Glucose and Fructose Fermentation by Wine Yeasts in Media Containing Structurally Complex Nitrogen Sources

Glucose and fructose fermentations by industrial yeasts strains are strongly affected by both the structural complexity of the nitrogen source and the availability of oxygen. In this study two Saccharomyces cerevisiae industrial wine strains were grown, under shaken and static conditions, in a media containing either a) 20% (w/v) glucose, or b) 10% (w/v) fructose and 10% (w/v) glucose or c) 20% (w/v) fructose, all supplemented with nitrogen sources varying from a single ammonium salt (ammonium sulfate) to free amino acids (casamino acids) and peptides (peptone). Data suggest that a complex structured nitrogen source is not submitted to the same control mechanisms as those involved in the utilization of simpler structured nitrogen sources, and mutual interaction between carbon and nitrogen sources, including the mechanisms involved in the regulation of aerobic/anaerobic metabolism, may play an important role in defining yeast fermentation performance and the differing response to the structural complexity of the nitrogen source, with a strong impact on fermentation performance.     

Altered Patterns of Maltose and Glucose Fermentation by Brewing and Wine Yeasts Influenced by the Complexity of Nitrogen Source

Maltose and glucose fermentations by industrial brewing and wine yeasts strains were strongly affected by the structural complexity of the nitrogen source. In this study, four Saccharomyces cerevisiae strains, two brewing and two wine yeasts, were grown in a medium containing maltose or glucose supplemented with a nitrogen source varying from a single ammonium salt (ammonium sulfate) to free amino acids (casamino acids) and peptides (peptone). Diauxie was observed at low sugar concentration for brewing and wine strains, independent of nitrogen supplementation, and the type of sugar. At high sugar concentrations altered patterns of sugar fermentation were observed, and biomass accumulation and ethanol production depended on the nature of the nitrogen source and were different for brewing and wine strains. In maltose, high biomass production was observed under peptone and casamino acids for the brewing and wine strains, however efficient maltose utilization and high ethanol production was only observed in the presence of casamino acids for one brewing and one wine strain studied. Conversely, peptone and casamino acids induced higher biomass and ethanol production for the two other brewing and wine strains studied. With glucose, in general, peptone induced higher fermentation performance for all strains, and one brewing and wine strain produced the same amount of ethanol with peptone and casamino acids supplementation. Ammonium salts always induced poor yeast performance. The results described in this paper suggest that the complex nitrogen composition of the cultivation medium may create conditions resembling those responsible for inducing sluggish/stuck fermentation, and indicate that the kind and concentration of sugar, the complexity of nitrogen source and the yeast genetic background influence optimal industrial yeast fermentation performance.     

Effect of Sugar Catabolite Repression in Correlation with the Structural Complexity of the Nitrogen Source on Yeast Growth and Fermentation

Biomass and ethanol production by industrial Saccharomyces cerevisiae strains were strongly affected by the structural complexity of the nitrogen source during fermentation in media containing galactose, and supplemented with a nitrogen source varying from a single ammonium salt (ammonium sulfate) to free amino acids (casamino acids) and peptides (peptone). Diauxie was observed at low galactose concentrations independent of nitrogen supplementation. At high sugar concentrations altered patterns of galactose utilisation were observed. Biomass accumulation and ethanol production depended on the nature of the nitrogen source and were different for baking and brewing ale and lager strains. Baking yeast showed improved galactose fermentation performance in the medium supplemented with casamino acids. High biomass production was observed with peptone and casamino acids for the ale brewing strain, however high ethanol production was observed only in the presence of casamino acids. Conversely, peptone was the nitrogen supplement that induced higher biomass and ethanol production for the lager brewing strain. Ammonium salts always induced poor yeast performance. The results with galactose differed from those obtained with glucose and maltose which indicated that supplementation with a nitrogen source in the peptide form (peptone) was more positive for yeast metabolism, suggesting that sugar catabolite repression has a central role in yeast performance in a medium containing nitrogen sources with differing levels of structural complexity.     

