what reason(s) have you not consumed kombucha?”. A total of 60% of these participants reported liking the taste of kombucha; however, they had not consumed it in the last 6 months. Overall, 61% of participants responded that they generally liked kombucha. Reasons for consuming kombucha included its taste, probiotic or health benefits, trendiness, and availability. Those that reported not consuming kombucha regularly mentioned that they did not like the taste, cost, carbonation, had not tried it before, or did not have it readily available. Samples prepared in this study were intentionally not carbonated. Carbonation is considered an oral irritant and can affect the overall liking of a beverage [ 24 ]. Since one of the objectives of this study focused on reducing the added sugar content of kombucha, it is useful to understand whether consumers are aware of the added sugar content in commercial kombucha beverages. Participants were asked to identify the amount of added sugar typically found in a serving of kombucha. Based on the market assessment of commercial kombucha beverages, the average amount per serving is 13 g. In the present study, 11% of participants selected 20 g, with 34% correctly selecting the average amount of added sugar (13 g), whereas 55% thought kombucha contained less than 13 g. This suggests that most consumers perceive a typical kombucha product to contain less added sugars. It is important to note, however, that not all participants were regular drinkers of kombucha.

3.2.2. Overall Liking

The overall liking of the kombucha samples is presented in Figure 2 . Separate ANOVA models examined the effect of sucrose concentrations on overall liking for each fermentation temperature. For 20 °C, there was a significant relationship between sucrose and overall liking [F(2, 309) = 9.58; p < 0.0001]. The post hoc test revealed differences between 5% and 10%, as well as 5% and 7.5% sucrose concentrations ( p < 0.05), without other significant differences between the three sucrose levels ( Figure 2 a). Similarly, there was a significant relationship between sucrose and the overall liking for samples fermented at 30 °C [F(2, 309) = 3.45; p = 0.033]. The post hoc test results revealed significant differences between 5% and 7.5% sucrose ( Figure 2 b). To test whether temperature had a significant relationship with reported liking, t -tests were conducted at each sucrose concentration. There was a significant difference at each sucrose concentration ( p < 0.05), with samples fermented at 20 °C liked significantly more at each sucrose concentration than those fermented at 30 °C. In other words, the greatest variation in liking was observed across fermentation temperatures. All room temperature batches were rated near like slightly to like moderately on the hedonic scale, whereas 30 °C samples were rated from “dislike slightly” to “dislike moderately” ( Figure 2 ).

Mean liking scores (±SEM) for kombucha with 5%, 7.5% and 10% ( w / v ) fermented at ( a ) 20 °C and ( b ) 30 °C.

A repeated-measures ANOVA was conducted to determine the interaction between temperature and sucrose concentration on overall liking. The model revealed no significant interaction effect ( p = 0.07); however, there was a significant effect of sucrose [F(2, 618) = 8.9; p = 0.0002)] and temperature [F(1, 618) = 425.7; p < 0.0001].

3.2.3. Sensory Attributes

Participants rated the perceived intensity of sweetness, sourness, astringency, vinegar flavor, apple flavor, carbonation, citrus flavor, and yeast aroma or flavor. Mean reported intensities are reported in Figure 3 .

Sensory profile of the kombucha beverages fermented at 20 °C and 30 °C (lines: dashed and solid) with 5%, 7.5%, and 10% sucrose (colors: green, orange, purple). Mean intensity ratings reported on general visual analog scale (gVAS) for eight flavor attributes.

A forward stepwise regression was conducted to determine the attributes that are significantly associated with reported overall liking. The model revealed that sweetness, sourness, astringency, vinegar flavor, apple flavor, and yeast flavor were significantly associated with overall liking ( Table 1 ) and that these attributes explained 51.8% of the variability in overall liking. Sweetness intensity had the strongest relationship with overall liking, with the second most influential attribute being astringency, which negatively influenced overall liking. Apple flavor was the only other attribute with a significant positive relationship with overall liking, with sourness, vinegar, and yeast flavor negatively influencing overall liking.

Summary of stepwise forward regression model. The model determined which variables were significant in overall liking scores. All attributes reported a p -value < 0.05.

The reported attribute intensities were assessed to be driven by sucrose concentration and fermentation temperatures. Separate ANOVA models were used to identify the effect of sucrose and temperature and their interaction on the reported intensity of each flavor attribute. The effect of sucrose on each of the flavor intensities showed statistical significance (all p < 0.0001) for the perception of sweetness and apple flavor ([F(2, 618) = 16.1] and [F(2, 618) = 5.6], respectfully). Temperature, in contrast, was significantly associated with the rated intensity of sourness [F(1, 618) = 451.9], vinegar flavor [F(1, 618) = 433.1], sweetness [F(1, 618) = 310.5], astringency [F(1, 618) = 247.8], yeast flavor [F(1, 618) = 57.9], apple flavor [F(1,618) = 48.4], lemon flavor [F(1, 618) = 47.8], and carbonation [F(1, 618) = 27.5] (all p < 0.0001). There was a significant interaction between fermentation temperature and sucrose concentration on the reported sweetness intensity [F(2, 618) = 7.4; p = 0.0006], with no other attributes demonstrating a significant interaction.

Measured titratable acidity was significantly associated with the reported sourness and astringency. A linear model determined that titratable acidity explained 39% of the variability in sourness ( Figure 4 ) and 26.6% of the variability in astringency ( p < 0.0001). A t -test revealed that the reported titratable acidity for samples fermented at 30 °C were significantly higher than samples fermented at 20 °C ( p < 0.0001).

A comparison between mean sourness intensity ratings (±SEM) and total titratable acidity (g/L) for kombucha samples. Intensity ratings were collected using a gVAS, general visual analog scale. Different shapes represent temperature (20 °C, circle and 30 °C, triangle) and different colors represent sucrose concentration (5%, green; 7.5%, orange; and 10%, purple).

4. Discussion

Fermentation conditions influence the flavor profile [ 3 , 5 ]; yet there is minimal published research on the consumer perception of kombucha [ 5 ]. This study extends our understanding of how fermentation temperature and sucrose concentration influence kombucha’s physical and sensory characteristics.

The pH, brix, and titratable acidity in kombucha drive the sensory profile of the fermented tea [ 5 , 19 , 25 ]. As expected, the decrease in pH coincided with an increase in titratable acidity in all samples. The pH of kombucha generally ranges from 2.5–3.5, with a pH lower than 2.5 indicating the greater microbial depletion of sucrose and the potential for increased ethanol concentrations. Since the pH of the higher-temperature samples, 30 °C, decreased below 2.5, and some below 2.0, the fermentation was halted at 11 days, and alcohol by volume percentages was calculated. A decrease in brix by the end of the fermentation period was not observed, indicating that initial sucrose concentrations exceeded microbial needs in the fermentation process.

A higher fermentation temperature resulted in an increase in qualitative fermentation rates, producing more organic acids (higher titratable acidity), and a faster decline in the pH. The higher-temperature kombucha was liked significantly less than the lower-temperature kombucha samples. The dislike of samples was driven by higher perceived astringency, sourness, and vinegar flavor. This is potentially linked to a significant association between titratable acidity and reported astringency. It is acknowledged that participants may have difficulty differentiating between sensations, specifically, astringency and sour, and possibly vinegar flavor [ 26 ]. However, these sensations correspond to a lower pH and higher titratable acidity. Andreson and colleagues (2022) reported a correlation between titratable acidity and perceived sourness for commercial kombucha beverages [ 25 ]. Current findings regarding the relationship between the fermentation temperature and sensory profile are supported by a previous study, which concluded that fermentation temperature impacts the sensory profile, noting that a higher fermentation temperature was associated with an increased perceived intensity for sourness, puckering, pungent, astringent, and vinegar flavor [ 18 ]. Moreover, this previous report concluded that fermentation temperature explains more variation in the reported intensity of sensory attributes, with sucrose concentration still having a significant, albeit less, impact on the sensory profile. This aligns with the present study, as fermentation temperature and sucrose concentrations independently influence the sensory profile, with the interaction between factors [ 18 ].Prior work has assessed the chemical composition of kombucha, including quantifying specific acid content [ 9 , 16 ], antioxidant activity [ 9 , 12 ], and polyphenol content [ 9 , 11 ], which traditionally has been used to predict the flavor profile. However, more empirical evidence is needed to validate this relationship between instrumental measures and consumer’s perceptions for kombucha [ 27 ]. Future studies linking chemical analysis with sensory profiles will benefit researchers and the industry.

Perceived astringency is modulated by adding sweet compounds and sweet-related flavors [ 28 , 29 ]. Therefore, one could rationalize that the addition of sucrose would suppress or reduce the perceived astringency and potential sourness. However, prior work demonstrated that the successful suppression of astringency was achieved for lower intensity levels. For the kombucha tested here, participants reported astringency and astringency-related sensations (sour and vinegar) as the dominant sensations reported for the higher-temperature samples, and the addition of sucrose did not appear to be effective at reducing the perceived astringency. Due to higher astringency, sourness, and vinegar sensations negatively influencing overall liking, it is important to identify the ideal pH and titratable acidity to ensure acceptable amounts of these sensations.

The perceived sweetness was the strongest predictor for reported overall liking across all samples. Kombucha fermented at 20 °C produced higher perceived sweetness compared to 30 °C, regardless of sucrose concentration. Sucrose concentrations appeared to drive differences in perceived sweetness and apple flavor but no other attributes, which are less pronounced than studies reporting kombucha’s sensory profile. Phetxumphou et al. (2023) report that lower sucrose concentrations produced beverages with a higher perceived intensity of astringent, yeasty aroma, fizzy, and sour attributes [ 18 ]. In addition, authors have reported that sucrose concentrations influenced perceived sweetness and sweet-related attributes (e.g., honey) along with fruity-related flavors (e.g., apple, berry, and grape) [ 18 ]. The perceived sweetness was the strongest predictor for reported overall liking across all samples. Kombucha fermented at 20 °C produced higher perceived sweetness compared to 30 °C, regardless of sucrose concentration. Previous studies have reported that sucrose concentrations were important for the perception of taste- and flavor-related sensations. Similar to the present study, Phetxumphou et al. (2023) reported that lower sucrose concentrations produced beverages that had a higher perceived intensity of astringent, yeasty aroma, fizzy, and sour attributes [ 18 ]. In addition, authors reported that sucrose concentrations influenced perceived sweetness and sweet-related attributes (e.g., honey) along with fruity-related flavors (e.g., apple, berry, and grape) [ 18 ]. One possible reason for this difference is the tea used for fermentation, with the present study examining black tea. As a result, the interaction between sucrose and other tea types or base beverages is likely to result in the formation of different sensory profiles.

The present study examines sucrose concentration and fermentation temperature on the physical and sensory characteristics. These parameters, along with tea type and fermentation time, influence the composition and metabolism of microorganisms [ 30 ]. Changes in the microbial community likely drive this effect, as different microbial compositions are associated with physical characteristics and chemical composition, including pH, titratable acid, reducing sugars, polyphenols, acetic acid, ethanol content, and flavor volatiles [ 31 , 32 , 33 ]. The chemical composition of kombucha, including acid content [ 9 , 16 ], antioxidant activity [ 9 , 12 ], and polyphenol content [ 9 , 11 ], are driven by these fermentation conditions, which drive the final flavor profile [ 27 ]. To ensure a stable fermentation, it has been recommended that the initial microorganisms are important to produce desirable characteristics [ 34 , 35 , 36 ] and that the microbial composition is an important factor contributing to the sensory profile [ 27 ]. There is a need for additional studies to examine the interaction between fermentation conditions and microbial community on the physical and sensory characteristics. A greater scientific understanding will help identify the ideal parameters for producing products with a desired sensory profile and health benefits.

Due to recommendations for limiting added sugar and considering consumer preference, the beverage industry should consider reducing the amount of sucrose added. For the sucrose amounts tested in the present study, the lower sucrose concentration (5%) was significantly less than 7.5% and 10% sucrose for the samples fermented at 20 °C. Nonetheless, the lowest sucrose concentration tested was still liked, rated at ‘like slightly’. One potential strategy for increasing perceived sweetness is the addition of sweet-related flavors (e.g., fruit and honey). Additional research is needed to determine if a greater reduction could be achieved on an industrial scale. Based on study findings, there was no difference in liking ratings between 7.5% and 10% ( Figure 2 a), suggesting a reduced added sugar can be achieved while maintaining liking. While formulations may need to be modified for larger-scale production, upscaling the current formulation, a reduction from 10% to 7.5% sucrose would translate to roughly a 6 g reduction in added sugar per serving (250 mL). The present study highlights the impact of these fermentation parameters on unflavored black tea under controlled conditions at a laboratory scale.

Prior work suggests that the optimal temperature for fermentation is 22–28 °C [ 10 ]. While higher temperatures tend to yield faster ferments [ 37 ], monitoring the titratable acidity and pH is important, as these parameters appear to drive perceived astringency and sourness, with higher intensities negatively impacting overall liking. While fermentation typically lasts 7 to 14 days, the present study stopped fermentation on day 11, it is possible that if the fermentation had ended earlier, especially in the case of samples fermented at 30 °C, it might have resulted in a lower production of titratable acidity and higher pH levels and higher liking ratings. Identifying the acceptable amount of titratable acidity, driven by fermentation temperature and the ideal sucrose concentration, is important for producing a desirable sensory profile.

5. Conclusions

The fermentation temperature and sucrose concentrations impact the development of novel sensorial attributes in kombucha. While both fermentation temperature and sucrose concentration were significantly associated with kombucha’s reported overall liking and sensory profile, temperature had a greater influence than sucrose concentration. Kombucha fermented at a lower temperature received higher liking ratings, driven by the perceived sweetness and apple flavor. The higher fermentation temperature sped up the fermentation rate, producing a higher titratable acidity and lower pH, which was associated with higher astringency and sourness intensity ratings and lower overall liking. While astringency is an authentic sensation of kombucha, higher astringency is likely suppressing the sweetness, which was not overcome by the higher sucrose levels. Results of the present study suggest that 7.5% sucrose provides similar liking of 10% sucrose, but lowering the sucrose to 5% negatively impacted liking. Future studies could examine the possibility of adding sweet-related flavors to improve the overall liking of a lower-sucrose kombucha beverage. In summary, this study highlights the importance of sucrose and fermentation temperature on the development of the sensorial properties of kombucha.

Acknowledgments

The authors thank Amanda J. Kinchla (UMass Amherst) for use of laboratory instruments and helpful discussions. Various members of the Nolden and Sela Lab groups are acknowledged for helpful discussions, and assistance in study recruitment and execution.

Funding Statement

A.A.N. receives funding from the Department of Food Science at University of Massachusetts Amherst and The National Institute of Food and Agriculture, U.S. Department of Agriculture, under Hatch project number MAS00491. D.A.S. receives funding from the U.S. Department of Agriculture, under Hatch project number MAS00556. The contents are solely the responsibility of the authors and do not necessarily represent the official views of the USDA or NIFA.

Author Contributions

Conceptualization, D.A.S., A.A.N. and G.C.; methodology, D.A.S. and A.A.N.; formal analysis, G.C. and A.A.N.; investigation, G.C.; resources, D.A.S. and A.A.N.; data curation, G.C. and A.A.N.; writing—original draft preparation, G.C.; writing—review and editing, A.A.N., G.C. and D.A.S.; visualization, G.C. and A.A.N.; supervision, D.A.S. and A.A.N. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

All protocols were reviewed and approved by the University of Massachusetts Amherst Institutional Review Board for Human Research (IRB #4169).

Data Availability Statement

Conflicts of interest.

