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.     

Citric acid production from sucrose by recombinant Yarrowia lipolytica using semicontinuous fermentation

The genetically modified yeast strain Yarrowia lipolytica H222‐S4(p67ICL1)T5 is able to utilize sucrose as a carbon source and to produce citric and isocitric acids in a more advantageous ratio as compared to its wild‐type equivalent. In this study, the effect of pH of the fermentation broth (pH 6.0 and 7.0) and proteose‐peptone addition on citric acid production by the recombinant yeast strain were investigated. It was found that the highest citric acid production occurred at pH 7.0 without any addition of proteose‐peptone. Furthermore, two process strategies (fed‐batch and repeated fed‐batch) were tested for their applicability for use in citric acid production from sucrose by Y. lipolytica. Repeated fed‐batch cultivation was found to be the most effective process strategy: in 3 days of cycle duration, approximately 80 g/L citric acid was produced, the yield was at least 0.57 g/g and the productivity was as much as 1.1 g/Lh. The selectivity of the bioprocess for citric acid was always higher than 90% from the very beginning of the fermentation due to the genetic modification, reaching values of up to 96.4% after 5 days of cycle duration.     

Oxygen requirements for growth and citric acid production of Yarrowia lipolytica

During continuous cultivation of Yarrowia lipolytica N 1, oxygen requirements for growth and citric acid synthesis were found to depend on the iron concentration in the medium. A coupled effect of oxygen and iron concentrations on the functioning of the mitochondrial electron transport chain in Y. lipolytica N 1 was established. Based on the results obtained in continuous culture, conditions for citric acid production in a batch culture of Y. lipolytica N 1 were proposed. At relatively low pO(2) value and a high iron concentration, citric acid accumulation was as high as 120 g l(-1); the specific rate of citric acid synthesis reached 120 mg citric acid (g cells h)(-1). The mass yield coefficient was 0.87 and the energy yield coefficient was 0.31.     

柠檬酸清液发酵的补料工艺研究进展

柠檬酸是一种重要的食品添加剂。微生物批次发酵是当前国内外柠檬酸企业的主流生产方式,而更高生产强度的补料发酵工艺开发逐渐成为行业领域的关注热点。本文分别对不同菌种发酵的补料工艺进行比较,从补料培养基、补料起始点、补料控制方式等角度介绍各自的补料工艺控制,为柠檬酸工业发酵的补料工艺提出可行性建议。     

产琥珀酸重组解脂酵母发酵副产物乙酸的代谢控制

琥珀酸是一种高附加值的有机酸,广泛用于食品、化工和农药领域。解脂酵母Yarrowia lipolytica作为新型强健的非传统酵母,近年来逐渐吸引了研究者的注意。前期通过基因敲除琥珀酸脱氢酶基因构建了一株产琥珀酸的重组解脂酵母PGC01003。由于糖酵解和TCA循环流量不协调,PGC01003分泌大量副产物乙酸,限制了琥珀酸产量的进一步提高。为降低乙酸的溢出,实现自然低pH值发酵生产琥珀酸,首先干扰旁路代谢,异源表达来自鼠沙门氏菌的乙酰辅酶A合酶,乙酸的产量下降至4.6 g/L,比对照降低了24.6%。而基因敲除乙酰辅酶A水解酶基因得到的重组菌PGC11505,发酵96 h乙酸分泌量只有0.4 g/L,琥珀酸产量提高到7.0 g/L,琥珀酸的转化率为0.30 g/g,为进一步构建高产琥珀酸的细胞工厂奠定基础。     

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 by Aspergillus niger using two downgraded Algerian date varieties

In the present work, the GHARS and the MECH DEGLA downgraded date varieties were used in a fermentation medium in order to produce citric acid by the Aspergillus niger. The biochemical characteristics of the dates were investigated, along with the chemical and physical characteristics of the solutions of both samples. The analyzed parameters included the moisture and sugar content, the ash residual, the pH values, and the electrical conductivity. The effect of the following fermentation parameters was studied: initial pH, temperature, incubation period, and methanol. For the GHARS and MECH DEGLA date varieties respectively, the ash residual measured at 1.90% and 2.47%. For each date variety, the moisture and total sugars were measured at 11.59% and 85%, for the GHARS, and 12.82% and 80.47% for the MECH DEGLA. Citric acid production using either of the two varieties of dates showed a high yield in a short time.The obtained results showed that the highest production of citric acid by both medium of dates was achieved at the initial pH value of 3.0, temperature 30 °C, and an incubation period of 8 days. Also, the maximum amount of citric acid was produced when both mediums contained 4% of methanol. Both varieties of dates showed a good yield for the citric acid and can be used as a culture medium since they are economic and ensure good growth for the Aspergillus niger.     

Preliminary genetic studies on a citric acid producing strain of Saccharomycopsis lipolytica

The genetics of a strain of Saccharomycopsis lipolytica which accumulates citric acid have been studied. The results show that a mating system exists in the organism although no diploid was isolated.     