Very high gravity sucrose fermentation by Brazilian industrial yeast strains: effect of nitrogen supplementation

In this study three industrial strains were inoculated into natural sugar cane juice containing 22, 30 and 35% (w/v) sucrose supplemented with nitrogen sources with differing structural complexities, which varied from a single ammonium salt (ammonium sulphate) to peptides (peptone), under aerobic and anaerobic conditions. At 30 °C, in shaken cultures, it was found that efficient sucrose utilization occurred only in media supplemented with a nitrogen source. In general, under agitation, supplementation with peptone led to more efficient fermentation compared with ammonium sulphate supplementation, with higher biomass accumulation and maintenance of cell viability. In a 35% (w/v) sucrose fermentation, under conditions with an inoculation of low cell density, nitrogen supplementation was required to obtain complete sucrose utilization, suggesting the possibility of producing wines with higher amounts of ethanol under working conditions that approach the limit of yeast alcohol tolerance. The results in this study have industrial relevance and they indicate that, under appropriate environmental and nutritional conditions, the commercial Brazilian yeast strains studied can efficiently use sugar, with high cell viability, even during very high gravity sucrose fermentation conditions. Copyright © 2012 The Institute of Brewing & Distilling     

Evaluation of Brazilian ethanol production yeasts for maltose fermentation in media containing structurally complex nitrogen sources

Maltose and glucose fermentations are strongly affected by the structural complexity of the nitrogen source and by the presence of oxygen. In this study five industrial Saccharomyces cerevisiae strains were grown in synthetic medium, containing maltose or glucose, supplemented with different nitrogen sources, with or without agitation. All strains were able to grow and efficiently ferment glucose, but not all strains were able to grow and ferment maltose well. Peptone and ammonium sulfate induced improved fermentation for all strains and conditions. Under agitation, as expected, higher biomass accumulation was detected. Casamino acids supplementation induced efficient maltose fermentation for all of the strains under aerated conditions, but differing maltose utilization patterns were observed for the static cultures. Copyright © 2012 The Institute of Brewing & Distilling     

Structural Complexity of the Nitrogen Source and Influence on Yeast Growth and Fermentation

The structural complexity of the nitrogen source strongly affects both biomass and ethanol production by industrial strains of Saccharomyces cerevisiae, during fermentation in media containing glucose or maltose, and supplemented with a nitrogen source varying from a single ammonium salt (ammonium sulfate) to free amino acids (casamino acids) and peptides (peptone). Diauxie was observed at low glucose and maltose concentrations independent of nitrogen supplementation. At high sugar concentrations diauxie was not easily observed, and growth and ethanol production depended on the nature of the nitrogen source. This was different for baking and brewing ale and lager yeast strains. Sugar concentration had a strong effect on the shift from oxido‐fermentative to oxidative metabolism. At low sugar concentrations, biomass production was similar under both peptone and casamino acid supplementation. Under casamino acid supplementation, the time for metabolic shift increased with the glucose concentration, together with a decrease in the biomass production. This drastic effect on glucose fermentation resulted in the extinction of the second growth phase, probably due to the loss of cell viability. Ammonium salts always induced poor yeast performance. In general, supplementation with a nitrogen source in the peptide form (peptone) was more positive for yeast metabolism, inducing higher biomass and ethanol production, and preserving yeast viability, in both glucose and maltose media, for baking and brewing ale and lager yeast strains. Determination of amino acid utilization showed that most free and peptide amino acids present, in peptone and casamino acids, were utilized by the yeast, suggesting that the results described in this work were not due to a nutritional status induced by nitrogen limitation.     

Impact of ammonium additions on volatile acidity, ethanol, and aromatic compound production by different Saccharomyces cerevisiae strains during fermentation in controlled synthetic media

Variations in ethanol, volatile acidity, and aromatic compounds produced by different Saccharomyces cerevisiae strains were studied in controlled synthetic medium (CSM). Different amounts of assimilable nitrogen (in the form of ammonium sulfate) were added at two fermentation stages (i.e. the beginning of fermentation and the halfway point). There were significant differences in the amount of ethanol produced when ammonia was added to the CSM, although this depended on the yeast strain used. When assimilable nitrogen was added, ethanol production either increased (with Fermicru AR2 and Stellevin NT116 yeast strains) or decreased (with LW LVCB CT1+ yeast strain). The degree of variation also depended on the time that the ammonia was added, with differences of up to 0.7% (v/v). Adding ammonium to the CSM always resulted in lower volatile acidity in the fermentation product. Different yeast strains ( P < 0.0001) and varying amounts of ammonium produced significant differences. Maximum impact – up to 70% less volatile acidity – was obtained using the Stellevin NT116 yeast strain and adding 280 mgN/L ammonium at the halfway point in fermentation. Acetoin production increased at higher concentrations of added ammonium, most markedly when it was added halfway through fermentation. The total amount of esters increased when 140 mgN/L was added to all three yeasts strains tested, irrespective of the fermentation stage. Smaller amounts of higher alcohols were produced following larger ammonium additions, especially at the beginning of fermentation.     

