Biosensor online control of citric acid production from glucose by Yarrowia lipolytica using semicontinuous fermentation

Our study aimed at the development of an effective method for citric acid production from glucose by use of the yeast Yarrowia lipolytica. The new method included an automated bioprocess control using a glucose biosensor. Several fermentation methodologies including batch, fed‐batch, repeated batch and repeated fed‐batch cultivation were tested. The best results were achieved during repeated fed‐batch cultivation: Within 3 days of cycle duration, approximately 100 g/L citric acid were produced. The yields reached values between 0.51 and 0.65 g/g and the selectivity of the bioprocess for citric acid was as high as 94%. Due to the elongation of the production phase of the bioprocess with growth‐decoupled citric acid production, and by operating the fermentation in cycles, an increase in citric acid production of 32% was achieved compared with simple batch fermentation.     

Laboratory-scale production of acetoin plus diacetyl by Enterobacter cloacae ATCC 27613

Conditions for the laboratory-scale production of acetoin plus diacetyl by Enterobacter Cloacae ATCC 27613 were studied. Thirty-five g acetoin plus diacetyl/50 g sucrose were obtained when fermentation was carried out in 2. 5 liter medium containing 12.5 g peptone and 12. 5 g yeast extract, at pH 7.0, in a 5 liter conical flask on a shaker (240rpm) at 28–30°C for 48 hr. Recovery of pure diacetyl was 85% of the total plus diacetyl.     

Kinetics of kojic acid fermentation byAspergillus flavus link S44-1 using sucrose as a carbon source under different pH conditions

Kojic acid production byAspergillus flavus strain S44-1 using sucrose as a carbon source was carried out in a 250-mL shake flask and a 2-L stirred tank fermenter. For comparison, production of kojic acid using glucose, fructose and its mixture was also carried out. Kojic acid production in shake flask fermentation was 25.8 g/L using glucose as the sole carbon source, 23.6 g/L with sucrose, and 6.4 g/L from fructose. Reduced kojic acid production (13.5 g/L) was observed when a combination of glucose and fructose was used as a carbon source. The highest production of kojic acid (40.2 g/L) was obtained from 150 g/L sucrose in a 2 L fermenter, while the lowest kojic acid production (10.3 g/L) was seen in fermentation using fructose as the sole carbon source. The experimental data from batch fermentation and resuspended cell system was analysed in order to form the basis for a kinetic model of the process. An unstructured model based on logistic and Luedeking-Piret equations was found suitable to describe the growth, substrate consumption, and efficiency of kojic acid production byA. flavus in batch fermentation using sucrose. From this model, it was found that kojic acid production byA. flavus was not a growth-associated process. Fermentation without pH control (from an initial culture pH of 3.0) showed higher kojic acid production than single-phase pH-controlled fermentation (pH 2.5, 2.75, and 3.0).     

Induction and Citric Acid Productivity of Fluoroacetate-sensitive Mutant Strains of Candida lipolytica

To establish a novel process for the economical production of citric acid from n-paraffins by yeast, attempts were made to obtain some mutant strains capable of producing citric acid in higher yield without (+)-isocitric acid.

From among the mutant strains derived from Candida lipolytica ATCC 20114, which produced citric acid and (+)-isocitric acid in the ratio of about 60:40 from n-paraffins, a citrate non-utilizing mutant strain, K-20, and a fluoroacetate-sensitive mutant strain , S-22, were selected on the basis of high citric acid and low (+)-isocitric acid productivity.

The mutant strain S-22 showed extremely poor growth in a medium containing sodium citrate as the sole carbon source and extremely high sensitivity to fluoroacetate. The production ratio of citric acid and (+)-isocitric acid by the mutant strain was changed to 97:3, and the yield of the citric acid from n-paraffins, charged to the fermentation medium, reached 145%(w/w).     