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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• Published: 19 August 2024

The antimicrobial effects of silver nanoparticles obtained through the royal jelly on the yeasts Candida guilliermondii NP-4

• Seda Marutyan   ORCID: orcid.org/0000-0002-8019-9093 1 ,
• Hasmik Karapetyan   ORCID: orcid.org/0000-0001-5394-6500 1 , 2 ,
• Lusine Khachatryan 1 ,
• Anna Muradyan   ORCID: orcid.org/0009-0002-0408-8508 1 ,
• Syuzan Marutyan   ORCID: orcid.org/0009-0007-3250-2100 1 , 2 ,
• Anna Poladyan   ORCID: orcid.org/0000-0002-8240-2382 1 , 2 &
• Karen Trchounian   ORCID: orcid.org/0000-0002-6624-8936 1 , 2 , 3  

Scientific Reports volume  14 , Article number:  19163 ( 2024 ) Cite this article

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• Biochemistry
• Microbiology

The effect of silver nanoparticles (Ag NPs) obtained in the presence of royal jelly (RJ) on the growth of yeast Candida guilliermondii NP-4, on the total and H + -ATPase activity, as well as lipid peroxidation process and antioxidant enzymes (superoxide dismutase (SOD), catalase) activity was studied. It has been shown that RJ-mediated Ag NPs have a fungicide and fungistatic effects at the concentrations of 5.4 µg mL −1 and 27 µg mL −1 , respectively. Under the influence of RJ-mediated Ag NPs, a decrease in total and H + -ATPase activity in yeast homogenates by ~ 90% and ~ 80% was observed, respectively. In yeast mitochondria total and H + -ATPase activity depression was detected by ~ 80% and ~ 90%, respectively. The amount of malondialdehyde in the Ag NPs exposed yeast homogenate increased ~ 60%, the catalase activity increased ~ 70%, and the SOD activity—~ 30%. The obtained data indicate that the use of RJ-mediated Ag NPs have a diverse range of influence on yeast cells. This approach may be important in the field of biomedical research aimed at evaluating the development of oxidative stress in cells. It may also contribute to a more comprehensive understanding of antimicrobial properties of RJ-mediated Ag NPs and help control the proliferation of pathogenic fungi.

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Introduction.

Currently, nanoparticles (NPs) are considered as effective medicine used in the treatment of many diseases, which are a good alternative, especially to antibiotics. Metallic NPs, particularly silver ones, are promising for use in medicine as therapeutic, antimicrobial, diagnostic agents, and drug-delivery enhancement agents. They are applied in the fight against various diseases, in cancer therapy, for diagnostic testing, in the treatment of HIV and AIDS 1 , etc. Polymeric NPs are hard colloidal particles, the sizes of which are usually in the range of 10–100 nm. They consist of polymer particles attached to the active ingredient and are usually surrounded by a surfactant that stabilizes the NP 2 .

NPs have several advantages: they are small, they protect the body from the side effects of the drug they carry, and they affect the target, ensuring the accuracy and safety of the effect. They are controllable and can combine different drugs. Due to their small size and the large contact surface, medicinal NPs exhibit high solubility and therefore high bioavailability. On the other hand, drugs containing proteins or nucleic acids require more innovative carrier systems to increase their efficacy and protect against unwanted degradation 3 , 4 . So the use of metal NPs as drug carriers is most applicable in modern medicine from the point of view of their targeted delivery, controlled release of drugs, and control of their removal from the body.

The antimicrobial effect of NPs is based on the following processes: cell membrane rupture/degradation, generation of reactive oxygen species (ROS), change in bacterial cell membrane permeability, and induction of intracellular antimicrobial effects, including interactions with DNA and proteins 4 . Numerous studies have shown that silver nanoparticles synthesized using various plant extracts, are beneficial due to their low cost, eco-friendliness, and simplicity of production. They exhibit antimicrobial effects by inhibiting the growth of bacteria 5 , 6 and yeasts 7 , making them potentially valuable in combating pathogenic fungi. However, the molecular and biochemical mechanisms of the effect of NPs on microbes, particularly on yeasts, have not been fully revealed.

The antifungal effect of Ag NPs is currently well-established 8 , 9 , 10 , 11 , 12 , 13 . Ag NPs derived from various biological sources have shown an inhibitory effect on the growth of over 30 different strains of Candida yeasts, creating inhibition zones of varying diameters 8 , 9 , 10 , without exhibiting cytotoxicity toward human cells 11 . It is believed that Ag NPs damage the yeast cell walls of different Candida strains 8 , penetrate the cells, and accumulate in the cytoplasm. This accumulation leads to the inhibition of yeast cell division, germ tube and biofilm formation, and the secretion of various hydrolytic enzymes (such as phospholipases, proteinases, and lipases) 9 , 12 . Additionally, the increase in intracellular Ag + concentration generates and accumulates reactive oxygen species (ROS), which leads to apoptosis. Ag NPs also alter the distribution of K + ions within different Candida species cells, affecting plasma membrane H + -ATPase activity. Furthermore, Ag NPs penetrate the mitochondria, reducing the activity of copper-dependent cytochrome c oxidase and consequently decreasing the rate of cellular respiration. However, these findings are preliminary, and further research is needed to elucidate the molecular and biochemical mechanisms underlying the antifungal effect of Ag NPs. More importantly, further studies should also focus on understanding the mechanisms of Candida yeast resistance to Ag NPs 13 .

From an ecological point of view, it is important to develop safe methods of obtaining NPs based on a redox reaction when metal ions are reduced to form stable NPs 14 . The particular interest has the way of "green synthesis" of NPs, which is an eco-friendly and biocompatible process, and uses plant extracts, yeast, or bacteria 15 . It should be noted that the green synthesis of metal nanoparticles has several advantages compared to traditional methods of chemical synthesis: it is eco-friendly, fast, cost-effective, and, most importantly, it does not use chemically hazardous reducing agents 14 .

Royal Jelly (RJ) served as a reducing and oxidizing agent in the green synthesis technology of colloidal Ag NPs 16 . The antioxidant activity of RJ is due to the flavonoids and phenolic compounds present in it 17 . Armenian bee products, including RJ, have unique properties 16 , which gives them an advantage to be used for medical purposes, and as a source of obtaining of metal NPs. Ag NPs affect yeast membranes, leading to changes in their ultrastructure and membrane 18 , 19 . On the other hand, Ag NPs can affect on the lipid peroxidation processes 13 leading to the development of oxidative stress 20 and to additional generation of ROS which can also lead to the inactivation of various macromolecules, including DNA, proteins and several enzymes. To repair the damages of these biologically important macromolecules, cells need additional energy, the main source of which in cells is the mitochondrial ATP-syntase, the hydrolysis of ATP by the enzyme ATPase. It can also act as an ATPase, hydrolyzing ATP into ADP and phosphoric acid and releasing energy that is used in repair processes 21 .

Yeast mitochondrial ATP-synthase is an enzyme complex composed of 30 different subunits 22 . They are grouped into the hydrophilic F 1 and membrane-bound F 0 sites, the subunits of which are mainly encoded by the nuclear genome. Only a fraction of subunits of the F 0 site are of mitochondrial origin 23 . The F 0 site of yeast mitochondrial ATP-synthase is embedded in the inner membrane of mitochondria and has a much more complex structure than the bacterial enzyme; most subunits of this site have no bacterial homologues 22 . Yeast ATP-synthase subunits, participating in the formation of the enzyme dimer in mitochondria, contribute to the development of cristae in the inner membrane of mitochondria. In addition, the composition of subunits and catalytic mechanism of the yeast ATP-synthase enzyme are very similar to the enzyme complex of humans and other eukaryotes 24 . Therefore, yeasts are a suitable model to understand the structure and functions of mitochondrial ATP-synthases in eukaryotes at the evolutionary molecular level, as well as to study the state of this enzyme both under normal physiological conditions and under different diseases and stress conditions. These studies may contribute to the development of genetic engineering strategies, such as gene therapy, for the treatment of various inherited human mitochondrial diseases.

ATP-synthase has an important role as a molecular drug target and may be useful for the development of antimicrobial and antitumor agents in medical practice, as well as for the development of pesticides and insecticides in agriculture, with yeasts being highly applicable as a suitable model for this purpose 22 , 23 , 24 .

Therefore, it is of great interest to study the changes in the activity of antioxidant enzymes and total and H + -ATPases in yeast homogenates and mitochondria under the influence of Ag NPs, which may contribute to the clarification of the mechanism of the antimicrobial effect of Ag NPs.

The primary aim of this study is to synthesize silver nanoparticles (Ag NPs) using Royal Jelly (RJ) through a green synthesis method and investigate their effects on the yeast species Candida guilliermondii NP-4. The study focuses on evaluating the impact of RJ-mediated Ag NPs on yeast growth, specifically looking at the alterations in the activity of total and H + -ATPases and antioxidant enzymes in yeast cells under the influence of these nanoparticles. In the presence of RJ, the synthesis of silver nanoparticles occurs significantly faster, resulting in AgNPs that maintain their stability for an extended period, even at room temperature.

This study aims to advance the understanding of the antimicrobial properties and biochemical interactions of RJ-mediated Ag NPs, contributing to the development of new therapeutic and biotechnological applications.

Materials and methods

Yeasts, their growth and survival.

The yeasts Candida guilliermondii NP-4 (the lab wild-type strain) 25 were incubated in minimal saline medium 26 (0.2 mM NH 4 HPO 4 , 0.5 mM (NH 4 ) 2 PO 4 , 0.6 mM K 2 SO 4 , 0.8 mM MgSO 4 , 100 mM glucose, pH 5.5) on a shaker (BioSan Shaker-Incubator ES-20/80, Latvia, 200–250 rpm), which provided necessary aeration. The yeasts were grown at 30 °C, for 24 h, after the yeast biomass was harvested by centrifugation (3500× g , 10 min), and the biomass amount was determined spectrophotometrically at the OD 590 (GENESIS 10S UV–VIS, Thermo Fisher Scientific Inc., USA). Yeast survival was recorded after 24 h incubation at 30 °C in a liquid medium (see above). The parameters of growth (duration of latent (lag) period, and specific growth rate (µ) were determined by hourly measurements of optical density (OD 590 ) of the culture medium with native and exposed to the RJ-mediated Ag NPs yeasts. µ (h −1 ) was determined at the interval when the logarithm of OD varied linearly during the time by equation dN/dt = μN(1 − N/N max ), where N is the biomass amount of the cells at time t, N max is the maximum amount of biomass (at the stationary phase) as described 26 ; duration of latent (lag) phase (t d ) was determined graphically, as described 25 .

Obtaining Ag NPs in the presence of RJ

Ag NPs were obtained by green synthesis in the presence of RJ (Armenia, Kotayk region). The NP synthesis method was adapted from earlier reports 16 , 27 . RJ solution of 5 mg mL −1 concentration was prepared in double-distilled water and mixed with 0.01 M silver nitrate solution at a 1:1 ratio. Afterward, the solution was kept under constant stirring at room temperature for ~ 6 h. Due to the reduction of silver ions, color change was observed.

Characterization of obtained Ag NPs

Ag NP characterization was performed by applying UV–Vis spectroscopy, dynamic light scattering (DLS), and transmission electron microscopy (TEM) in combination with selected area electron diffraction (SAED). UV–Vis absorption spectra were recorded in the wavelength range of 280 and 720 nm (GENESYS 10S UV–Vis, Thermo Scientific, USA). The hydrodynamic radius and polydispersity level of the Ag NP sample were studied using DLS. Measurements were performed, as described earlier with some modifications 14 . For DLS characterization, 20-fold dilutions of Ag NP samples were applied and monitored for ~ 30 min (Spectrosize 300, XtalConcepts, Germany; laser wavelength: 660 nm). Software supporting the CONTIN algorithm was used to analyze collected autocorrelation functions. For TEM and SAED analysis, a drop of Ag NP sample dried on a copper grid and a 200 kV acceleration voltage were applied 16 . These experiments and data collection were performed in the XBI Biolab (European XFEL) 28 .

Study of changes in growth dynamics of yeasts under the influence of RJ-mediated Ag NPs

The growth dynamics of C. guilliermondii NP-4 under the influence of RJ-mediated Ag NPs were studied by hourly recording the yeast biomass in cultural medium without Ag NPs and containing RJ-mediated Ag NPs in different concentrations (1.6–108.0 μg mL −1 ) during 24 h of growth. The fungicidal and fungistatic concentrations of Ag NPs were determined: the concentration of Ag NPs when the growth of yeast is suppressed by more than 90% was taken as the fungicidal concentration, and the concentration of Ag NPs when the growth of yeast is suppressed by 50% was taken as the fungistatic concentration 29 .

Isolation of yeast mitochondria and determination of ATPase activity

The mitochondria from yeasts C. guilliermondii NP-4 were isolated by 30 . Frozen (− 10 °C) yeast biomass was pressed with a French press, then the mitochondria from yeast homogenates were isolated by differential centrifugation in 0.25 M sucrose in the following sequence: 3000× g for 20 min; supernatant was centrifuged by 15,000× g for 15 min; then the supernatant was centrifuged by 30,000× g , for 20 min. The resulting precipitate is the mitochondrial fraction.

ATPase activity in yeast homogenates and mitochondria was assessed by measuring the quantity of inorganic phosphate (Pi) generated using 5 mM ATP at pH 7.5, Focea et al. 31 , in an assay mixture containing 50 mM Tris–HCl buffer (pH 7.5) with 1 mM MgSO 4 . The activity was expressed as µg P i mg −1 protein. Spectrophotometric determination of P i was conducted according to the method described earlier 32 . N, N'- dicyclohexylcarbodiimide (DCCD) was utilized as a F O F 1 yeast ATPase inhibitor, following the protocol established by Stewart et al. 33 .

Determination of antioxidant enzymes activities

The activity of catalase (EC 1.11.1.6) was assessed in yeast homogenates following the method outlined by Chen et al. 34 . It was quantified as the amount of hydrogen peroxide (H 2 O 2 ) decomposed per milligram of protein (μmol H 2 O 2  mg −1 protein). The activity of superoxide dismutase (SOD, EC 1.15.1.1) was determined based on its ability to inhibit the rate of adrenaline self-oxidation, as described by Carbone et al. 35 . SOD activity was assayed by its ability to inhibit the auto-oxidation of adrenaline, determined by the increase in the absorbance at 480 nm and at 30 °C. The reaction was carried out in 50 mM L −1 sodium carbonate buffer, pH 10.2, and was initiated by the addition of 0.1 mM L −1 adrenaline.

Quantitative determination of malondialdehyde

The concentration of malondialdehyde (MDA) in yeast homogenates was assessed through spectroscopic measurement of the optical density (OD) of the reaction products with 2-thiobarbituric acid, as described by Mejia-Barajas et al. 36 . The quantity of MDA was determined and expressed as nM per mg −1 protein.

Statistical analysis

The statistical processing of the obtained results and the construction of graphs were carried out with the GraphPad Prism 9 program 30 . Experiments were repeated five times. The results are represented as means ± SD. Standard errors, as well as the validity of the differences between different series of experiments, were evaluated by Student's validity criteria (P), results were considered reliable if P < 0.05.

Characteristics of Ag NPs

Ag NPs’ absorbance peak was observed at ~ 430 nm (Fig.  1 a), consistent with an earlier study 16 . In terms of heterogeneity and dimensions of NPs, a polydispersity index (PDI) of ~ 46.2% and an average hydrodynamic radius of 58.25 ± 9.7 nm were determined for particles in aqueous solution (Fig.  1 b). Further, the TEM analysis (Fig.  1 c) demonstrated the round shape of Ag NPs, but still with some variance in morphology.

The characteristics of RJ-mediated Ag NPs: ( a ) UV–Vis absorption spectra, ( b ) hydrodynamic radii evolution, ( c ) TEM (dilution: 1:90), and ( d ) SAED pattern.

Effect of RJ-mediated Ag NPs on growth of yeasts C.guilliermondii NP-4

Under the influence of RJ-mediated Ag NPs, growth of yeasts C. guilliermondii NP-4 is suppressed resulting in growth inhibition zones in Petri dishes (Fig.  2 ). With the increase of Ag NPs concentration, the diameter and surface area of yeast growth suppression zones increase (Table 1 ) showing the antifungal effect of RJ-mediated Ag NPs.

Inhibition zones of yeasts C. guilliermondii NP-4 grown in the presence of RJ-mediated Ag NPs: A—1.62 µg mL −1 , B—2.7 µg mL −1 , C—5.4 µg mL −1 , D—10.8 µg mL −1 .