The influence of nitrate on the physiology of the yeast Dekkera bruxellensis grown under oxygen limitation

A previous study showed that the use of nitrate by Dekkera bruxellensis might be an advantageous trait when ammonium is limited in sugarcane substrate for ethanol fermentation. The aim of the present work was to evaluate the influence of nitrate on the yeast physiology during cell growth in different carbon sources under oxygen limitation. If nitrate was the sole source of nitrogen, D. bruxellensis cells presented slower growth, diminished sugar consumption and growth‐associated ethanol production, when compared to ammonium. These results were corroborated by the increased expression of genes involved in the pentose phosphate (PP) pathway, the tricarboxylic acid (TCA) cycle and ATP synthesis. The presence of ammonium in the mixed medium restored most parameters to the standard conditions. This work may open up a line of investigation to establish the connection between nitrate assimilation and energetic metabolism in D. bruxellensis and their influence on its fermentative capacity in oxygen‐limited or oxygen‐depleted conditions. Copyright © 2013 John Wiley & Sons, Ltd.     

Biosynthesis of higher alcohol flavour compounds by the yeast Saccharomyces cerevisiae: impact of oxygen availability and responses to glucose pulse in minimal growth medium with leucine as sole nitrogen source

Higher alcohol formation by yeast is of great interest in the field of fermented beverages. Among them, medium‐chain alcohols impact greatly the final flavour profile of alcoholic beverages, even at low concentrations. It is widely accepted that amino acid metabolism in yeasts directly influences higher alcohol formation, especially the catabolism of aromatic and branched‐chain amino acids. However, it is not clear how the availability of oxygen and glucose metabolism influence the final higher alcohol levels in fermented beverages. Here, using an industrial Brazilian cachaça strain of Saccharomyces cerevisiae, we investigated the effect of oxygen limitation and glucose pulse on the accumulation of higher alcohol compounds in batch cultures, with glucose (20 g/l) and leucine (9.8 g/l) as the carbon and nitrogen sources, respectively. Fermentative metabolites and CO₂/O₂ balance were analysed in order to correlate the results with physiological data. Our results show that the accumulation of isoamyl alcohol by yeast is independent of oxygen availability in the medium, depending mainly on leucine, α‐keto‐acids and/or NADH pools. High‐availability leucine experiments showed a novel and unexpected accumulation of isobutanol, active amyl alcohol and 2‐phenylethanol, which could be attributed to de novo biosynthesis of valine, isoleucine and phenylalanine and subsequent outflow of these pathways. In carbon‐exhausted conditions, our results also describe, for the first time, the metabolization of isoamyl alcohol, isobutanol, active amyl alcohol but not of 2‐phenylethanol, by yeast strains in stationary phase, suggesting a role for these higher alcohols as carbon source for cell maintenance and/or redox homeostasis during this physiological phase. Copyright © 2014 John Wiley & Sons, Ltd.     

Performance of indigenous yeasts in the processing of Chinese strong‐flavoured liquor during spontaneous mixed solid‐state or submerged fermentation

To explore the in situ metabolic characteristics of yeasts involved in the spontaneous fermentation process of Chinese strong‐flavoured liquor, a comparison was conducted between solid‐state fermentation (SSF) and submerged fermentation (SmF) when supplemented with 24 indigenous yeast strains, with a focus on the production of ethanol and a broad range of volatile compounds responsible for the characteristics of Chinese strong‐flavoured liquor. Under the various experimental conditions, the 24 indigenous yeast strains showed different influences on the mixed fermentation system. The fluctuations caused by different yeast strains in the mixed system were less than those caused by the different fermentation modes relative to the formation of flavour compounds. SSF was found to be more suitable for the production of ethanol, methanol and ethyl lactate, whereas SmF was more suitable for the production of 10 higher alcohols, four esters and four acids. This study revealed the relationships amongst the indigenous yeasts, SSF, and the distinctive flavour profiles of Chinese strong‐flavoured liquor. This work provides evidence of the existence of internal stability in spontaneous SSF, thereby facilitating a better understanding of the fermentative mechanism in the SSF process for Chinese strong‐flavoured liquor production Copyright © 2015 The Institute of Brewing & Distilling     