Biosynthesis of citric acid by Yarrowia lipolytica repeat-batch culture on ethanol

After analysis of batch culture and identification of the ways for prolongation of citric acid active synthesis by yeast, repeat-batch (RB) cultivation was suggested. Yarrowia lipolytica strain RB cultivation was studied and optimal conditions for cultivation selected. It was shown that when applying RB cultivation, better results were obtained than for batch cultivation. The activity of the culture remained stable after cultivation for more than 700 h. Comparative analysis of enzyme activities confirmed the regularity of the effect described, as the activity of practically of all the enzymes participating in ethanol oxidation and citric acid biosynthesis remained stable over time during RB cultivation. Advantages of RB cultivation for the production of citric acid by yeast are discussed. Received: 1 March 1999 / Received revision: 28 June 1999 / Accepted: 5 July 1999     

Influence of Glucose and Saturated Free-Fatty Acid Mixtures on Citric Acid and Lipid Production by Yarrowia lipolytica

In the present report, the effect of glucose and stearin (substrate composed by saturated free-fatty acids) on the production of biomass, reserve lipid, and citric acid by Yarrowia lipolytica ACA-DC 50109 was investigated in nitrogen-limited cultures. Numerical models that were used in order to quantify the kinetic behavior of the above Yarrowia lipolytica strain showed successful simulation, while the optimized parameter values were similar to those experimentally measured and the predictive ability of the models was satisfactory. In nitrogen-limited cultures in which glucose was used as the sole substrate, satisfactory growth and no glucose inhibition occurred, although in some cases the initial concentration of glucose was significantly high (150 g/l). Citric acid production was observed in all trials, which was in some cases notable (final concentration 42.9 g/l, yield 0.56 g per g of sugar consumed). The concentration of unsaturated cellular fatty acids was slightly lower when the quantity of sugar in the medium was elevated. In the cases in which stearin and glucose were used as co-substrates, in spite of the fact that the quantity of cellular lipid inside the yeast cells varied remarkably (from 0.3 to 2.0 g/l – 4 to 20% wt/wt), de novo fatty acid biosynthesis was observed. This activity increased when the yeast cells assimilated higher sugar quantities. The citric acid produced was mainly derived from the catabolism of sugar. Nevertheless, citric acid yield on sugar consumed and citrate specific production rate, as evaluated by the numerical model, presented substantially higher values in the fermentation in which no fat was used as glucose co-substrate compared with the cultures with stearin used as co-substrate.     

Ethanol addition enhances acid treatment to eliminate Lactobacillus fermentum from the fermentation process for fuel ethanol production

Fermentation is one of the most critical steps of the fuel ethanol production and it is directly influenced by the fermentation system, selected yeast, and bacterial contamination, especially from the genus Lactobacillus. To control the contamination, the industry applies antibiotics and biocides; however, these substances can result in an increased cost and environmental problems. The use of the acid treatment of cells (water‐diluted sulphuric acid, adjusted to pH 2·0–2·5) between the fermentation cycles is not always effective to combat the bacterial contamination. In this context, this study aimed to evaluate the effect of ethanol addition to the acid treatment to control the bacterial growth in a fed‐batch system with cell recycling, using the industrial yeast strain Saccharomyces cerevisiae PE–2. When only the acid treatment was used, the population of Lactobacillus fermentum had a 3‐log reduction at the end of the sixth fermentation cycle; however, when 5% of ethanol was added to the acid solution, the viability of the bacterium was completely lost even after the first round of cell treatment. The acid treatment +5% ethanol was able to kill L. fermentum cells without affecting the ethanol yield and with a low residual sugar concentration in the fermented must.

Significance and Impact of the Study

In Brazilian ethanol‐producing industry, water‐diluted sulphuric acid is used to treat the cell mass at low pH (2·0) between the fermentative cycles. This procedure reduces the number of Lactobacillus fermentum from 10⁷ to 10⁴ CFU per ml. However, the addition of 5% ethanol to the acid treatment causes the complete loss of bacterial cell viability in fed‐batch fermentation with six cell recycles. The ethanol yield and yeast cell viability are not affected. These data indicate the feasibility of adding ethanol to the acid solution replacing the antibiotic use, offering a low cost and a low amount of residue in the biomass.     