Analysis of literature data showed that when growing bacteria in the presence of Ag NPs at a concentration of 80 μg mL −1 , complete and irreversible inhibition of cell growth is observed, while a concentration of 20 μg mL −1 of Ag(0) leads to partial suppression and prolonged delay in growth phases of E. coli 37 . At the same time, Ag NPs inhibit the growth of yeast Candida albicans by 90% at the concentration of 40 μg mL −1 18 . We investigated the effect of RJ-mediated AgNPs on the yeasts C. guilliermondii NP-4 in a wider range of concentrations, by adding AgNPs to the growth medium at a concentration of 1.62–108 μg mL −1 and followed the growth dynamics during the first 24 h of growth when the stationary phase of their life cycle is confirmed 25 .

The addition of RJ-mediated Ag NPs to the growth medium of yeasts decreases the specific growth rate of C. guilliermondii NP-4 (Fig.  3 ), which increases as the concentration of RJ-mediated Ag NPs increases. In parallel, the amount of yeast biomass also decreases in the 24 th  h of growth (stationary phase) (Fig.  3 ). The results show that a concentration of 27 μg mL −1 of Ag NPs inhibited the growth of C. guilliermondii NP-4 more than 90%, which confirms their fungicidal effect (Table 1 ). Whereas, a fungistatic effect was demonstrated at a concentration of 5.4 μg mL −1 of RJ-mediated Ag NPs, when yeast growth was inhibited by 50% 28 .

Growth dynamics (OD) of yeasts C.guilliermondii NP-4 under influence of RJ-mediated Ag NPs of different concentrations. (n = 5, ***p < 0.001, ****p < 0.0001). Yeasts were grown in the presence of RJ-mediated Ag NPs in growth medium by different concentrations: 1.62 µg mL −1 —27 µg mL −1 . (n = 5, ***p < 0.001, ****p < 0.0001).

We conducted a study on the influence of RJ and AgNO 3 on yeast growth as a control (data not shown). The results obtained showed that, as expected, RJ has no antifungal effect; instead, being a rich food, it stimulates yeast growth. RJ, which contains oxidizing and reducing components, simply acts as a surfactant source that allows for the synthesis of silver nanoparticles. Additionally, to confirm that the antifungal effect of AgNPs due to their silver content, we studied the antifungal effect of silver nitrate at the same concentration. The results obtained showed that AgNO 3 exhibits the same cytotoxic effect as silver nanoparticles synthesized in the presence of RJ (data not shown). Therefore, despite AgNO3 and AgNPs having a similar cytotoxic effect on yeasts, we recommend the use of AgNPs for antifungal fight because they can be applied more selectively without harming other cells of the organism.

Effect of RJ-mediated Ag NPs on the ATPase activity of the yeast C. guilliermondii NP-4

In yeast homogenate and mitochondria treated with different concentrations of Ag NPs (Fig.  4 ), the total and DCCD-sensitive ATPase activities were investigated. Thus, in the NPs-treated yeast homogenate, the total ATPase activity decreased compared to control, and the degree of inhibition increased linearly with increasing RJ-mediated Ag NPs concentration. Thus, under the influence of silver nanoparticles with a concentration of 2.7 μg mL −1 , the ATPase activity in the homogenate decreased ~ 40%, at a concentration of 3.78 μg mL −1 —~ 80%, and at a concentration of 5.4 μg mL −1 —~ 90%. The same effect was also observed in the case of H + -ATPase activity. In the homogenate of yeasts, under the influence of Ag NPs at a concentration of 2.7 μg mL −1 , the activity of DCCD-sensitive ATPase decreased ~ 50%, at a concentration of 3.78 μg mL −1 —~ 60%, and at a concentration of 5.4 μg mL −1 —~ 80%. Thus, at low concentrations of Ag NPs, the suppression of total ATPase activity is more noticeable than that of H + -ATPase activity, and at a fungistatic concentration, the H + -ATPase activity is more strongly depressed than the total ATPase activity.

The total and DCCD-sensitive ATPase activity in homogenates and in mitochondria of yeasts C. guilliermondii NP-4 grown in the presence of different concentrations of RJ-mediated Ag NPs: ( A ) yeast homogenates, ( B ) yeast mitochondria (n = 5, **p < 0.01, ***p < 0.001, ****p < 0.0001).

In the case of yeast mitochondria, at low concentrations of Ag NPs (2.7–3.78 μg mL −1 ), the decrease in total and DCCD-sensitive ATPase activity does not exceed 30% compared to intact yeast mitochondria, and at the fungistatic concentration (5.4 μg mL −1 ) enzyme activity depression is observed ~ 80% and 90%, respectively. The inhibition of ATPase activity in the plasma and mitochondrial membranes of yeast at a fungistatic concentration of RJ-mediated Ag NPs is notably greater for the DCCD-sensitive ATPase. In both instances, this suppression surpasses the reduction observed in total ATPase activity.

Effects of RJ-mediated Ag NPs on lipid peroxidation processes and antioxidant enzyme activities in yeasts C. guilliermondii NP-4

A marker of lipid peroxidation and oxidative stress development in the living organism is malondialdehyde (MDA), which is formed during the breakdown of polyunsaturated lipids by ROS 20 . Therefore, to assess the state of lipid peroxidation processes in yeast under the influence of RJ-mediated Ag NPs, the amount of MDA in the cells was first studied (Fig.  5 ). By the increase of concentration of Ag NPs in the yeast growth medium of yeasts C. guilliermondii NP-4, the amount of MDA in the yeast homogenate increased, and its highest amount (an increase of more than 60%) was observed at a concentration of 10.8 µg mL −1 compared to the intact yeasts. At the same time, the catalase activity was increased in yeasts exposed to Ag NPs, and the highest activity was recorded at the concentration of Ag NPs 5.4 µg mL −1 —~ 70%. A further increase of RJ-mediated Ag NPs concentration (up to 10.8 µg mL −1 ) led to a decrease in catalase activity, although it was still ~ 30% higher than in non-exposed yeasts. The suppression of enzyme activity in the presence of a certain amount of RJ-mediated Ag NPs is caused by an increase in the amount of free radicals, which can cause changes in protein structures, in particular, they can oxidize the porphyrin hemes of catalase, deactivating the latter 38 . At the same time, no significant change in SOD activity was observed in yeasts C. guilliermondii NP-4 under the influence of low concentrations of Ag NPs, and at concentrations of 5.4 μg mL −1 and 10.8 μg mL −1 , an increase in SOD activity was observed by 25% and 30%, respectively.

Effect of different concentrations of RJ-mediated Ag NPs on MDA content ( A ), catalase ( B ) and SOD ( C ) activity in yeasts C. guilliermondii NP-4 (n = 5, ***p < 0.001, ****p < 0.0001).

Ag NPs affect yeast membranes, leading to changes in their ultrastructure and membrane permeability 18 , 19 , as well as lipid peroxidation processes 13 . Currently, silver nanoparticles obtained with the presence of Royal Jelly (RJ-mediated Ag NPs) are of great interest, especially in the case of RJ from the Armenian region, because Ag NPs mediated with Armenian RJ show stability over a long period 16 .

RJ-mediated Ag NPs exhibit a strong antifungal effect on the yeasts C. guilliermondii NP-4. They suppress the growth of yeast: zones of growth inhibition of yeasts appear on Petri dishes; the specific growth rate of yeast in the logarithmic phase decreases; and the amount of biomass of yeast in the stationary phase decreases, too. In addition, the processes of lipid peroxidation in yeast cells are strengthened, which is expressed by the increase in the amount of MDA, as well as the activities of antioxidant enzymes catalase and SOD were increased. This proves that under the influence of Ag NPs, oxidative stress develops in yeasts, which is one of the possible mechanisms of the antimicrobial effect of Ag NPs 20 . Oxidative stress, depending on the concentration of Ag NPs, can have different developments (Fig.  6 ). Ag NPs (Ag(0)), penetrating the cell, turn into Ag + ions, which cause additional generation of ROS and contribute to the activation of lipid peroxidation processes as described in Fig.  6 . As a result, the amount of MDA increases (Fig.  5 ), which is a sufficient basis 39 to claim that oxidative stress has developed in yeasts C. guilliermondii NP-4. On the other hand, the activity of antioxidant enzymes also increases under the influence of Ag NPs (Fig.  5 ). Oxidative stress can also lead to the inactivation of various proteins, including enzymes (Fig.  6 ). At the same time, our data show that under the influence of RJ-mediated Ag NPs on the yeasts C. guilliermondii NP-4 a more pronounced increase is observed for the enzyme catalase than in the case of SOD. Probably, this phenomenon can be explained by the fact that as a result of SOD activity, hydrogen peroxide is also generated, the neutralization of which is facilitated by catalase40 40 , 41 , so a higher level of the latter's activity is observed in the cell. In any case, the increase in catalase activity is a protective reaction that helps to overcome the harmful effects of oxidative stress in cells.

The working mechanism by which RJ-mediated Ag NPs influence on the ATPase activity and lipid peroxidation (LP) in yeasts C. guilliertmondii NP-4.

It should be noted that catalase is always present in those systems where electron transfer occurs with the participation of cytochromes, and leads to the formation of hydrogen peroxide, that is, its activity is combined with the operation of the electron transport chain (ETC) (Fig.  6 ). On the other hand, the F O F 1 -ATP-synthase enzyme is a part of the respiratory chain and the process of oxidative phosphorylation. Under appropriate conditions ATP synthase acts as ATPase, generating proton motive force and providing the cell with the necessary energy. ATPase is a membrane-bound enzyme, as several transport systems, including the structure of Na + -K + -ATPase, mediates the transport of many compounds across the membrane at the expense of ATP 42 . Ag NPs can cause cell death by blocking Na + transport mechanisms and disrupting the Na + -homeostasis in cells 43 which can lead to the change of ion fluxes and regulation of enzyme activity, leading to the death of yeast cells. Thus, it can be assumed that the antifungal effect of Ag NPs is mediated by the following mechanism: Ag NPs in the plasma and mitochondrial membranes of yeasts lead to a decrease in total and DCCD-sensitive ATPase activity.

One of the ways of enzyme inactivation is most likely related to the disturbances of phosphorylation-dephosphorylation processes, which is associated with the change of ATPase activity in yeast under the influence of Ag NPs. In particular, the sharp decrease in total and H + -ATPase activity under the influence of fungistatic concentration (5.47 µg mL −1 ) of RJ-mediated Ag NPs ~ 80 and 90%, respectively, indicates that the amount of ATP in the treated cells is reduced as a result of inhibition of ATP hydrolysis, which, in turn, can cause a violation of the phosphorylation of enzymes and lead to a decrease in their activity, because ATP, in addition to being a source of energy, also performs many other functions in the cell. In particular, ATP is a source of phosphate and is included in the processes of phosphorylation and activation of various enzymes, including protein kinases.

In addition, a decrease in H + -ATPase activity, especially in mitochondria, leads to ATP deficiency in cells, which in turn can cause the disruption of various manifestations of cellular activity, inhibit growth and reproduction processes, and prevent stages of plastic exchange. Both processes result in metabolic disturbances, and the amount of yeast biomass accumulated in the stationary phase of growth decreases.

In summary, the data obtained allow us to conclude that the use of RJ-mediated Ag NPs have multifaceted influence on yeasts C. guilliermondii NP-4 and could hold promise for biomedical research, enabling the evaluation of cellular oxidative stress levels, the perturbation of ion transport mechanisms, and potential disruptions in enzyme phosphorylation-dephosphorylation processes. So, it can contribute to deeper understanding of the antifungal influence of RJ-mediated Ag NPs, as well as to control the growth of pathogenic fungi.

These data hold significant potential in advancing genetic engineering approaches, including gene therapy, for addressing diverse inherited human mitochondrial diseases. Moreover, ATP-synthase emerges as a crucial molecular target for drug development, promising advancements in antimicrobial and antitumor agents within medical applications. Additionally, its relevance extends to agriculture, where it could facilitate the development of pesticides and insecticides. The versatility of yeast as a model organism further underscores its utility in these endeavors, offering valuable insights into therapeutic and agricultural innovation.

Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

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Acknowledgements

We thank Dr. Robin Schubert from European XFEL GmbH for the TEM analysis of Ag NPs.

This work was supported by the Higher Education and Science Committee of MESCS in the frames of Research Project № 21 T-1F300 and Basic Support to the Research Institute of Biology, Yerevan State University.

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All authors contributed to the study’s conception and design. The investigations and analysis of results were carried out by SM, HK, LK, AM, SM. SM, AP, HK drafted the manuscript. SM and KT directed the project, corrected and finalized the manuscript. All authors participated in the revision and approval of the final version of the manuscript.

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Marutyan, S., Karapetyan, H., Khachatryan, L. et al. The antimicrobial effects of silver nanoparticles obtained through the royal jelly on the yeasts Candida guilliermondii NP-4. Sci Rep 14 , 19163 (2024). https://doi.org/10.1038/s41598-024-70197-w

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Performance of different saccharomyces strains on secondary fermentation during the production of beer.

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1. Introduction

2. materials and methods, 2.1. yeast strains, media, and culture conditions, 2.2. cell concentration and viability determination, 2.3. vitality determination, 2.4. foam stability, 2.5. apparent extract (ae), alcohol concentration, and ph measurement, 2.6. volatile analysis, 2.7. sensory evaluation of beer, 2.8. sample preparation for proteomic analysis, 2.9. lc-ms/ms, 2.10. data processing and analysis, 3. results and discussion, 3.1. growth of yeast strains during bottle conditioning, 3.2. impact of yeast strain on beer characteristics, 3.3. yeast viability and its impact on foam stability during bottle conditioning, 3.4. impact of yeast strain on volatile profile, 3.5. proteomic profiling to investigate key protein changes that are associated with decreased viability, 4. conclusions, supplementary materials, author contributions, institutional review board statement, informed consent statement, data availability statement, acknowledgments, conflicts of interest.

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Dilmetz, B.A.; Brar, G.; Desire, C.T.; Meneses, J.; Klingler-Hoffmann, M.; Young, C.; Hoffmann, P. Performance of Different Saccharomyces Strains on Secondary Fermentation during the Production of Beer. Foods 2024 , 13 , 2593. https://doi.org/10.3390/foods13162593

Dilmetz BA, Brar G, Desire CT, Meneses J, Klingler-Hoffmann M, Young C, Hoffmann P. Performance of Different Saccharomyces Strains on Secondary Fermentation during the Production of Beer. Foods . 2024; 13(16):2593. https://doi.org/10.3390/foods13162593

Dilmetz, Brooke A., Gurpreet Brar, Christopher T. Desire, Jon Meneses, Manuela Klingler-Hoffmann, Clifford Young, and Peter Hoffmann. 2024. "Performance of Different Saccharomyces Strains on Secondary Fermentation during the Production of Beer" Foods 13, no. 16: 2593. https://doi.org/10.3390/foods13162593

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• Open access
• Published: 20 August 2024

Activation of the yeast Retrograde Response pathway by adaptive laboratory evolution with S-(2-aminoethyl)-L-cysteine reduces ethanol and increases glycerol during winemaking

• Víctor Garrigós 1 ,
• Cecilia Picazo 1 ,
• Emilia Matallana 1 &
• Agustín Aranda 1  

Microbial Cell Factories volume  23 , Article number:  231 ( 2024 ) Cite this article

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Global warming causes an increase in the levels of sugars in grapes and hence in ethanol after wine fermentation. Therefore, alcohol reduction is a major target in modern oenology. Deletion of the MKS1 gene, a negative regulator of the Retrograde Response pathway, in Saccharomyces cerevisiae was reported to increase glycerol and reduce ethanol and acetic acid in wine. This study aimed to obtain mutants with a phenotype similar to that of the MKS1 deletion strain by subjecting commercial S. cerevisiae wine strains to an adaptive laboratory evolution (ALE) experiment with the lysine toxic analogue S-(2-aminoethyl)-L-cysteine (AEC).