Evaluation of a Brazilian Fuel Alcohol Yeast Strain for Scotch Whisky Fermentations

Traditionally, distilling companies in Scotland have employed a very limited number of yeast strains in the production of alcohol for Scotch whiskies. Recent changes such as the decline in availability of brewers' yeast as a secondary yeast strain and the availability of yeast in different formats (e.g., dried and cream yeast as alternatives to compressed yeast) have promoted interest in alternative Scotch whisky distilling yeasts. In previous work, we investigated different strains of yeasts, specifically Brazilian yeasts which had been isolated from and used in fuel alcohol distilleries. One of the Brazilian yeasts (CAT 1) showed a comparable fermentation performance and superior stress tolerance compared with a standard commercial Scotch whisky distilling yeast (M Type). The Brazilian CAT 1 yeast isolate was further assessed in laboratory scale fermentations and subsequent new make spirit was subjected to sensory analyses. The spirits produced using the Brazilian strain had acceptable flavour profiles and exhibited no sensory characteristics that were atypical of Scotch whisky new make spirit. This study highlights the potential of exploiting yeast biodiversity in traditional Scotch whisky distillery fermentation processes.     

Isolation and Characterisation of Sri Lankan Yeast Germplasm and Its Evaluation for Alcohol Production

Use of inferior yeast cultures represents one of the reasons for low fermentation efficiencies in Sri Lankan alcohol distilleries that use sugarcane molasses. The present study isolated and characterised yeast strains found in natural environments in Sri Lanka and evaluated their performance under laboratory conditions in an effort to select superior strains for industrial fermentations. Yeasts were characterised based on morphological and physiological features such as sugar fermentation and nitrate assimilation. Ethanol production, alcohol tolerance and growth rate of the most promising strains were monitored following laboratory fermentations of molasses. Over a thousand yeast cultures were collected and screened for fermentative activity and a total of 83 yeast isolates were characterised as higher ethanol producers. Most of these belonged to the genus Saccharomyces. Certain strains produced over 10% (v/v) alcohol in molasses media during 72 h laboratory fermentations. Only two strains, SL‐SRI‐C‐102 and 111, showed an appreciable fermentation efficiency of about 90%. The latter strain produced the highest level of ethanol, 11% (v/v) within a 48 h fermentation and exhibited improved alcohol tolerance when compared with the baker's yeast strains currently used in Sri Lankan alcohol distilleries. This study highlights the benefits of exploiting indigenous yeasts for industrial fermentation processes.     

Improving the performance of industrial ethanol‐producing yeast by expressing the aspartyl protease on the cell surface

The yeasts used in fuel ethanol manufacture are unable to metabolize soluble proteins. The PEP4 gene, encoding a vacuolar aspartyl protease in Saccharomyces cerevisiae, was either secretively or cell‐surface anchored expressed in industrial ethanol‐producing S. cerevisiae. The obtained recombinant strains APA (expressing the protease secretively) and APB (expressing the protease on the cell wall) were studied under ethanol fermentation conditions in feed barley cultures. The effects of expression of the protease on product formation, growth and cell protein content were measured. The biomass yield of the wild‐type was clearly lower than that of the recombinant strains (0.578 ± 0.12 g biomass/g glucose for APA and 0.582 ± 0.08 g biomass/g glucose for APB). In addition, nearly 98–99% of the theoretical maximum level of ethanol yield was achieved (relative to the amount of substrate consumed) for the recombinant strains, while limiting the nitrogen source resulted in dissatisfactory fermentation for the wild‐type and more than 30 g/l residual sugar was detected at the end of fermentation. In addition, higher growth rate, viability and lower yields of byproducts such as glycerol and pyruvic acid for recombinant strains were observed. Expressing acid protease can be expected to lead to a significant increase in ethanol productivity. Copyright © 2010 John Wiley & Sons, Ltd.     