Repeated batch production of citric acid from sugarcane molasses using recycled solid-state surface culture ofAspergillus niger

Summary The recycled solid-state surface fermentation (SSF) culture ofAspergillus niger KCU520 was used for repeated batch production of citric acid from sugarcane molasses. The rate of citric acid production was doubled, reducing the fermentation time to half, compared to the normal single cycle batch submerged or surface fermentation process. About 80% sugar was converted to citric acid in five-day batch fermentation and three batches were carried out with the same fungal mat without any significant loss of productivity.     

Improvement of docosahexaenoic acid fermentation from Schizochytrium sp. AB‐610 by staged pH control based on cell morphological changes

Schizochytrium sp. AB‐610 accumulates relatively higher amount of DHA‐rich lipid in the cells, and it was found that DHA yield was closely related to the cell morphology and pH value during fermentation period. DHA production from Schizochytrium sp. AB‐610 in fed‐batch fermentation was investigated and four growth stages were clarified as lag stage, balanced growth stage, lipid accumulation stage, and lipid turnover stage, based on the morphologic observation and key parameters changes. Then a simple strategy of two‐stage pH control was developed, in which pH 7.0 was kept until 12 h after the end of balanced growth stage, and then shifted to 5.0 for the rest period in fermentation. A maximal DHA production of 11.44g/L was achieved. This approach has advantage of easy scaling up for industrial DHA fermentation from Schizochytrium sp. cells.     

Improved production of citric acid by Yarrowia lipolytica using oleic acid as the oxygen‐vector and co‐substrate

Yarrowia lipolytica is able to secrete large amounts of citric acid (CA), which is greatly affected by the dissolved oxygen concentration (DOC) in the fermentation medium. In this study, oleic acid was selected as oxygen‐vector to improve DOC during CA fermentation. When 2% (v/v) of oleic acid was added to the culture broth, higher DOC (>42.1%) was determined throughout the CA synthesis phase. The yield of CA reached a maximum of 32.1 g/L (25.4% higher than the control) and the biomass was 8.8 g/L. The substrate uptake rate, products formation rate and key enzyme activities were also determined, and the results indicated that CA synthesis was strengthened with oleic acid addition. Furthermore, it was detected that oleic acid could be assimilated by the cells, which means that oleic acid could be served both as oxygen‐vector and co‐substrate for CA synthesis by Y. lipolytica. In a bioreactor with working volume of 3 L, the highest concentration of CA reached to 36. 4 g/L in the presence of 2% (v/v) oleic acid after 192 h of fermentation. These results confirmed that oleic acid could be applied in the large‐scale production of CA by Y. lipolytica.     

Optimization of citric acid production from a carrot juice-based medium by <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> using response surface methodology

In this study, diluted and fortified carrot juice was used for modelling and optimization of citric acid production by a new mutant strain, Yarrowia lipolytica K-168. Protein concentrate obtained from fine flour -a byproduct of semolina production- was used as a nitrogen source in the fermentation medium. Interactive effects of selected independent variables, initial total sugar concentration, initial pH, initial concentration of protein concentrate obtained from fine flour of semolina and temperature, on the growth and citric acid production of the yeast were investigated. An experimental design including 30 experiments was conducted by using the method of central composite design. Modelling the effects of these independent variables on maximum citric acid concentration, maximum citric acid production rate, citric acid yield, the ratio of maximum citric acid concentration to maximum isocitric acid concentration and specific growth rate were performed by response surface methodology. The variations of all of the responses with the independent variables were defined by a quadratic model. Numeric optimization was performed by using the desireability function. The conditions with 190.83 g/L initial sugar concentration, 5.90 initial pH, 0.07 g/L initial concentration of fine flour protein concentrate and 27.86 °C were determined as optimal conditions for citric acid production. The maximum citric acid concentration reached to 80.53 g/L in optimal conditions.     