In laboratory-scale wine fermentation, isolated AEC-resistant mutants overproduced glycerol and reduced acetic acid. In some cases, ethanol was also reduced. Whole-genome sequencing revealed point mutations in the Retrograde Response activator Rtg2 and in the homocitrate synthases Lys20 and Lys21. However, only mutations in Rtg2 were responsible for the overactivation of the Retrograde Response pathway and ethanol reduction during vinification. Finally, wine fermentation was scaled up in an experimental cellar for one evolved mutant to confirm laboratory-scale results, and any potential negative sensory impact was ruled out.

Conclusions

Overall, we have shown that hyperactivation of the Retrograde Response pathway by ALE with AEC is a valid approach for generating ready-to-use mutants with a desirable phenotype in winemaking.

The wine industry must be constantly evolving to keep up with changing trends. Nowadays, wine consumers are demanding new sensory profiles, and markets are driven by higher aromatic intensity, freshness, full-bodied and ripe fruit flavour profiles and lower alcohol content [ 42 , 56 ]. However, current climate change leads to the overripening of grapes at harvest, increasing the sugar content and lowering the acidity, hence producing high-alcohol wines that lack freshness [ 29 , 39 ]. Fermentation of grape juice by yeasts can be targeted to address all these issues. The most frequently used species in the industry is Saccharomyces cerevisiae due to its fermentative power and adaptation to the winemaking conditions, resulting in rapid and predictable fermentation. The success of S. cerevisiae lies in its ability to adapt metabolically to the changing environment of the industrial processes to which it is subjected [ 37 ]. Nutrient signalling pathways are the main molecular systems responsible for controlling growth and stress response by sensing the presence or absence of nutrients outside and inside the cell [ 8 ]. The main pathways in S. cerevisiae , common to all eukaryotes, are the TOR pathway (which senses mainly nitrogen) and Protein Kinase A (that responds to the presence of glucose).

A phenomic analysis of mutations in nutrient signalling pathways in a haploid wine yeast strain revealed the key relevance of PKA and TORC1 during winemaking [ 52 ]. Under the same conditions, deletion of MKS1 led to an increase in glycerol and a decrease in ethanol and acetic acid [ 19 ]. This phenotype was consistent in several commercial wine strains, as well as in brewer's and baker's yeasts [ 19 ]. MKS1 encodes a repressor of the Retrograde Response (RR), a signalling pathway that communicates mitochondrial dysfunction to the nucleus (reviewed in Jazwinski [ 26 ]). When mitochondrial function is impaired, the complex formed by the transcription factors Rtg1 and Rtg3 translocates to the nucleus and triggers the induction of a broad array of target genes [ 48 ]. The subcellular location of the Rtg1/3 complex is controlled by the repressor Mks1 and the activator Rtg2 [ 12 , 34 ]. RR-targets include genes encoding mitochondrial and peroxisome enzymes that divert cytosolic pyruvate and acetyl-CoA to citrate and then to α-ketoglutarate, a precursor for glutamate/glutamine and lysine biosynthesis (reviewed in Jazwinski [ 26 ]). Due to its role in nitrogen metabolism, RR is repressed by the TOR complex in conditions of abundance through its negative regulator Mks1 [ 9 ]. Thus, the MKS1 deletion mutant exhibits hyperactivated Retrograde signalling and increased expression of RR targets and most of the LYS genes involved in lysine biosynthesis [ 12 ], making it more tolerant to a toxic analogue of lysine called S-aminoethyl-L-cysteine (AEC,also called thialysine [ 16 ].

Nutrient signalling pathways are good targets for improving the production of metabolites of interest. For example, genetic manipulation of TOR components in wine yeasts or chemical inhibition by the herbicide glufosinate have been used to increase glycerol production during winemaking [ 54 , 55 ]. In S. cerevisiae , glycerol plays a major role in redox homeostasis and osmotic stress resistance [ 2 ] and contributes positively to the quality of wine [ 63 ]. Much work has been done on genetic manipulation to redirect metabolism towards increased glycerol production and thus away from ethanol accumulation [ 10 , 38 , 40 , 45 , 57 ] reviewed in [ 22 ]. Due to redox unbalances, this type of manipulation leads to an undesired increase in acetic acid, and additional genetic manipulations are necessary to reduce it [ 4 , 14 ]. However, the use of Genetically Modified Organisms (GMOs) for the food industry still has the main disadvantage of consumer rejection, in addition to strict production and labelling regulations. Consequently, there is great interest in using alternative approaches to improve the properties of wine yeast strains without genetic modification. Adaptive Laboratory Evolution (ALE), which is based on long-term adaptation under environmental or metabolic constraints, is a non-GMO alternative and has been described as a powerful tool in modern industrial biotechnology [ 47 ]. ALE has been successfully used in wine yeast to reduce ethanol production and increase glycerol levels by applying osmotic pressure [ 50 ], but also to achieve higher fermentation rates and enhanced production of aroma compounds [ 3 ]. Recently, it has been reported as a promising strategy to obtain strains that do not increase volatile acidity during winemaking under aerobic conditions [ 23 ]. In this work, we described a workflow (Fig.  1 ) to generate AEC-resistant S. cerevisiae wine strains by ALE and to select those that resemble the previously characterized mks1 ∆ phenotype in winemaking. Several evolved strains achieved reduced acetic acid production, increased glycerol levels and reduced ethanol in laboratory-scale vinifications, and mutations in RTG2 were identified as responsible. One of them was further investigated for reproducing this relevant industrial phenotype at the pilot scale.

Overview of the experimental design described in this study. Steps and experimental procedures for generating new AEC-resistant mutants with improved winemaking phenotypes from S. cerevisiae wine strains. Adaptive laboratory evolution – Commercial S. cerevisiae wine strains MAE, TAE and EAE were serially transferred (up to 29 transfers) into fresh SD media supplemented with 35 mg/L of AEC. Tolerance to AEC in plate – Individual clones isolated from evolved populations were tested for resistance to AEC by spot-analysis to confirm their suitability for further steps. Microvinification – The winemaking phenotype of clones isolated from the evolved populations was analysed by laboratory-scale fermentations in natural grape must. Target gene expression—The expression of Retrograde Response target genes was analysed in those isolated clones that showed a better phenotype in winemaking. Whole-genome sequencing – The whole genomes of selected clones were sequenced to identify possible mutations causing the phenotype of interest. Phenotypic testing of gene mutations—The identified mutations were cloned into an expression vector in yeast to confirm causality. Pilot-scale vinification—Wine fermentations were scaled up to verify the suitability of an isolated mutant in a relevant environment

Materials and methods

Strains and culture conditions.

The Saccharomyces cerevisiae commercial wine strains MAE, TAE and EAE (Lallemand, Co., Canada) were used for adaptive laboratory evolution (ALE) experiments, and the haploid wine strain C9 [ 59 ] was used for genetic manipulation. The complete list of strains used in this work is available in Additional file 1 .

For standard propagation and genetic manipulation, yeasts were cultured in YPD media (10 g/L yeast extract, 20 g/L peptone and 20 g/L glucose) at 30 °C and 180 rpm. For the selection of yeast strains with the kan MX dominant marker, YPD medium supplemented with 200 mg/L geneticin was used. Minimal medium SD (1.7 g/L yeast nitrogen base without amino acids, 5 g/L ammonium sulphate, 20 g/L glucose) or SC (the same as SD but supplemented with the indicated amount of drop-out powder (Formedium)) supplemented with 2 g/L cycloheximide was used to select the transformants with cycloheximide resistance. The solid media were prepared by the addition of 20 g/L agar before sterilization.

ALE experiments and growth tests were performed in SD media supplemented with 35 mg/L of 2-aminoethyl-L-cysteine (AEC), denoted as SD + AEC. Microfermentation experiments were conducted in red grape juice (Bobal variety) sterilized overnight with 500 mg/L dimethyl dicarbonate in cold. For pilot-scale fermentations, 50 kg of tempranillo grapes were used at Vitec, Wine Technology Centre (Falset, Spain).

The Escherichia coli NZYα strain (NzyTech) was used to maintain and amplify the cloned plasmids. E. coli cells were propagated in LB media (10 g/L of tryptone, 5 g/L of yeast extract and 5 g/L of NaCl) supplemented with 100 mg/L of ampicillin to maintain plasmids at 37 °C and 220 rpm.

• Adaptive laboratory evolution

The S. cerevisiae industrial wine strains MAE, TAE and EAE were used as the original parental strains for the ALE experiments. For ALE, a single colony of the parental strain was inoculated into 5 mL of SD media and grown overnight. This culture was used to inoculate 125 mL flasks containing 25 mL of SD + AEC (35 mg/L) at an initial OD 600 of 0.1. The cultures were grown at 30 °C and 180 rpm until they reached the stationary phase before being transferred into fresh medium every 2–3 days at an initial OD 600 value of 0.1. The number of generations through evolution was calculated by the following equation: n = log 2 (N 0 / N t ), where n is the number of generations, N 0 is the initial OD 600 and N t is the OD 600 at time t [ 62 ]. After 8 and 29 transfers, cryostocks of the cultures were prepared, and single clones were isolated by plating 1 µL of the culture on SD + AEC agar. Interdelta analysis of the randomly selected colonies was conducted according to Legras and Karst [ 30 ] to determine whether there was any contamination.

Microfermentation experiments

For the microvinification experiments, cells from 2-day cultures in YPD medium were inoculated at an OD 600 value of 0.1 into conical centrifuge tubes with 30 mL of red grape juice (Bobal variety), a gift from Bodegas Murviedro (Requena, Spain). Fermentations were performed at 24 °C with low shaking (50 rpm). The vinification process was followed by taking aliquots of the supernatant every 2–3 days and measuring the consumption of reducing sugars with DNS (dinitro-3,5-salicylic acid) according to Miller’s method [ 46 ]. The supernatant was used for metabolite measurement at the end of fermentation. Glycerol and acetic acid were measured with commercial kits (Megazyme Ltd., Bray, Ireland). The enzymatic quantification of ethanol was performed by spectrophotometric detection at 340 nm of NADH formed during the oxidation of ethanol to acetaldehyde by the enzyme alcohol dehydrogenase. The assay was performed in 0.2 M glycine—0.3 M Tris buffer (pH 9.7) supplemented with 2 mM NAD + and 20 U/mL yeast alcohol dehydrogenase in a final volume of 1 mL, and 200 μL of sample was added (appropriate dilution).

Pilot-scale fermentations

Pilot-scale fermentations were performed at Vitec’s experimental cellar (Falset, Spain). The selected yeast strains were grown in liquid YPD for 48 h at 28 °C and then transferred to Pyrex bottles for growth in 1 L of YPD media. From this volume, total yeast was counted by optic microscopy to inoculate at a concentration of 2 × 10 6  cells/mL the non-sterile must from 50 kg of Tempranillo grapes. Vinifications were carried out in triplicate and independent vats. Alcoholic fermentation was monitored by daily control of density and temperature and by studying the total and viable yeast population. At the end of fermentation, aliquots of the different fermentations were transferred to YPD plates to control the implantation of the inoculated strain, and the following parameters were analysed: density, sugars (D-glucose/D-fructose), alcoholic strength, total tartaric acidity, volatile acidity, pH, L-malic acid, and glycerol. The analyses were performed according to the protocols established by the Compendium of International Methods of Wine and Must Analysis OIV (2011).

Aroma and taste analysis were carried out by a panel of 10 wine experts who are part of Vitec's accredited tasting panel. The samples were presented simultaneously in a different order for each expert based on a Latin square experimental design. All the samples were served at room temperature and the panellists were not informed about the nature of the samples to be evaluated.

Plasmid construction

The plasmids and primers used herein are listed in Additional files 2 and 3, respectively. For cloning the different alleles of the RTG2 , LYS20 and LYS21 genes, each gene containing its promoter and terminator was PCR-amplified from the genomic DNA of the parental and selected evolved strains using Phusion DNA polymerase (Thermo Scientific). The primers RTG2-X/RTG2-B, LYS20-X/LYS20-B, and LYS21-X/LYS21-B were used for RTG2 , LYS20 and LYS21 amplification, respectively. PCR products and the pCUP1pNuiHA kanMX CEN plasmid were digested with the restriction enzymes XhoI and BamHI, gel-checked and purified using mi-PCR Purification Kit (Metabion). pCUP1pNuiHA kanMX CEN was a gift from Nils Johnsson (Addgene plasmid #131168; http://n2t.net/addgene:131168 ; RRID:Addgene_131168) [ 13 ]. Then, the ligation reactions were carried out using T4 DNA Ligase Kit (NzyTech). The E. coli -positive transformants were selected and the plasmids were PCR-checked and sequenced.

Yeast genetic manipulation

The recyclable kan MX selection marker from plasmid pUG6 [ 24 ] was used to perform gene disruptions. This marker contains flanking loxP sites to excise it by employing the Cre recombinase from plasmid YEp-cre-cyh [ 11 ]. Yeast cells were transformed by the PEG/LiAc method according to [ 21 ]. Routinely, 200–500 ng of plasmid DNA and 0.5–1 µg of linear DNA were used per transformation. All transformants were selected on the corresponding selective agar media.

Relative gene expression level quantification by real-time PCR

To quantify the relative expression levels of the indicated genes, yeast cells were first cultivated in 5 mL of YPD media overnight and transferred to 50 mL of fresh YPD media. Then, they were cultured to an OD 600 of 0.6–0.8. The cells were harvested, and total RNA was extracted [ 6 ]. The obtained RNA was reverse transcribed using NZY First-Strand cDNA Synthesis Kit (Nzytech). Described pair of primers CIT2f/CIT2r, DLD3f/DLD3r and ACT1f/ACT1r [ 49 ] were used to amplify CIT2 and DLD3 , and the housekeeping gene ( ACT1 ) was used as an internal control. Quantitative PCR using NZYSpeedy qPCR Green Master Mix (Nzytech) was performed with a QuantStudio 3 instrument following the manufacturer’s instructions. Each reaction was carried out in triplicate, and the reported Ct value was reported as the average of triplicate samples. Transcript levels were calculated using the 2 −ΔΔCt method [ 35 ].

DNA extraction and sequencing

YeaStar Genomic DNA Kit (Zymo Research) was used to extract genomic DNA from 5 mL of overnight yeast YPD culture using the protocol recommended by the manufacturer. The quality and concentration of the extracted DNA were assessed with a NanoDrop One spectrophotometer (Thermo Scientific). Sequencing, library preparation and subsequent bioinformatics analysis were carried out by the company Novogene. The genomic DNA was randomly sheared into short fragments. The obtained fragments were end repaired, A-tailed and further ligated with Illumina adapter. The fragments with adapters were PCR amplified, size selected, and purified. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. The quantified libraries were pooled and sequenced on the Illumina HiSeq ™ platform. The data recorded in the image files were first transformed to sequence reads by base calling with CASAVA software. The effective sequencing data were aligned with the reference sequences through BWA software (parameters: mem -t 4 -k 32 -M), and the mapping rate and coverage were determined according to the alignment results. Data mapping was performed against the S288c genome (GCF_000146045.2). The mapping rate, depth and coverage statistics are described in Additional file 4 , together with the accession numbers for the genomes of the evolved strains deposited in NCBI. This table contains the unique SNPs and InDels of the evolved strains, with the mutations of interest highlighted. Non-synonymous polymorphisms were identified in the CDS regions of selected genes by visual comparison to the parental strains. Mutations in both the parental and evolved strains were excluded from further analysis.