Fermentative capacity of Kluyveromyces marxianus and Saccharomyces cerevisiae after oxidative stress

Volatile compound production during alcoholic fermentation has been studied in the production of many beverages. Temperature, yeast strain, nutrients and pH have been identified as important factors in the production of volatile compounds. In addition, other factors could influence this production during the fermentation process as well. Oxidative stress could occur during yeast biomass production because oxygen is an essential nutrient that is added to the growth medium. The fermentation parameters and the volatile compound production of one Saccharomyces cerevisiae strain (MC4) and two Kluyveromyces marxianus strains (OFF1 and SLP1) were evaluated in relation to fermentation parameters after oxidative stress induced by hydrogen peroxide or menadione. These yeasts were compared with S. cerevisiae W303–1A and showed significant differences in ethanol production, ethanol yield and maximum ethanol production rate. K. marxianus (OFF1) showed better fermentative capacity after oxidative stress. The higher alcohol production decreased after oxidative stress by >35% after 72 h fermentation time, and the amyl alcohol decreased at a higher level (>60%); however, the isobutanol production increased after oxidative stress between 1.5 and 4 times. The yeasts produced significant concentrations of esters however ethyl lactate, ethyl caprylate and the ethyl caproate were not detected in the control fermentation, while in the stress fermentation they accounted for up to 3 mg/L. These results demonstrate that oxidative stress can play an important role in the final aroma profile; but it is necessary to guarantee adequate yeast growth to obtain the volatile compounds desired. Copyright © 2017 The Institute of Brewing & Distilling     

发酵条件对5种产酯酵母酒精发酵和产酯的影响

该实验探讨了供氧情况、发酵pH和温度对各种产酯酵母酒精发酵和产酯的影响。 结果表明,供氧情况对产酯酵母的酒精发酵和产酯均有显著影响,其中对克鲁斯假丝酵母的酒精发酵影响最大,对球拟酵母产酯影响最大。 发酵pH对产酯酵母的酒精发酵和产酯有一定影响,其中卡特多菲毕赤酵母的最适产酒pH为5.0,克鲁斯假丝酵母和汉逊酵母最适产酒pH为4.0,球拟酵母最适产酒pH 为6.0～7.0;上述4种产酯酵母的最适产酯pH均为6.7,高产酯酿酒酵母的最适产酯pH为6.0～7.0。 温度对各种酵母的酒精发酵和产酯有一定影响,其中卡特多菲毕赤酵母、克鲁斯假丝酵母、汉逊酵母和球拟酵母的最适产酒温度均为28 ℃;卡特多菲毕赤酵母的最适产酯温度为20 ℃,克鲁斯假丝酵母、汉逊酵母和高产酯酿酒酵母为24 ℃,球拟酵母为28 ℃。     

Production of sensory compounds by means of the yeast Dekkera bruxellensis in different nitrogen sources with the prospect of producing cachaça

The distilled spirit made from sugar cane juice, also known as cachaça, is a traditional Brazilian beverage that in recent years has increased its market share among international distilled beverages. Several volatile compounds produced by yeast cells during the fermentation process are responsible for the unique taste and aroma of this drink. The yeast Dekkera bruxellensis has acquired increasing importance in the fermented beverage production, as the different metabolites produced by this yeast may be either beneficial or harmful to the end‐product. Since D. bruxellensis is often found in the fermentation processes carried out in ethanol fuel distillation in Brazil, we employed this yeast to analyse the physiological profile and production of aromatic compounds and to examine whether it is feasible to regard it as a cachaça‐producing microorganism. The assays were performed on a small scale and simulated the conditions for the production of handmade cachaça. The results showed that the presence of aromatic and branched‐chain amino acids in the medium has a strong influence on the metabolism and production of flavours by D. bruxellensis. The assimilation of these alternative nitrogen sources led to different fermentation yields and the production of flavouring compounds. The influence of the nitrogen source on the metabolism of fusel alcohols and esters in D. bruxellensis highlights the need for further studies of the nitrogen requirements to obtain the desired level of sensory compounds in the fermentation. Our results suggest that D. bruxellensis has the potential to play a role in the production of cachaça. Copyright © 2014 John Wiley & Sons, Ltd.     

Amino acid uptake by wild and commercial yeasts in single fermentations and co-fermentations

Musts require nitrogen-containing compounds in order to ensure yeast development. This study examined the nitrogen-nutrient requirements of two commercial yeasts and three wild strains isolated from inoculated fermentations. The results showed that wild strains generally consumed lower amounts of amino acids than commercial yeasts. Most amino acids were assimilated during the exponential growth phase; only a few – including asparagine and histidine – were metabolized until the end of fermentation. The study also sought to determine whether industrial drying affected yeast nitrogen requirements.     