Fermentation of kraft black liquor for the production of citric acid by Candida tropicalis

Summary The production of citric acid by batch fermentation with the yeast strain Candida tropicalis ATCC 20240 was chosen as a potential process for the valorization of kraft black liquor. The effect of nitrogen concentration was studied and direct bioconversion of acetate to citrate was achieved when no nitrogen was supplemented to the medium. The use of kraft black liquor's acetate as a potential substrate for citric acid production was investigated. The acid precipitated liquor was highly inhibitory when its concentration was above 25% of the fermentation broth content. The yields of citric acid at low concentrations of kraft black liquor (5% and 15%) were the same as those recorded in synthetic acetate medium. Other organic acids present in the liquor may affect the yields and rates of citric acid production over acetate. Substrate uptake rates and product formation rates were lower, however, in comparison to synthetic media. The utilization of immobilized biomass improved the process parameters on kraft black liquor and enhanced the fermentation capabilities.     

Acetic acid production from whey lactose by the co-culture ofStreptococcus lactis andClostridium formicoaceticum

Summary Acetic acid was produced from anaerobic fermentation of lactose by the co-culture ofStreptococcus lactis andClostridium formicoaceticum at 35° C and pHs between 7.0 and 7.6. Lactose was converted to lactic acid, and then to acetic acid in this mixed culture fermentation. The overall acetic acid yield from lactose was about 95% at pH 7.6 and 90% at pH 7.0. The fermentation rate was also higher at pH 7.6 than at pH 7.0. In batch fermentation of whey permeate containing about 5% lactose at pH 7.6, the concentration of acetic acid reached 20 g/l within 20 h. The production rate then became very slow due to end-product inhibition and high Na⁺ concentration. About 30 g/l acetate and 20 g/l lactate were obtained at a fermentation time of 80 h. However, when diluted whey permeate containing 2.5% lactose was used, all the whey lactose was converted to acetic acid within 30 h by this mixed culture.     

Simultaneous production of sugars and ethanol from inulin rich-extracts in a chemostat

Summary Incomplete fermentation of inulin-containing extracts by Saccharomyces diastaticus allows the simultaneous production of ethanol and syrups with increased fructose content. The yeast strain used ferments sucrose and inulin small polymers but does not easily ferment inulin large polymers. After batch fermentation a production of 62.5 g/L ethanol and 75 g/L of sugars containing up to 94 % fructose can be obtained. A continuous fermentation was performed in a chemostat permitting the adjustment of both productions according to the dilution rate with a maximal ethanol productivity of 3.9 g/L.h.     

Engineering of unconventional yeast Yarrowia lipolytica for efficient succinic acid production from glycerol at low pH

Yarrowia lipolytica is considered as a potential candidate for succinic acid production because of its innate ability to accumulate citric acid cycle intermediates and its tolerance to acidic pH. Previously, a succinate-production strain was obtained through the deletion of succinate dehydrogenase subunit encoding gene Ylsdh5. However, the accumulation of by-product acetate limited further improvement of succinate production. Meanwhile, additional pH adjustment procedure increased the downstream cost in industrial application. In this study, we identified for the first time that acetic acid overflow is caused by CoA-transfer reaction from acetyl-CoA to succinate in mitochondria rather than pyruvate decarboxylation reaction in SDH negative Y. lipolytica. The deletion of CoA-transferase gene Ylach eliminated acetic acid formation and improved succinic acid production and the cell growth. We then analyzed the effect of overexpressing the key enzymes of oxidative TCA, reductive carboxylation and glyoxylate bypass on succinic acid yield and by-products formation. The best strain with phosphoenolpyruvate carboxykinase (ScPCK) from Saccharomyces cerevisiae and endogenous succinyl-CoA synthase beta subunit (YlSCS2) overexpression improved succinic acid titer by 4.3-fold. In fed-batch fermentation, this strain produced 110.7 g/L succinic acid with a yield of 0.53 g/g glycerol without pH control. This is the highest succinic acid titer achieved at low pH by yeast reported worldwide, to date, using defined media. This study not only revealed the mechanism of acetic acid overflow in SDH negative Y. lipolytica, but it also reported the development of an efficient succinic acid production strain with great industrial prospects.     