Adaptive laboratory evolution of S. cerevisiae wine strains and mutant screening

We have previously demonstrated that the deletion of the Retrograde Response repressor MKS1 increases glycerol and reduces acetic acid production in S. cerevisiae wine strains under winemaking conditions [ 19 ]. After these results, our interest focused on obtaining ready-to-use strains with the same phenotype but without genetic manipulation. Deletion of MKS1 has been reported to promote lysine biosynthesis, resulting in increased tolerance to its toxic analogue S-(2-aminoethyl)-L-cysteine (AEC), also called thialysine [ 16 ]. This phenotype was also found to be true in wine strains of S. cerevisiae [ 19 ]. Taking advantage of this phenotype, an adaptive laboratory evolution experiment was designed to generate evolved populations of three commercial S. cerevisiae wine strains with increased tolerance to thialysine. The ALE experiment was planned for three different strains to prove that is a reliable and reproducible method that could be applied to any yeast of interest. First, the native tolerance of these strains to AEC was determined by measuring growth after 48 h in SD media supplemented with different concentrations of this compound. A concentration of 35 mg/L AEC was selected for ALE as it reduced yeast growth by ~ 50% (data not shown), thus leaving room for improvement without completely inhibiting yeast growth.

Initially, the ALE experiment in SD + AEC was conducted for the MAE strain. Two single colonies were used as seeds for two independent replicates of the experiment (eMAE 1, eMAE 2). Due to the rapid adaptation of the strain under the studied conditions, single colonies of each replicate were isolated on SD + AEC (35 mg/L) plates after 8 transfers. The ALE experiments were stopped after 29 transfers due it was found that there was no change in maximum cell density (Fig.  2 A). In both replicates, a similar number of cumulative generations was achieved over time (Fig.  2 B). The same strategy was followed for the TAE and EAE strains, and the experiment was performed in parallel with a single replicate of each strain. Adaptation of EAE was faster and reached a higher number of cumulative generations than TAE (Additional file 5 A and 5B).

Adaptive laboratory evolution experiments and AEC resistance of individual clones isolated from each evolved population were monitored. A Generations obtained in each of the transfers performed during the directed evolution of the two replicates (eMAE 1, eMAE 2) of the MAE strain and B accumulated generations over the course of the experiment. The black arrows indicate 8 and 29 transfers, the time points at which individual clones were isolated from the evolved population. C Spot growth analysis to test AEC tolerance of several clones isolated from MAE, TAE and EAE evolution after 8 and 29 transfers. A 5 μl volume of each serially diluted culture (from 10 –1 to 10 –4 ) was spotted on SD plates supplemented or not with 35 mg/L of AEC. All plates were incubated at 30 °C for 48 h. Information regarding the progress of the adaptive evolution experiment of strains TAE and EAE, as well as the thialysine resistance of each isolated clone, can be found in Additional file 1

Within each ALE experiment, five random individual clones were picked from culture plates and increased tolerance to AEC was confirmed. Figure  2 C shows a spot analysis of one clone from each directed evolution experiment, after 8 and 29 transfers, compared to its parental strain and its respective MKS1 null mutant. After 8 transfers, it was already observed that these clones had greater tolerance to AEC than their parental strains. This phenotype was compatible with hyperactivation of the Retrograde Response pathway, as these strains were even more tolerant than the MKS1 mutants. This phenotype was also observed for the five tested clones from each ALE experiment (Additional file 5 C ).

Phenotypic characterization of the evolved mutants during wine fermentation

In order to study whether the evolved clones show a phenotype compatible with the MKS1 null mutant in wine fermentation [ 19 ], microvinification experiments were conducted on red grape must with the most promising clones.

Figure  3 shows the microfermentation experiment with individual clones isolated from the evolution of the TAE strain. Five clones selected after 8 (Fig.  3 A) and five after 29 transfers (Fig.  3 B) showed a slower fermentation profile than the parental strain, although they completed the consumption of sugars. Metabolites of oenological interest were measured on the last day of fermentation. After 8 transfers, only clone eTAE 8a showed significant differences in final acetic acid and glycerol levels compared to those of the parental strain. The glycerol content of eTAE 8a was 1.26-fold higher than that of the TAE strain (Fig.  3 C), and the acetate levels were 0.76-fold lower (Fig.  3 D), but no significant differences in ethanol production were detected (Fig.  3 E). However, after 29 transfers all the evolved clones showed an increase in glycerol up to 1.98-fold (Fig.  3 C), a decrease in acetic acid up to 0.37-fold (Fig.  3 D) and up to 0.85-fold decrease in ethanol (Fig.  3 E) compared to those of the parental strain.

Winemaking performance of five individual clones from the TAE-evolved population in natural red must. A Reducing sugar consumption (from the initial concentration of 250 g/L of the natural red grape must, Bobal variety) during fermentation in clones isolated after 8 transfers and B after 29 transfers. C Glycerol, D acetic acid and E ethanol produced at the end of fermentation with the individual clones isolated after 8 transfers (left) and 29 transfers (right). Fermentation was carried out in triplicate, and the average and standard deviation are provided. Significant differences (*p < 0.05, Student’s t-test) between the isolated clones and their parental strains are shown. Information regarding the residual sugars at the end of fermentation, as well as the metabolites of oenological interest produced by the clones isolated from the MAE, EAE and TAE evolved populations, where significant differences with respect to the parental strain have been recorded, can be found in Additional file 6

Regarding the clones isolated from the MAE and EAE ALE experiments, Additional file 6 shows those in which significant differences in acetate, glycerol and/or ethanol were recorded compared to the parental strain. After 8 and 29 transfers of MAE evolution, several clones exhibited reduced acetate levels and increased glycerol production compared to those of the parental strain. However, only clones eMAE 29-2a and eMAE 29-2c (obtained after 29 transfers) showed a significant decrease in ethanol production. From the evolution of EAE, after 29 transfers, only two clones (eEAE 29j and eEAE 29o) were obtained that showed higher glycerol and lower acetate levels than the parental strain, although with no significant differences in ethanol.

Activation status of the retrograde pathway in evolved mutants

After verifying that the selected evolved mutants showed increased resistance to AEC (Fig.  2 c and Additional file 5 C) and a similar phenotype to the MKS1 null mutant in winemaking (more glycerol, less acetate and sometimes less ethanol) ([ 19 ], Fig.  3 and Additional file 6 ), the next step was to study whether they also had a hyperactivated Retrograde pathway.

For this purpose, the evolved mutants with the most differential phenotypes in winemaking were selected. After 8 transfers, eMAE 8-1b, eMAE 8-2d and eTAE 8a were selected because they showed the greatest reduction in acetic acid and increase in glycerol compared to their parental strain. For the same reasons, eEAE 29j and eEAE 29o were selected after 29 transfers, and eMAE 29-2c and eTAE 29 l, as they also showed reduced ethanol production. From exponential growing cells in glucose-rich medium (conditions in which the Retrograde pathway is repressed [ 28 , 32 ]), quantitative real-time PCR was performed to verify the relative expression levels of two canonical RR marker genes, CIT2 and DLD3 [ 7 , 31 ]. CIT2 encodes the peroxisomal isoform of citrate synthase, and DLD3 is a 2-hydroxyglutarate transhydrogenase that produces lactate from pyruvate. Both enzymes promote α-ketoglutarate synthesis in cells with mitochondrial dysfunction [ 1 , 33 ].

Among the clones selected after 8 transfers for MAE and TAE, only eTAE 8a showed a slightly higher increase in CIT2 relative expression than did the parental strain, but there was no difference in DLD3 expression (Fig.  4 A). Contrary to our assumption, CIT2 transcription levels in eMAE 8-1b and eMAE 8-2d were significantly lower than those in MAE (Fig.  4 B), so the phenotype of these clones in winemaking was not linked to overactivated Retrograde signalling. However, after 29 transfers, all the selected mutants showed increased expression of CIT2 and DLD3 , although to a lesser extent for eEAE 29j (Fig.  4 C).

mRNA levels of the Retrograde Response targets CIT2 and DLD3 in selected mutants. A Relative expression levels in selected clones isolated after 8 and 29 transfers from the evolution of MAE, B from the evolution of TAE and C from the evolution of EAE. Cells are isolated from an exponential culture in rich medium YPD, where RR targets are repressed. The data are presented as averages of three independent experiments with standard errors. Significant differences (*p < 0.05, Student’s t-test) between the isolated clones and their parental strains are shown

Whole-Genome sequencing of selected evolved mutants

To investigate the genetic basis behind the obtained phenotypes, we performed whole-genome sequencing on the mutants with higher RR expression, namely, eMAE 29-2c, eTAE 29 l, eEAE 29j and eEAE 29o. The eMAE 8-1b mutant was also included with the aim of identifying mutations, independent of the Retrograde pathway, causing its interesting phenotype in winemaking. Due to the differences between industrial strains and the reference S. cerevisiae genome and because commercial strains are diploid while laboratory strains are used and sequenced as haploid strains, it was difficult to perform a direct comparison. Lists for the unique exonic non-synonymous SNPs and in/del compared to each parental strain, that was sequenced in parallel, were obtained (Additional file 4 ). A visual analysis ignoring the subtelomeric regions that exhibited the most changes, mainly focusing on the genes involved in the Retrograde pathway and lysine biosynthesis, was performed (Fig.  5 A). Figure  5 B shows an overview of the relevant genes mutated in the selected evolved strains. As all mutant strains contained mutations compatible with the phenotype observed, the rest of the mutations, which were mostly present in heterozygosis (and therefore are less stable), were excluded. Mutations in RTG2 , which encodes the RR activator Rtg2, seem of outstanding relevance due to their presence in three of the five sequenced strains. Moreover, the variety of mutations found in this gene is a further indication of its importance in AEC resistance. In strains eMAE 29-2c and eTAE 29 l there were two homozygous mutations resulting in two different amino acid changes, while in strain eEAE 29o there was another different amino acid substitution, but in this case, it was heterozygous.

Targeted search for mutations in genes involved in RR-dependent lysine biosynthesis. A Summary of the pathways, genes and metabolites involved in lysine biosynthesis. RR-dependent gene expression is based on a dynamic interaction between Rtg2 and Mks1. When the RR pathway is inactive, Mks1p dissociates from Rtg2p and interacts with the 14–3-3 protein Bmh1/2 to inhibit Rtg1/3 nuclear translocation. Grr1-dependent degradation of free Mks1 ensures an efficient switch between the Rtg2-Mks1 and Bmh1/2-Mks1 complexes. The TCA cycle reactions that turn succinate into oxaloacetate are rendered inactive when Retrograde signalling occurs. However, the TCA cycle can be fuelled by citrate generated in the glyoxylate cycle, which requires only a source of acetyl-CoA. The β-oxidation of fatty acids or other sources may provide this acetyl-CoA. The TCA cycle can also be sustained by the anaplerotic conversion of pyruvate to oxaloacetate (OAA) in reactions initiated by pyruvate carboxylase. This over-supplementation of OAA and the connection to the glyoxylate cycle allows the TCA cycle to remain as a net source of α-ketoglutarate (α-KG) for lysine biosynthesis. The condensation of α-KG and acetyl-CoA, catalysed by Lys20/21 homocitrate synthases, is the first step of the lysine biosynthetic pathway in S. cerevisiae . According to [ 12 ], upregulated genes in the mks1 ∆ deletion mutant are indicated in green. ACS1 , acetyl-coenzyme A synthetase; PYC1 , pyruvate carboxylase; CIT1 , mitochondrial citrate synthase; ACO1/2 , aconitases; IDH1/2 , isocitrate dehydrogenases 1 and 2; CIT2 , peroxisomal citrate synthase; LYS20/21 , homocitrate synthases; LYS4 , homoaconitase; LYS12 , Homo-isocitrate dehydrogenase; ARO8 , aminotransferase; LYS2 , α-aminoadipate reductase; LYS9 and LYS21 , Saccharopine dehydrogenases. B Table of gene mutations found in selected clones from each independently evolved population

Mutations in the LYS20 and LYS21 genes were also found, as previously described in studies where AEC-resistant mutants were isolated [ 17 , 20 ]. These genes encode two paralogous homocitrate synthases, Lys20 and Lys21, which catalyse the first step of lysine biosynthesis, using 2-oxoglutarate and acetyl-CoA (Fig.  5 A). In strains evolved from EAE, the same mutation was found in LYS20 , but only in heterozygosis in the eEAE 29o strain. In eMAE 8-1b and eTAE 29 l, different homozygous mutations were found in LYS21 . In an attempt to isolate any of the two eEAE 29o mutations in homozygosis, the same adaptive evolution experiment with AEC was extended for an additional 29 transfers but without success (data not shown).

Analysis of isolated gene mutations

In order to find the genetic causes of the observed phenotypes, we selected evolved genes with homozygous mutations ( RTG2 – eMAE 29-2c, RTG2 – eTAE 29 l, LYS21 – eMAE 8-1b, LYS21 – eTAE 29 l, LYS20 – eEAE 29j) and cloned them into a centromeric plasmid under their own promoter. The resulting constructs were subsequently transformed into the corresponding rtg2∆ or lys20∆lys21∆ mutant versions of the haploid wine strain C9 (derived from the commercial strain L2056). The use of a haploid version facilitated the deletion of the genes and introduced another genetic background, which would reinforce the relevance of the mutations. Figure  6 shows a spot analysis test to verify the implication of the isolated mutations in AEC tolerance compared with the parental gene isolated from each genetic background (which in all cases was identical to the reference genome). Mutant alleles of RTG2 conferred greater resistance to AEC than did the parental alleles, and the effect was most severe in the case of the R30C mutation isolated from eMAE 29-2c. The lys20∆lys21∆ mutant strain was unable to grow on minimal medium given its inability to synthesize lysine, a defect that was alleviated by introducing at least one of the LYS20 or LYS21 versions, confirming their functional overlap. Again, the LYS20/21 alleles of the evolved strains conferred greater resistance to AEC than did the alleles of the parental strains. This effect is less apparent in LYS20, as the presence of the parental strain allele already confers some resistance to the toxic, demonstrating that this homocitrate synthase isoform exerts greater control over lysine synthesis, as described in previous works [ 43 , 44 ]. Furthermore, the AEC tolerance phenotype conferred by these mutations was observed when the C9 wild-type strain (and the lys20∆ and lys21∆ single mutants) was transformed, indicating the dominant nature of such mutations (Additional file 7 ).

Involvement of mutations identified in the RTG2 , LYS20 and LYS21 genes on the thialysine resistance phenotype. Spot growth analysis of C9 rtg2∆ and C9 lys20∆lys21∆ mutants containing the empty vector (+ vector), the RTG2 , LYS20 and LYS21 alleles of the parental strains, or the alleles carrying the mutations identified in eMAE 8-1b, eMAE 29-2c, eTAE 29 l and eEAE 29j evolved strains. A 5-μl volume of each serially diluted culture (from 10 –1 to 10 –4 ) was spotted on SD (untreated) plates or SD plates containing 200 mg/L geneticin with or without 35 mg/L AEC. All plates were incubated at 30 °C for 48 h

The next step was to determine whether these mutations were responsible for the phenotype in winemaking. For this purpose, microvinification experiments were carried out on red grape juice, restricted only to those mutations isolated in homozygosis that were also responsible for hyperactivation of the Retrograde Response (Fig.  7 ). The lys20∆lys21∆ strain with the empty plasmid ( lys20∆lys21∆ empty) was discarded for further measurements as it was unable to consume the sugars present in the must (Fig.  7 B), highlighting the importance of the amino acid lysine under winemaking conditions. The rest of the strains consumed all sugars, although with a slight delay in the case of the rtg2∆ empty plasmid strain, and the strains carrying the mutation in RTG2 of eMAE 29-2c (Fig.  7 A) and the mutation in LYS21 of eTAE 29 l (Fig.  7 B). Ethanol, acetic acid and glycerol levels were measured at the end of fermentation (Fig.  7 C). Compared with the C9 wild-type strain, the rtg2∆ strain showed no difference in glycerol or acetate levels, and there were no differences in ethanol levels either as described for Tempranillo variety grape juice [ 41 ] or in other genetic backgrounds [ 19 ]. The LYS21 mutation isolated from eTAE 29 l resulted in a slight increase in glycerol levels but did not decrease ethanol or volatile acidity. Mutations in RTG2 , isolated in eTAE 29 l and eMAE 29-2c, led to a reduction in ethanol and acetic acid levels, as well as an increase in glycerol content. Therefore, mutations in RTG2 were responsible for the winemaking phenotype of the evolved strains, while the LYS21 mutation from eTAE 29 l by itself did confer resistance to AEC but was not able to reduce volatile acidity or ethanol.