Formation of α-ketoglutaric acid by wine yeasts and its oenological significance

α-Ketoglutaric acid was measured enzymically in wines made in the laboratory from three grape varieties by pure cultures of 12 wine yeasts of the genus Saccharomyces. The results were confirmed with the same juices and 4 yeasts on pilot-plant scale in replicated 30 gallon lots. Mean values for the 12 yeasts ranged from 9 to 117 ppm (overall mean 53). In any one juice the yeasts differed by at least 10-fold in the amounts produced, and certain yeasts produced consistently high or low yields in all juices. The amounts of α-ketoglutaric acid produced depended somewhat on the grape juices used, even though these had comparable pH values, and a significant yeast-juice interaction occurred. The amount of α-ketoglutaric acid formed during fermentation at 15° was 60 per cent of that formed at 25°, and over twice as much was formed at pH 4.2 as at pH 3.0, using four yeast strains. Formation of α-ketoglutaric and pyruvic acids were not significantly correlated. The α-ketoglutaric acid content of 18 white table wines made under comparable conditions on pilot-plant scale from different grape varieties using the same yeast strain ranged from 38 to 152 ppm (mean 90). The significance of the results is discussed, particularly in relation to the binding of sulphur dioxide in wine, and recommendations are given on how to make wines which are low in α-ketoglutaric acid. Formation of α-ketoglutaric acid by three yeasts in a chemically defined medium was lower with increased amounts of nitrogen as ammonium sulphate and higher in the presence of L-glutamic acid, both being used separately as sole nitrogen sources. These findings are discussed in relation to the rǒle of α-ketoglutaric acid in nitrogen metabolism of yeasts.     

Improvement of Ethanol Production by an Industrial Yeast Strain via Multiple Gene Deletions

The genetic engineering of yeasts used in commercial processes can be both time-consuming and laborious. This is because industrial yeasts possess largely uncharacterised genomes, which frequently carry at least two copies of any gene. Such strains are usually devoid of auxotrophic or other genetic markers and this requires the incorporation of positively selectable (and often heterologous) genes into plasmids or other transforming DNA molecules. In this paper, we demonstrate that multiple gene deletions may be readily performed in industrial yeasts. Using a specially designed loxPkanMX4 gene replacement cassette, we deleted the two PET191 alleles essential to respiration in the diploid, high alcohol-producing, wine yeast, K1. The two integrated deletion cassettes, which rendered the respiratory-deficient mutant, K1 Δpet191ab, resistant to the antibiotic geneticin were then excised from the genome following the expression of a cre recombinase gene harboured on the multi-copy plasmid YEP351-cre-cyh. This plasmid was maintained in the mutant under the selective pressure of the antibiotic cycloheximide and then removed when both genes had been successfully deleted. Batch fermentations were performed in homebrew style for strains K1 and K1Δpet191ab and revealed a 40% higher volumetric ethanol production rate and a 9% higher ethanol ceiling for the mutant. This demonstrates that, because of their respiratory deficiency, nuclear petites are not subject to the Pasteur effect and so exhibit higher rates of fermentation. Furthermore, nuclear petites cannot metabolise the product of fermentation, ethanol, allowing higher ethanol titres to be achieved. We believe that the method of strain manipulation demonstrated here will be of interest to scientists in the alcoholic beverages industry, who wish to delete genes in production yeast strains, while simultaneously ensuring the removal of all foreign coding sequences.     

Medium Optimization of Gamma Linolenic Acid Production in Mucor rouxii CFR -G15 using RSM

Omega-6 polyunsaturated fatty acids has both pharmaceutical and neutraceutical importance. Central composite rotatable design (CCRD), along with response surface methodology (RSM), was employed to optimize the medium components for maximum production of gamma linolenic acid (GLA) from Mucor rouxii CFR-G15. Nutritional parameter such as carbon (glucose) and nitrogen (yeast extract and ammonium nitrate) sources were focused in this study to enhance the GLA production. The optimal conditions for maximizing GLA production (18.55%) were at 65 g glucose per liter; 3.5 g yeast extract per liter, and 0.5 g ammonium nitrate per liter. Thus, by using CCRD with RSM, it is quite possible to determine the accurate values of the fermentation parameters where maximum production of GLA is achieved.