Enhanced butyric acid tolerance and bioproduction by Clostridium tyrobutyricum immobilized in a fibrous bed bioreactor

Repeated fed‐batch fermentation of glucose by Clostridium tyrobutyricum immobilized in a fibrous bed bioreactor (FBB) was successfully employed to produce butyric acid at a high final concentration as well as to adapt a butyric‐acid‐tolerant strain. At the end of the eighth fed‐batch fermentation, the butyric acid concentration reached 86.9 ± 2.17 g/L, which to our knowledge is the highest butyric acid concentration ever produced in the traditional fermentation process. To understand the mechanism and factors contributing to the improved butyric acid production and enhanced acid tolerance, adapted strains were harvested from the FBB and characterized for their physiological properties, including specific growth rate, acid‐forming enzymes, intracellular pH, membrane‐bound ATPase and cell morphology. Compared with the original culture used to seed the bioreactor, the adapted culture showed significantly reduced inhibition effects of butyric acid on specific growth rate, cellular activities of butyric‐acid‐forming enzyme phosphotransbutyrylase (PTB) and ATPase, together with elevated intracellular pH, and elongated rod morphology. Biotechnol. Bioeng. 2011; 108:31–40. © 2010 Wiley Periodicals, Inc.     

Characteristics of the growth on rapeseed oil and synthesis of citric and isocitric acids by <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> yeasts

The native strain Yarrowia lipolytica VKMY-2373 grown in a complete medium exhibited the maximum lipase activity at the concentration of rapesseed oil of at least 5.0 g/l. In the course of yeast growth, no considerable changes were observed in the glycerol concentration, the proportions of the major free fatty acids formed via oil hydrolysis, or the fatty acid composition of oil. Under nitrogen limitation of cell growth, the accumulation of citric acids reached 77.1 g/l with predominance of isocitric acid at pH 6.0, whereas at pH 4.5, almost equal amounts of citric and isocitric acids were produced. Cultivation of the mutant strain Y. lipolytica N 1 at pH 4.5 resulted in the predominant accumulation of citric acid (66.6 g/l) with an insignificant amount of isocitric acid. In the period of intense acid synthesis, high production of lipase was observed.     

Ergosterol production from molasses by genetically modified <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Ergosterol is an economically important metabolite produced by fungi. Recombinant Saccharomyces cerevisiae YEH56(pHXA42) with increased capacity of ergosterol formation was constructed by combined overexpression of sterol C-24(28) reductase and sterol acyltransferase in the yeast strain YEH56. The production of ergosterol by this recombinant strain using cane molasses (CM) as an inexpensive carbon source was investigated. An ergosterol content of 52.6 mg/g was obtained with 6.1 g/l of biomass from CM medium containing 60 g/l of total sugar in 30 h in shake flask. The ergosterol yield was enhanced through the increasing cell biomass by supplementation of urea to a concentration of 6 g/l in molasses medium. Fermentation was performed in 5-l bioreactor using the optimized molasses medium. In batch fermentation, the effect of agitation velocity on ergosterol production was examined. The highest ergosterol yield was obtained at 400 rpm that increased 60.4 mg/l in comparison with the shake flask culture. In fed-batch fermentation, yeast cells were cultivated, firstly, in the starting medium containing molasses with 20 g/l of total sugar, 1.68 g/l of phosphate acid, and 6 g/l of urea (pH 5.4) for 5 h, then molasses containing 350 g/l of total sugar was fed exponentially into the bioreactor to keep the ethanol level in the broth below 0.5%. After 40 h of cultivation, the ergosterol yield reached 1,707 mg/l, which was 3.1-fold of that in the batch fermentation.