RTG2 mutations were responsible for increasing glycerol and decreasing acetic acid and ethanol during winemaking. A Laboratory-scale wine fermentations in natural grape must (Bobal variety) with an initial concentration of reducing sugars of 250 g/L were carried out with the strain C9, the rtg2∆ mutant carrying the empty vector or containing different RTG2 alleles B and the lys20∆lys21∆ mutant carrying the empty plasmid or containing the eTAE 29 l LYS21 allele were monitored by measuring sugar consumption. C Ethanol, acetic acid and glycerol levels produced at the end of fermentation by each strain. The experiments were performed in triplicate, and the means and standard deviations are provided. Significant differences (*p < 0.05, Student’s t-test) between the C9 strain and the mutants containing the plasmids are described in Additional file 2

Pilot-scale wine fermentation in experimental cellar

Since strain eTAE 29 l hyperactivated Retrograde signalling and showed the greatest ethanol reduction (as well as increased glycerol and reduced acetic acid levels) (Fig.  3 and Additional file 6 ), it was selected for evaluation of its potential for use under industrial conditions. Wine fermentations were carried out in an experimental cellar with 50 kg of red grapes (variety Tempranillo) to determine whether the results at a larger scale were consistent with those observed at the laboratory scale. Figure  8 shows the results obtained from the pilot-scale fermentations. As expected, the fermentation of eTAE 29 l was slower than that of its parental strain TAE (Fig.  8 A). However, eTAE 29 l produced significantly more glycerol (Fig.  8 B) and less acetic acid (volatile acidity) (Fig.  8 C), as described in the microfermentation experiments. In addition, it also achieved a significant reduction of 0.8% (v/v) in ethanol (Fig.  8 D). In addition, it increased the total acidity, measured analytically as tartaric acid (Fig.  8 E), but did not affect the levels of other acids, such as L-malic acid, or significantly reduced the final pH (data not shown). Finally, an organoleptic analysis was performed to rule out the possibility that the mutation produced any unpleasant secondary effects. Sensory analysis of the wines obtained after fermentation (Fig.  8 F) revealed that eTAE 29 l contributed a higher presence of red fruit, but less greenery and black, candied and dried fruit. In addition, the wines resulting from fermentation with eTAE 29 l were judged to have greater volume on attack, aromatic intensity, acidity and less dryness, although they had less tannic strength and a slight chemical touch. Despite the increase in glycerol levels by this strain, it did not affect the unctuousness of the wine. Overall, the aromatic intensity and global score were best for the evolved strain, so the mutations did not cause any undesired effects on the final product.

Exploring the suitability of the eTAE 29 l evolved strain in pilot-scale fermentations. A The progress of alcoholic fermentation by the evolved strain eTAE 29 l and its parent strain TAE was monitored by density control. B Glycerol, C acetic acid (expressed as volatile acidity), D ethanol and E total acidity were measured at the end of fermentation. F Sensory description of the wine samples . Fermentation was carried out in triplicate, and the average and standard deviations are provided. Significant differences (* p < 0.05, Student’s t-test) are shown

Global warming has various effects on the wine industry, including an increase in alcohol content. The framework of the present research is based on the idea of reducing ethanol levels during wine fermentation by improving yeasts, avoiding the drawbacks of excess volatile acidity and increasing glycerol production. To address these challenges, in recent years, there has been a growing trend to explore the potential of non- Saccharomyces yeasts [ 27 , 58 ]. However, the use of non-conventional yeasts requires additional inoculation strategies [ 5 ], as well as optimization of their production [ 51 ] and nutrient management [ 36 ], among other parameters. Therefore, direct breeding of S. cerevisiae wine strains is currently more straightforward, given its industrial know-how and the simplicity of the process.

The research of our laboratory has been focused on S. cerevisiae nutrient signalling pathways as a target for improvement. In line with our previous works, genetic engineering was used for generating mutants at key points in these pathways that, despite not being suitable for the market, can provide valuable phenotypic information that can be used later for targeted development of new strains [ 19 , 52 , 54 ]. Alternatively to genetic manipulation, chemical inhibitors of such pathways can also be used to test hypotheses and improve phenotypes [ 53 , 55 ] or to generate new ones through directed evolution strategies [ 23 ]. Recently, we reported that hyperactivation of the Retrograde Response pathway, by deletion of its repressor MKS1 , changes carbon metabolism by increasing glycerol and reducing ethanol during winemaking [ 19 ].

In this work, we developed an ALE-driven approach (Fig.  1 ) for obtaining non-recombinant S. cerevisiae commercial wine strains that reduce ethanol and acetic acid levels in winemaking, without giving up the beneficial characteristics provided by glycerol, as described for the mks1 ∆ mutant. By using this strategy, the resulting strains can be applied at the industrial level, as they are non-GMOs and therefore avoid regulatory constraints and poor consumer acceptance [ 60 ]. To this end, yeast cultures of three different commercial wine strains were serially transferred under the pressure of a constant concentration of the toxic lysine analogue AEC. This would lead to an increase in lysine synthesis, ideally as a result of hyperactivation of Retrograde signalling. In S. cerevisiae , the lysine biosynthesis pathway starts from α-ketoglutarate, and the production of this intermediate is increased in response to Retrograde stimulation. Therefore, MKS1 was first known as LYS80 , and its deletion increases tolerance to this poisonous lysine analogue [ 16 ].

After exploring the genomes of selected evolved strains with the expected phenotype, point mutations in genes involved in lysine biosynthesis ( LYS20 and LYS21 ) and/or Retrograde Response ( RTG2 ) pathways were detected (Fig.  5 B). In most cases, homozygous mutations were detected in these diploid strains. Therefore, we confirmed the AEC suitability to exert selective pressure at specific points along these pathways, as regardless of the experiment and genetic background, all commercial wine strains used showed similar patterns of evolutionary dynamics. LYS20 and LYS21 encode two paralogous homocitrate synthases, which catalyse the aldol condensation of α-ketoglutarate and acetyl-CoA to form homocitrate, the first step of the α-amino adipate pathway for lysine biosynthesis (Fig.  5 A). This enzymatic reaction is a rate-limiting step because the end-product, lysine, regulates the activity of homocitrate synthases via feedback inhibition [ 17 ]. The rational design of these homocitrate synthases has been described as a strategy for constructing new yeast strains with increased lysine productivity [ 25 ]. Hence, mutations in these genes could lead to a hyperactivated or retroinhibition-insensitive enzyme that diverts metabolic flux from pyruvate to lysine production, reducing acetyl-CoA and thus acetic acid. This could be the case for the eMAE 8-2d and eEAE 29j strains, with mutations in LYS21 and LYS20, respectively, which show a reduction in acetic acid levels (Additional file 6 ) without activating the Retrograde pathway (Fig.  4 ). As they are dominant mutations, they may cause defects in feedback inhibition, but further work is needed to test this hypothesis. Mutations in Ser385 of the LYS20 gene lead to extreme desensitization to feedback inhibition [ 25 ]. Our mutations are located close to that position, for instance, in Asn379 of LYS20 in eEAE 29j and eEAE 29o, which also points to a similar behaviour.

Surprisingly, all other strains showed mutations in RTG2 and none were found in the most obvious target, the MKS1 gene. Rtg2 is a cytoplasmic protein with an N-terminal ATP-binding domain that acts as a sensor of mitochondrial dysfunction and interacts directly with Mks1, allowing the translocation of Rtg1/3 transcription factors from the cytoplasm to the nucleus to activate the expression of RR target genes [ 18 , 34 ](Fig.  5 A). Thus, mutations in RTG2 could prevent or decrease the repression of Retrograde signalling equivalently to MKS1 deletion, that may be more pleiotropic, affecting other processes during the evolution experiment. Indeed, the eMAE 29-2c, eTAE 29 l and eEAE 29o strains showed hyperactivation of the CIT2 and DLD3 genes, which are subject to Retrograde regulation. CIT2 encodes the peroxisomal citrate synthase, a glyoxylate cycle enzyme that enables yeast to use two carbon compounds as its only carbon source. In respiration-deficient cells, CIT2 is overexpressed to increase citrate production from acetyl-CoA and oxaloacetate, providing the metabolic intermediates necessary for anabolic lysine or glutamate biosynthesis from α-ketoglutarate [ 33 ]. DLD3 , encoding a potential cytoplasmic isoform of D-lactate dehydrogenase, is also overexpressed in cells with dysfunctional mitochondria [ 7 ]. It has been described that Dld3 in fact acts as a FAD-dependent transhydrogenase that uses pyruvate as a hydrogen acceptor to convert D-2-hydroxyglutarate to α-ketoglutarate [ 1 ]. Therefore, during vinification, these strains could undergo an increase in glycerol levels at the expense of glycolytic flux, reducing ethanol without increasing volatile acidity, as pyruvate is being pushed towards α-ketoglutarate synthesis, consuming acetyl-CoA in the process (Fig.  3 , Additional file 6 ). Another explanation could be that, in these evolved strains, the ethanol produced during wine fermentation is taken up to supply most of the cytosolic and mitochondrial acetyl-CoA [ 61 ]. Further studies using omics approaches are needed to elucidate the metabolic fluxes of these mutants during grape must fermentation.

Regardless of the mutations accumulated by the evolved strains, all of them were more resistant to AEC than their respective ancestral strains (Fig.  2 , Additional file 5 C). However, for laboratory-scale vinifications, after 8 transfers of ALE, most of these strains did not give a final product with the desired characteristics (Fig.  3 , Additional file 6 ). It is expected that mutations are first produced in one of the alleles of these diploid strains and the mutation may be fixed in the other copy by gene conversion, due to the high homologous recombination activity of yeast. Alternatively, this may be because the selective pressure exerted by the AEC first forces mutations at the level of the lysine synthesis pathway and later at the Retrograde pathway. Our study shows that this can stabilize the mutations by extending the evolution experiment without the need to increase the concentration of the toxic agent. By studying the effect of the mutations described in the sequenced evolved strains, we observed that these mutations were indeed responsible for the AEC resistance phenotype in these strains (Fig.  6 ). Moreover, the effect of these alleles dominates over those of the wild-type strain (Additional file 7 ). As described, the Lys20 homocitrate synthase isoform exerts greater control over lysine synthesis [ 43 , 44 ]. Therefore, it is possible that the N379D mutation in one of the LYS20 copies of strain eEAE 29o was potent enough to decrease the AEC selective pressure. This would explain why eEAE 29o showed mutations in LYS20 and RTG2 in heterozygosis, even prolonging its evolution for another 29 transfers. This time, it would have been interesting to increase the AEC concentration during the evolution of this strain to exert greater selective pressure and fix the mutations in homozygosity.

Pilot-scale trials on Tempranillo grape must with the eTAE 29 l strain were in line with those carried out in the laboratory, achieving a 0.8% (v/v) reduction in ethanol. The wine produced was evaluated by a panel of experts and was assessed positively without detecting any significant defects (Fig.  8 ). During ALE experiments, yeast is under almost invariant conditions, so it is not uncommon to observe that evolved strains show lower performance or a different phenotype when returned to an industrially relevant environment. This may be due to genetic drift or offsets of genetic modifications [ 15 ]. Therefore, it was crucial to scale up the winemaking process and confirm that the overall good attributes of the eTAE 29 l strain obtained in this study were maintained.

Taken together, our study demonstrated that through adaptive evolution, it is possible to obtain non-genetically modified industrial S. cerevisiae wine strains with hyperactivated Retrograde signalling by accumulating mutations in RTG2 , the activator of this pathway. These evolved yeasts, by showing a diversification of carbon metabolism, give rise to low-alcohol wines with reduced volatile acidity but high glycerol content. This metabolic redistribution may lead to specific adverse effects, such as a decrease in the fermentation rate, which the industry should consider for its application. However, no defect in growth was detected (see the control plates on Fig.  2 C). To completely assess the utility of these strains for the wine industry, more pilot-scale studies on various grape must types and winemaking conditions, together with a sensory examination of the wines developed, are needed.

Understanding the functioning of nutrient signalling pathways in S. cerevisiae wine strains has the potential to aid in the development of new and improved strains resulting in a wine with better characteristics. Our study showed that adaptive evolution in the presence of AEC is a promising strategy for obtaining non-recombinant, low-ethanol-producing yeasts by increasing Retrograde signalling. Evolved mutants with an overactive Retrograde Response led to a reduction of volatile acidity in wine, as they allocate carbon skeletons to lysine production (from ketoglutarate) to acquire tolerance to its toxic analogue. In addition, these mutants overproduce glycerol at the expense of glycolytic flux, so they are also able to reduce ethanol during the fermentation of grape must. One mutant tested in an experimental cellar showed that yeast performance was improved without impairing the organoleptic characteristics of the final product.

Availability of data and materials

Data is provided within the manuscript or supplementary information files. Sequence data have been deposited in NCBI under accession codes PRJNA1105211, PRJNA1105213, PRJNA1105003, PRJNA1105208 and PRJNA1105207.

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Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. The authors would like to acknowledge the financial support from the Spanish Ministry of Science and Innovation through grant PID2021-122370OB-I00 (co-financed by FEDER funds) to EM and AA. VG is the recipient of a predoctoral grant from the University of València (Atracció de Talent Program), and CP is supported by Maria Zambrano postdoc contract (ZA21-068) from the Spanish Ministry of Universities.

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VG, EM and AA contributed to the study conceptualization and design. VG, CP and AA performed the experiments and the formal analysis. CP and AA supervised the project. VG wrote the first draft of the manuscript and elaborated all figures. All the authors have read and edited the final manuscript. EM and AA contributed to funding acquisition and project management.

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The authors declare that some of the results presented in this manuscript have also been included in the patent application PCT/ES2023/070344 (WO2023227820A1), whose co-inventors are Agustín Aranda, Emilia Matallana and Víctor Garrigós.

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Supplementary Information

Additional file 1: list of the strains used in this study., additional file 2: plasmids used in this work., additional file 3: primers used in this work, 12934_2024_2504_moesm4_esm.xlsx.

Additional file 4: Statistics of mapping, accession numbers, and unique SNPs and InDels for the five evolved strains. Relevant mutations in RTG2 and LYS20/21 are indicated in bold.

12934_2024_2504_MOESM5_ESM.pdf

Additional file 5: Monitoring adaptive laboratory evolution experiments of the TAE and EAE strains and thialysine-resistance characterization of all individual clones isolated after 8 and 29 transfers from the MAE, TAE and EAE evolved populations. (A) Generations obtained in each of the transfers performed during the directed evolution of the TAE and EAE strains and (B) accumulated generations throughout the experiment. The black arrows indicate 8 and 29 transfers, the time points at which individual clones were isolated from the evolved population. (C) Spot growth analysis to test the tolerance of each clone isolated from MAE, TAE and EAE evolutions after 8 and 29 transfers.

12934_2024_2504_MOESM6_ESM.docx

Additional file 6: Values of the main metabolites and residual sugars at the end of fermentation. Glycerol, acetic acid and ethanol levels of the isolated evolved clones after 8 and 29 transfers, that registered significative differences compared to the parental strain at the end of fermentations in sterilised natural red grape must (Bobal variety) with an initial concentration of reducing sugars of 250 g/L. The final values for residual sugars have also been indicated. Fermentation was carried out in triplicate, and the average and standard deviations are provided. Statistical differences (* p < 0.05, Student’s t-test) between the evolved clones and their parental strains are shown.

12934_2024_2504_MOESM7_ESM.pdf

Additional file 7: Involvement of mutations identified in the RTG2 , LYS20 and LYS21 genes on the thialysine resistance phenotype. Spot growth analysis of C9, C9 lys20∆ and C9 lys21∆ mutants containing the empty vector (+ vector), the RTG2 , LYS20 and LYS21 alleles of the parental strains, or the alleles carrying the mutations identified in eMAE 8-1b, eMAE 29-2c, eTAE 29l and eEAE 29j evolved strains. A 5-μl volume of each serially diluted culture (from 10 -1 to 10 -4 ) was spotted onto SD (untreated) plates or SD plates containing 200 mg/L geneticin with or without 35 mg/L AEC. All plates were incubated at 30°C for 48h.

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Garrigós, V., Picazo, C., Matallana, E. et al. Activation of the yeast Retrograde Response pathway by adaptive laboratory evolution with S-(2-aminoethyl)-L-cysteine reduces ethanol and increases glycerol during winemaking. Microb Cell Fact 23 , 231 (2024). https://doi.org/10.1186/s12934-024-02504-z

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Hydrothermal conditioning of oleaginous yeast cells to enable recovery of lipids as potential drop-in fuel precursors

• Shivali Banerjee 1 , 3 ,
• Bruce S. Dien 2 , 3 &
• Vijay Singh 1 , 3  

Biotechnology for Biofuels and Bioproducts volume  17 , Article number:  114 ( 2024 ) Cite this article

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Lipids produced using oleaginous yeast cells are an emerging feedstock to manufacture commercially valuable oleochemicals ranging from pharmaceuticals to lipid-derived biofuels. Production of biofuels using oleaginous yeast is a multistep procedure that requires yeast cultivation and harvesting, lipid recovery, and conversion of the lipids to biofuels. The quantitative recovery of the total intracellular lipid from the yeast cells is a critical step during the development of a bioprocess. Their rigid cell walls often make them resistant to lysis. The existing methods include mechanical, chemical, biological and thermochemical lysis of yeast cell walls followed by solvent extraction. In this study, an aqueous thermal pretreatment was explored as a method for lysing the cell wall of the oleaginous yeast Rhodotorula toruloides for lipid recovery.

Hydrothermal pretreatment for 60 min at 121 °C with a dry cell weight of 7% (w/v) in the yeast slurry led to a recovery of 84.6 ± 3.2% (w/w) of the total lipids when extracted with organic solvents. The conventional sonication and acid-assisted thermal cell lysis led to a lipid recovery yield of 99.8 ± 0.03% (w/w) and 109.5 ± 1.9% (w/w), respectively. The fatty acid profiles of the hydrothermally pretreated cells and freeze-dried control were similar, suggesting that the thermal lysis of the cells did not degrade the lipids.

This work demonstrates that hydrothermal pretreatment of yeast cell slurry at 121 °C for 60 min is a robust and sustainable method for cell conditioning to extract intracellular microbial lipids for biofuel production and provides a baseline for further scale-up and process integration.

Graphical abstract

Hydrothermal pretreatment was evaluated as a method for lysing oleaginous yeast cell walls.

Thermal lysis of a yeast slurry (7% w/v, dry cell weight basis) at 121 °C for 60 min. followed by solvent extraction yielded 84.6 ± 3.2% (w/w) of the total lipids.

Hydrothermal pretreatment of a yeast slurry (7% (w/v, dry cell weight basis)) at 170 °C for 10 min yielded 35.6 ± 0.6% (w/w) of the total lipids.

Conditioning yeast slurry at 121 °C for 60 min is proposed to be a green scalable method for cell lysis.

Introduction

Drop-in fuels are sustainable and renewable fuels that can be substituted directly (without engine modification) for either diesel [ 1 ], gasoline [ 2 ], or jet fuel [ 3 ]. Extensive research and development are ongoing to develop commercially viable drop-in fuels from biomass [ 4 , 5 , 6 , 7 , 8 ]. The present industrial routes to produce drop-in fuels are either to convert vegetable oil to green diesel or to ferment sugars to either ethanol or butanol and catalytically upgrade these to synthetic gasoline. The production of lipid-based biofuels is greatly influenced by the availability of lipids [ 9 ]. Single-cell (microbial) oil produced using lignocellulosic hydrolysates could provide an alternate feedstock for biofuel production [ 10 , 11 , 12 , 13 ]. These microbial lipids are obtained from a wide variety of microorganisms including yeast, bacteria, filamentous fungi, and microalgae that can accumulate lipids primarily in the form of triacylglycerols (TAGs) [ 14 ]. Oleaginous yeast allows for easier cultivation, higher cell densities, and better productivity than bacteria or fungi [ 15 ]. Oleaginous yeast cells can supplement plants as a source of oils because they have similar fatty acid profiles as those found in oil seeds. Furthermore, the use of agricultural resides (e.g., corn stover) or bioenergy crops grown on marginal farmlands are expected to increase oil production without impacting row cropland [ 16 , 17 ]. Oleaginous yeast is defined as yeast that can accumulate at least 20% of their dry weight in lipids and many species of yeast under optimized culture conditions exceed 50% (w/w) lipid contents. Some of the genera containing oleaginous representatives are Rhodotorula , Yarrowia , Lipomyces , Rhodosporidium, Cryptococcus , Candida , and Trichosporon [ 15 , 18 , 19 , 20 , 21 , 22 ]. Oleaginous yeast accumulates lipids from sugars when grown on nitrogen-limited media (high C/N ratio) [ 23 , 24 , 25 ]. The lipids in the yeast cells are stored as intracellular droplets and are used as an energy source, stress response, and cell growth [ 26 ].

Oil recovery methods developed for oilseeds do not work for yeast because of their tough cell walls. Therefore, cells need to be broken up first for solvent extraction of the lipids [ 27 , 28 ]. A variety of methods have been proposed for the recovery of single-cell oil [ 29 , 30 , 31 , 32 ]. However, much of the cell lysis research has focused on microalgae, which may not be directly applicable to oleaginous yeast cells because of the differences in cell wall composition [ 30 , 31 , 33 , 34 , 35 , 36 ]. Notably, cell lysis research has also focused on the recovery of proteins and other high-value cell components. The conditioning/pretreatment/disruption of yeast cell biomass removes or weakens the protective cell walls to make the intracellular lipids more accessible to solvent extraction, facilitating high lipid recovery yields. Cell conditioning methods are broadly classified into chemical, physical, and enzymatic methods. The most applied techniques include treatment using microwaves, ultrasounds, shear abrasion, maceration, high-pressure homogenization, and hydrolysis (acidic, basic, or enzymatic) [ 37 ]. An ideal  cell conditioning step would enable efficient solvent extraction without degrading the TAGs, would be scalable and economical [ 38 ]. This includes the use of solvents that are compatible with current industrial practices. It is also preferable to avoid the need for drying the yeast [ 39 ]. When recovering lipids from wet cell biomass, the cell disruption method, lipid accessibility, mass transfer, and emulsion are the major factors that control the scalability, economics, and sustainability of a process [ 31 , 40 , 41 ]. While numerous laboratory-based methods have been proposed, there is still a need to develop new processes that are driven by engineering and economic targets. Methods for effective extraction of lipids from wet cell biomass are required for competitive process economics [ 42 ].

Most of the research on oleaginous yeasts and their industrial applications are centered around species like Yarrowia lipolytica or Rhodotorula toruloides due to their ease of genetic manipulation and ease of growth leading to an improved understanding of their physiology with a possibility of studying and engineering them further [ 43 ]. In similar lines, this work aims to evaluate the potential of thermal lysis of cell walls of the oleaginous yeast strain Rhodotorula toruloides as an aqueous process of lipid recovery. The process is analogous to the aqueous extraction of distillers corn oil in the corn dry grind ethanol process. The efficacy of the aqueous thermal process has been compared with the conventional cell conditioning methods including acid-assisted thermal lysis and sonication.

Materials and methods

Microbial biomass production, pre-seed culture.

The yeast strain R. toruloides Y-6987 was generously provided by the ARS Culture Collection (NCAUR, Peoria, IL) and was maintained on YPD (1% yeast extract, 2% peptone, 2% dextrose) agar plates and incubated at 28 °C for approximately 48 h. The colonies were transferred from the plates to culture tubes containing 3 mL of YPD incubated at 28 °C overnight and mixed at 250 rpm.

Seed culture

The culture tube pre-culture (1 mL) was transferred to a 250 mL baffled flask filled with 50 mL of YPD and incubated at 28 °C for approximately 18 to 24 h with 250 rpm shaking. The optical cell density (A 600 ) of the pool seed culture was 30–36. The contents of the seed culture (~ 20 mL) were centrifuged and resuspended in sterile distilled water to an optical cell density of 50.

Fermentation

The concentrated pre-culture cells (A 600 ) were reinoculated into the production flasks (500 mL) containing 100 mL of the lipid production media (per L: 3 g peptone, 8 g yeast extract, and 100 g glucose). The production flasks were incubated at 28 °C for 3 to 5 days with 250 rpm shaking. Sampling was done to monitor the glucose levels in the fermentation media and the cells were harvested when the concentration of glucose was less than 1%. The cells were harvested by centrifuging the culture in 250 mL bottles followed by the washing of the cell pellets using deionized water. The harvested cell paste was then stored at − 80 °C for further experiments on oil recovery.

Determination of dry cell weight

To determine the dry weight, the oleaginous yeast cell paste/slurry (1 mL) was dried until a constant weight was achieved at 105 °C. The dry cell well was expressed in terms of grams of dry yeast per 100 mL of the cell paste/slurry.

Estimation of total lipids in the microbial cells

The total lipids present in the cell biomass were estimated by a modified method [ 44 ]. Briefly, 5 mL of the cell slurry was mixed with 10 mL of isopropanol and 15 mL of hexane in a 50 mL screw top tube. The mixture was sonicated using a probe sonicator (Misonix XL-2000 Ultrasonic Liquid Processors) twice (for 1 min each) to disintegrate the microbial cell wall. Further, the slurry was shaken with a wrist action shaker (HB-1000 Hybridizer, UVP LLC, Upland, CA) for 10 min at room temperature. Then 16 ml of sodium sulfate solution (6.7%, w/v) was added and shaken for 10 min. The reaction mixture was centrifuged for 20 min at 5000 rpm, and the top phase was carefully transferred with a pipette to a pre-weighed screw-capped tube. The solvent was evaporated under a gentle stream of nitrogen and the lipid was weighed on an analytical balance.

To compare the accuracy of the method, a comparative estimation was made by extracting the lipids from the freeze-dried cell biomass using a conventional Soxhlet extractor. Briefly, 1 g of freeze-dried oleaginous yeast cells (in a Whatman filter paper bag) were subjected to Soxhlet extraction at 65–70 °C with hexane as the extracting solvent for 8 h. The microbial lipids selectively extracted into the hexane (150 mL) during the percolation process were recovered after the vaporization of solvent in a rotary evaporator. The yield of the recovered lipids was then calculated gravimetrically.

Cell conditioning methods

Figure  1 represents the schematic flow diagram of the different conditioning strategies for lysing the oleaginous yeast cells followed by the recovery of extracted lipids. A brief description of the cell conditioning methods is as follows:

Flow diagram of the different conditioning strategies for lysing the oleaginous yeast cells followed by the recovery of extracted lipids

Hydrothermal pretreatment in an autoclave

The microbial cell wall is known to disintegrate by autoclaving. This aspect was used in lysing the oleaginous yeast cells for recovering lipids. Autoclave was the choice of equipment to ascertain the scalability of the developed process. Briefly, 5 mL of oleaginous yeast slurry was loaded in autoclavable Schott bottles (20 mL). The dry cell weight in the slurry was ~14% (w/v) which was diluted with deionized water to achieve a dry cell weight of ~7% (w/v) to study the effect of solid loading on the recovery of lipids. The cell slurries were autoclaved at 121 °C for 30, 60, and 90 min. After cell conditioning, the reaction mixture was collected in a 50 mL screw top tube for extracting the lipids.

Acid-assisted hydrothermal pretreatment in an autoclave

Acid-assisted thermal lysis was carried out by loading 5 mL of yeast cell slurry (14%, w/v) in autoclavable Schott bottles (20 mL). The slurry was diluted using deionized water and hydrochloric acid (HCl) to make up a dry cell weight of 7% (w/v) and a total HCl concentration of 1% (v/v) in the final cell slurry mixture (10 mL). The reaction mixture was subjected to hydrothermal pretreatment in an autoclave at 121 °C for 60 min. The post-conditioning mixture was collected in a 50 mL screw top tube for extracting the lipids.

Around 5 mL of the diluted cell slurry (7% w/v dry cell wt.) was taken in a 50 mL screw top tube and was subjected to sonication using a probe sonicator (Misonix XL-2000 Ultrasonic Liquid Processors) for 5 min. The lipids released after cell disruption were recovered and quantified.

Hydrothermal pretreatment in a sand bath

The wet microbial cell slurry (14%, w/v) was loaded in tube reactors and diluted with deionized water to achieve a dry cell weight of 7% (w/v). Hydrothermal pretreatment was carried out in a fluidized sand bath (IFB-51 Industrial Fluidized Bath, Techne Inc., Burlington, NJ, USA). The loaded reactors were immersed in the sand bath and heated to 130, 150, 170, and 190 °C with 10 min residence time. The time 10 min was chosen based on the previous studies conducted for the hydrothermal pretreatment of bioenergy crops [ 6 , 45 , 46 ] with the idea to subject the microbial cell slurry to hydrothermal pretreatment along with these lignocellulosic crops in an integrated biorefinery. A thermocouple (Penetration/Immersion Thermocouple Probe Mini Conn, McMaster-Carr, Robbinsville, NJ, USA) connected to a datalogger thermometer was used to measure the internal temperature of the tube reactor. After the residence time of 10 min, the autohydrolysis reaction was stopped by submerging the reactor vessel in cold water to rapidly decrease the temperature below 50 °C. The reaction mixture was collected in a 50 mL screw top tube for further recovery of the extracted lipids.

Recovery of lipids

After the cell conditioning, the reaction mixture was collected in 50 mL screw top tubes followed by the addition of isopropyl alcohol (10 mL) and hexane (15 mL). The reaction mixture was shaken with a wrist action shaker (HB-1000 Hybridizer, UVP LLC, Upland, CA) for 10 min at room temperature. Then 16 mL of sodium sulfate solution (6.7%, w/v) was added and shaken for 10 min. The reaction mixture was centrifuged for 20 min at 5000 rpm, and the top phase was carefully transferred with a pipette to a pre-weighed screw-capped tube. The solvent was evaporated under a gentle stream of nitrogen and the recovered lipid was quantified gravimetrically.

The recoverable lipid content was calculated using Eq. ( 1 ):

where w 1 is the lipid recovered by the pretreatment while w 2 is the total lipid present in the whole yeast cell.

As depicted in the schematic flow diagram (Fig.  1 ), another strategy for recovering the extracted lipids at an industrially relevant scale includes centrifugation of the conditioned/pretreated cell slurry at 10,000 rpm for 20 min to separate the solid cell debris and the supernatant. The cell debris, supernatant, and emulsion were analyzed for the total lipid content in them.

Fatty acid profile analysis

The extracted lipid samples were mixed with 2 mL of fresh hexane and 0.2 mL of 2 N KOH. The transesterification step was carried out according to the method previously published [ 47 ]. The samples were then run using the following conditions on the GC: injection volume = 1 μL; inlet = splitless mode; inlet temp = 240 °C; mobile phase = H 2  with 7.5 psi; detector temp = 280 °C; H 2  flow = 35 mL/min; airflow = 400 mL/min; oven temp = 140 °C, then ramp 15 °C/min to 240 °C, and hold for 2.5 min. The peaks were identified using two commercial reference standards (Nu-Chek Prep, product nos. 17A and 20A).

Statistical analysis

All the experiments were conducted in triplicates and the results have been expressed as mean ± standard deviation. One-way analysis of variance (ANOVA) was conducted to determine the statistical significance of the response ( p  < 0.05). The difference in % lipid recovery by different pretreatment methods was evaluated using a one-way ANOVA. Means were compared using Tukey’s test at a 95% confidence interval. Two-way ANOVA with a general linear model was conducted to determine the statistical significance of pretreatment time (min) and the % dry cell weight of yeast slurry on % recovery of lipids ( p  < 0.05). Minitab Statistical software version 21 (Pennsylvania State University, USA) was used to analyze the data.

Results and discussion

Yeast lipid contents.

Hexane and isopropyl alcohol were evaluated as potential co-solvents for measuring total lipid contents in wet cell slurries as an alternative to the conventional Bligh and Dyer method because it avoids the use of methanol and chloroform. Both methanol and chloroform are rated as undesirable and dangerous to use because of their health and environmental impacts [ 48 ]. The total lipid content of the R. toruloides cells was estimated to be 50.8 ± 1.9% (w/w) using ultrasound disruption followed by extraction with a solvent mixture of hexane and isopropyl alcohol (Fig.  2 ). However, the total lipid content (in the freeze-dried yeast cells) measured using the conventional Soxhlet extraction (with hexane as the extracting solvent) was 39.6 ± 1.4% (w/w). The difference in lipid contents can be attributed to the fact that the sonication of the oleaginous yeast cells (before extracting with hexane and isopropyl alcohol) disrupts the cell wall through the cavitation phenomenon, whereas the Soxhlet extraction works by diffusion without cell wall disruption [ 49 , 50 ]. Further, isopropyl alcohol also allows for the recovery of polar (membrane) lipids versus the conventional Soxhlet method solely using hexane. In addition to lipids, the extractant includes carotenoids because of this red yeast. R. toruloides is a natural producer of carotenoids, including β-carotene, torulene, and torularhodin. This is desirable because these molecules are valued by the chemical, pharmaceutical, feed, and cosmetics industries [ 51 , 52 ].

Lipid yields in % cell weight of R. toruloides extracted from wet cell slurry (with hexane and isopropyl alcohol) and freeze-dried cells (with hexane). The data represents the average of three independent experiments and the error bars indicate the standard deviation

Recovery of microbial lipids

The harvested yeast cells were concentrated in a 14.1 ± 0.1% (w/v) slurry based on dried weight. Two different concentrations of cell slurry were used during the conditioning procedures including ~14% and ~7% (w/v).

While recovering lipids from oleaginous microbes, the scalability and economics of the process have been the key parameters. In particular, the thermal cell lysis in an autoclave was explored for recovering microbial lipids because it does not require adding chemicals or drying/freezing of the cells, both of which would add expense. Autoclaving is known to partially solubilize cell wall polysaccharides in hot water [ 53 ]. Preliminary results reveal that the hydrothermal pretreatment of the cell slurry (14%, w/v) at 121 °C for 30 to 90 min led to a lipid yield of greater than 63% w/w. The autoclave took 30–45 min to both reach the desired temperature and to cool down (to 60 °C). The statistical model contains two main effects, viz, pretreatment time (min) at 121 °C and dry cell concentration in yeast slurry (%) and their interaction. Both main effects were significant ( p value < 0.05), but not their interaction ( p value > 0.05) (Table  1 ). The linear model fitted the model well with R 2 and adjusted R 2 values of 94.9% and 92.8%, respectively. The maximum lipid recovery yield (~ 84%, w/w) obtained using a 7% (w/v) yeast slurry pretreated for 60 and 90 min at 121 °C was statistically similar (Table  2 ). The higher lipid recovery could be attributed to the higher severity of the pretreatment due to prolonged heating. The contour plot represents the % recovery of lipids as a function of yeast dry cell weight concentration in the slurry and pretreatment time (at 121 °C). It shows that the lipid recovery was greater than 80% (w/w) of the total lipids for a pretreatment time of 50 to 90 min and yeast cell slurry concentration of 7 to 11% (w/v) (Fig.  3 ).

Contour plot showing the relative recovery of lipids from R. toruloides at different pretreatment time (min) and dry cell weight in yeast slurry (%, w/v) at 121 °C

The lipid yield at a yeast concentration of 7% w/v using acid-assisted hydrothermal pretreatment at 121 °C for 60 min is 109.5 ± 1.9% (w/w) (Fig.  4 a). When heating was combined with an acid (1% v/v HCl), the thermal lysis of yeast cells led to greater cell wall disruption, which increased the recovery of intracellular lipids. Likely, acid hydrolyzes both the cell wall and protein layer protecting the lipid droplets. Xiao et al. reported that the hydrothermal pretreatment of Phaffia rhodozyma at 121 °C (0.1 Mpa, 15 min) with low acid concentration (HCl, 0.5 M) was found to be efficient in disrupting the cell wall leading to an astaxanthin extractability of 84.8 ± 3.2% [ 54 ]. Likewise treating Candida  sp. LEB-M3 in an autoclave (at 121 °C, 101 kPa) for 15 min with HCl (2 M) also resulted in a very high lipid recovery (155.0 ± 4.1% lipids) [ 37 ]. It is notable that a higher lipid yield was observed than the actual microbial lipid content of the cells.

a Lipid recovery yields obtained after applying different cell lysis techniques to R. toruloides and b fractionation of the microbial lipids recovered from the pretreated solid cell debris + emulsion and the supernatant post-centrifugation. The data represent the average of three independent experiments, and the error bars indicate the standard deviation. The labels on the x -axis are abbreviated as HT hydrothermal, AA acid-assisted, SN sonication

The lipid recovery yields obtained with the sonication of the yeast slurry (7%, w/v) was 99.8 ± 0.03% (w/w) when extracted with hexane and isopropyl alcohol as the binary solvent system. Sonication works on the cavitation phenomenon wherein the bubbles formed during the rarefaction phase collapse at the compression phase, which gives a violent shock wave that generates pressure and heat and, ultimately, cell disruption. The lipid recovery from oleaginous microbes using sonication is affected by the time, temperature, cell concentration, frequency, power, and solvent system and has been studied in detail in the literature [ 50 , 55 , 56 , 57 ]. In the present study, sonication at 30 °C for 5 min was evaluated as the conventional cell conditioning method for recovering total lipids from R. toruloides with hexane and isopropyl alcohol as co-solvents.

A lipid balance was performed on the disrupted yeast cells to determine how much of the lipids are recoverable in the liquid phase and the residual lipids in the disrupted yeast cells. The pretreated cell slurry was centrifuged to separate the cell debris from the supernatant to quantify the amount of lipids recovered in the liquid phase (Fig.  4 b). The ideation of this sequential aqueous process of cell conditioning followed by centrifugation was adapted from the aqueous recovery of distillers corn oil in the corn dry grind ethanol process that has been commercialized [ 58 , 59 ]. The hydrothermal pretreatment of the cell slurry at 121 °C for 30, 60, and 90 min led to a recovery of 18.1, 20.0, and 21.8%, respectively, of lipids in the supernatant after centrifugation. While 21.2 and 26.2% of the free lipids were recovered in the supernatant for the acid-assisted hydrothermal pretreatment and sonication-assisted pretreatment, respectively. The remaining 75–80% of the lipids were trapped in the residual cake of pretreated cells left after centrifugation and the emulsion.

The hydrothermal pretreatment of yeast cell slurry at elevated temperatures in a fluidized heat bath was used to evaluate the possibility of reducing the heating time by treating at higher temperatures. Lipids were recovered after hydrothermal pretreatment at 130 to 190 °C for 10 min (Fig.  5 ). The maximum lipid yield was only 35.6 ± 0.6% (w/w) of total lipids by pretreating the yeast slurry at 170 °C for 10 min. Heating at both 150 and 190 °C were similar to each other and significantly lower than 170 °C. The worst lipid yield (16.9 ± 1.8%, w/w) was measured when the yeast cells were treated at 130 °C for 10 min. In contrast, hydrothermal pretreatment at 121 °C for 60 min yielded 84.5 ± 3.2% (w/w) of total lipids. These results highlight the impact of pretreatment time on the extent of cell disruption and eventually the lipid yields. Kruger et al. studied the effect of thermal cell lysis on lipid recovery from oleaginous yeast at 170 and 220 °C [ 60 ]. The highest lipid recovery yield of 62.9% was obtained when the cells were subjected to thermal lysis at 220 °C for 60 min at a cell solid loading of 16%. The high-temperature pretreatment strategies (130–190 °C for 10 min) were tested so that an integrated system could be designed to pretreat the microbial lipid slurry along with the cellulosic biomass such as sugarcane, miscanthus, sorghum, and newer generation bioenergy crops that are being developed to accumulate lipids in the vegetative tissues [ 6 , 45 , 46 , 61 ]. These wild-type and transgenic bioenergy crops could be cultivated on marginal lands and used to produce drop-in fuels that reduce dependency on fossil fuels and food crops [ 62 ]. Combined processing of the bioenergy crops and the microbial cell slurry would be a novel approach and would eventually improve the process economics by reducing the number of unit operations in an integrated biorefinery. However, the recovery of extracted microbial lipids and the separation of the pretreated bioenergy crops would be challenging and would need further research.

Lipid recovery yields obtained after applying hydrothermal pretreatment for 10 min. Each bar is the mean of three independent experiments, and the error bars indicate the standard deviation

Fatty acid profile

The dominant fatty acids synthesized by R. toruloides are palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), and linoleic acid (C18:2) with other fatty acids accounting for < 15% of the total. The fatty acid profiles of the lipids recovered by various methods of cell conditioning are listed in Table  3 . The palmitic, stearic, oleic, and linoleic acid contents in all the extracts are essentially the same, which suggests that the cell conditioning did not degrade the lipids. This is especially important for manufacturing biodiesel because the fuel properties are influenced by the fatty acid composition, including fatty acid chain length, saturation, and unsaturation. The ignition quality of biodiesel can be measured by cetane number, which increases for longer chain length of the fatty acids and decreases with increased branching [ 63 , 64 ]. Polyunsaturated fatty acids reduce stability, cloud point, and cetane number and increase NOx emission, while the long-chain saturated fatty acids increase cloud point and cetane number, and improve oxidative stability with a significant reduction in NOx emission [ 65 , 66 ]. The lipids recovered by each of the cell conditioning methods hold the potential to be converted into biodiesel due to their major fraction being oleic acid (C18:1) which is considered to be the most desirable fatty acid for biodiesel production [ 67 ].

The method selected for conditioning the cell slurry of R. toruloides for solvent extraction was critical for determining the final lipid yield. The maximum lipid yield (109.5 ± 1.5% (w/w)) was obtained for the acid-assisted hydrothermal pretreatment of yeast slurry at 121 °C for 60 min. That the recovery was greater than 100% indicates that this method was more efficient than the standard lipid recovery method. In the absence of acid, the hydrothermal pretreatment at 121 °C for 60 min led to a lipid recovery of 84.6 ± 3.2% (w/w) with organic solvents. In practice, the yeast cells can be concentrated into a slurry and heated using steam in a tank. However, the hydrothermal pretreatment of the cell slurry at 121 °C for 30, 60, and 90 min led to a recovery of 18.1, 20.0, and 21.8%, respectively, of lipids in the supernatant after centrifugation (can be recovered without using any organic solvent). The remaining 79–80% of the lipids were trapped in the residual cake of pretreated cells left after centrifugation and the emulsion. This is analogous to the aqueous recovery of lipids in the corn dry grind ethanol process that has been commercialized. The high lipid yield and the green nature of the process make it a promising method for lysing yeast cells and recovering lipids at an industrially relevant scale. The use of a mild temperature is critical because hydrothermal pretreatment of the cells at 170 °C for 10 min reduced the recovery to 35.6 ± 0.6% (w/w) of the total lipids. The highlighting feature of this method of cell conditioning is that it could be combined with the pretreatment of bioenergy crops in an integrated biorefinery. However, the recovery of extracted microbial lipids and the separation of the pretreated bioenergy crops would be challenging. The different cell conditioning methods render the extractable lipids intact, making them suitable for biofuel production. The extracted lipids contain a high fraction of palmitic, stearic, oleic, and linoleic acid chains which can be readily upgraded to green diesel fuel. Future research is needed to determine the composition of the residual yeast cell mass to determine its value (e.g., as a feed protein source). The end application of the cell residue could be decided after assessing the effect of temperature and acid on the residual proteins and carbohydrate fractions. Further, a detailed technoeconomic study is required to ascertain the applicability of the process at a commercial level.

Data availability

Data will be made available on request.

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Acknowledgements

The authors thank Ms. Thompson for assistance with the oleaginous yeast cultures. Dr. Dien and Ms. Thompson received financial support from the U.S. Department of Agriculture, Agricultural Research Service, United States (CRIS Numbers 5010-41000-189 or 190-00D). The mention of trade names or commercial products in this article is solely for the purpose of providing scientific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer.

This work was funded by the DOE Center for Advanced Bioenergy and Bioproducts Innovation (U.S. Department of Energy, Office of Science, Biological and Environmental Research Program under Award Number DE-SC0018420). Any opinions, findings, and conclusions or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the views of the U.S. Department of Energy.

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Banerjee, S., Dien, B.S. & Singh, V. Hydrothermal conditioning of oleaginous yeast cells to enable recovery of lipids as potential drop-in fuel precursors. Biotechnol Biofuels 17 , 114 (2024). https://doi.org/10.1186/s13068-024-02561-x

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DOI : https://doi.org/10.1186/s13068-024-02561-x

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Production, purification and characterization of phytase from Pichia kudriavevii FSMP-Y17and its application in layers feed

• Biotechnology and Industrial Microbiology - Research Paper
• Published: 20 August 2024

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• Ritu Sharma 1 ,
• Arpana Mittal 1 ,
• Varun Gupta 2 &
• Neeraj K. Aggarwal   ORCID: orcid.org/0000-0003-3515-1207 1  

Introduction

Phytase, recognized for its ability to enhance the nutritional value of phytate-rich foods, has has gained significant prominence. The production of this enzyme has been significantly boosted while preserving economic efficiency by utilizing natural substrates and optimizing essential factors. This study focuses on optimizing phytase production through solid-state fermentation and evaluating its effectiveness in enhancing nutrient utilization in chicken diets.

The objective is to optimize phytase production via solid-state fermentation, characterize purified phytase properties, and assess its impact on nutrient utilization in chicken diets. Through these objectives, we aim to deepen understanding of phytase's role in poultry nutrition and contribute to more efficient feed formulations for improved agricultural outcomes.

Methodology

We utilized solid-state fermentation with Pichia kudriavzevii FSMP-Y17 yeast on orange peel substrate, optimizing variables like temperature, pH, incubation time, and supplementing with glucose and ammonium sulfate. Following fermentation, we purified the phytase enzyme using standard techniques, characterizing its properties, including molecular weight, optimal temperature and pH, substrate affinity, and kinetic parameters.

The optimized conditions yielded a remarkable phytase yield of 7.0 U/gds. Following purification, the enzyme exhibited a molecular weight of 64 kDa and displayed optimal activity at 55 °C and pH 5.5, with kinetic parameters (Km = 3.39 × 10 –3  M and a V max of 7.092 mM/min) indicating efficient substrate affinity.

The addition of purified phytase to chicken diets resulted in significant improvements in nutrient utilization and overall performance, including increased feed intake, improved feed conversion ratio, enhanced bird growth, better phosphorus retention, and improved egg production and quality. By addressing challenges associated with phytate-rich diets, such as reduced nutrient availability and environmental pollution, phytase utilization promotes animal welfare and sustainability in poultry production.

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Abbreviations

Diethylaminoethyl cellulose

Ethylenediaminetetraacetic acid

Feed conversion ratio

β-mercaptoethanol

Polyacrylamide gel electrophoresis

Sodium dodecyl sulphate

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The laboratories used for this research were provided by Kurukshetra University in Kurukshetra, for which the authors are grateful.

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Ritu Sharma: Conceptualization, resources, methodology, writing—original draft. Arpana Mittal: experimentation, visualization, writing— review and editing. Varun Gupta: review and editing. Neeraj K. Aggarwal: formal analysis, supervision, writing—review and editing.

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Sharma, R., Mittal, A., Gupta, V. et al. Production, purification and characterization of phytase from Pichia kudriavevii FSMP-Y17and its application in layers feed. Braz J Microbiol (2024). https://doi.org/10.1007/s42770-024-01492-x

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Received : 04 October 2023

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DOI : https://doi.org/10.1007/s42770-024-01492-x

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