Effect of some metabolic inhibitors on citric acid production by Aspergillus niger

Stationary cultures of Aspergillus niger grown on a synthetic medium have been used to study the effect of some metabolic inhibitors on citric acid production. Addition of 0.05 to 1 mM sodium malonate or 0.01 to 0.1 mM potassium ferricyanide, iodoacetate, sodium azide, sodium arsenate or sodium fluoride stimulated citric acid production (3.6 to 45%), but not total titratable acids. Addition of higher concentrations (0.2 to 10 mM) of later inhibitors caused a marked inhibition of fungal growth and citric acid production. The implications of these preliminary findings are discussed.     

Oxygen uptake and citric acid production by Candida lipolytica Y 1095

The rates of oxygen uptake and oxygen transfer during cell growth and citric acid production by Candida lipolytica Y 1095 were determined. The maximum cell growth rate, 1.43 g cell/L . h, and volumetric oxygen uptake rate, 343 mg O(2)/L . h, occurred approximately 21 to 22 h after inoculation. At the time of maximum oxygen uptake, the biomass concentration was 1.3% w/v and the specific oxygen uptake rate was slightly greater than 26 mg O(2)/g cell . h. The specific oxygen uptake rate decreased to approximately 3 mg O(2)/g cell . h by the end of the growth phase.During citric acid production, as the concentration of dissolved oxygen was increased from 20% to 80% saturation, the specific oxygen uptake and specific citric acid productivity (mg citric acid/g cell . h) increased by 160% and 71%, respectively, at a biomass concentration of 3% w/v. At a biomass concentration of 5% w/v, the specific oxygen uptake and specific citric acid productivity increased by 230% and 82%, respectively, over the same range of dissolved oxygen concentrations.The effect of dissolved oxygen on citric acid yields and productivities was also determined. Citric acid yields appeared to be independent of dissolved oxygen concentration during the initial production phase; however, volumetric productivity (g citric acid/L . h) increased sharply with an increase in dissolved oxygen. During the second or subsequent production phase, citric acid yields increased by approximately 50%, but productivities decreased by roughly the same percentage due to a loss of cell viability under prolonged nitrogen-deficient conditions. (c) 1994 John Wiley & Sons, Inc.     

Investigation of vinegar production using a novel shaken repeated batch culture system

Nowadays, bioprocesses are developed or optimized on small scale. Also, vinegar industry is motivated to reinvestigate the established repeated batch fermentation process. As yet, there is no small‐scale culture system for optimizing fermentation conditions for repeated batch bioprocesses. Thus, the aim of this study is to propose a new shaken culture system for parallel repeated batch vinegar fermentation. A new operation mode — the flushing repeated batch — was developed. Parallel repeated batch vinegar production could be established in shaken overflow vessels in a completely automated operation with only one pump per vessel. This flushing repeated batch was first theoretically investigated and then empirically tested. The ethanol concentration was online monitored during repeated batch fermentation by semiconductor gas sensors. It was shown that the switch from one ethanol substrate quality to different ethanol substrate qualities resulted in prolonged lag phases and durations of the first batches. In the subsequent batches the length of the fermentations decreased considerably. This decrease in the respective lag phases indicates an adaptation of the acetic acid bacteria mixed culture to the specific ethanol substrate quality. Consequently, flushing repeated batch fermentations on small scale are valuable for screening fermentation conditions and, thereby, improving industrial‐scale bioprocesses such as vinegar production in terms of process robustness, stability, and productivity. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1158–1168, 2013     

Early‐stage sustainability assessment of biotechnological processes: A case study of citric acid production

Sustainability assessment using a life‐cycle approach is indispensable to contemporary bioprocess development. This assessment is particularly important for early‐stage bioprocess development. As early‐stage investigations of bioprocesses involve the evaluation of their ecological and socioeconomic effects, they can be adjusted more effectively and improved towards sustainability, thereby reducing environmental risk and production costs. Early‐stage sustainability assessment is an important precautionary practice and, despite limited data, a unique opportunity to determine the primary impacts of bioprocess development. To this end, a simple and robust method was applied based on the standardized life‐cycle sustainability assessment methodology and commercially available datasets. In our study, we elaborated on the yeast‐based citric acid production process with Yarrowia lipolytica assessing 11 different substrates in different process modes. The focus of our analysis comprised both cultivation and down‐stream processing. According to our results, the repeated batch raw glycerol based bioprocess alternative showed the best environmental performance. The second‐ and third‐best options were also glycerol‐based. The least sustainable processes were those using molasses, chemically produced ethanol, and soy bean oil. The aggregated results of environmental, economic, and social impacts display waste frying oil as the best‐ranked alternative. The bioprocess with sunflower oil in the batch mode ranked second. The least favorable alternatives were the chemically produced ethanol‐, soy oil‐, refined glycerol‐, and molasses‐based citric acid production processes. The scenario analysis demonstrated that the environmental impact of nutrients and wastewater treatment is negligible, but energy demand of cultivation and down‐stream processing dominated the production process. However, without energy demand the omission of neutralizers almost halves the total impact, and neglecting pasteurization also considerably decreases the environmental impact.     

Batch and repeated batch production of l(+)-lactic acid by Enterococcus faecalis RKY1 using wood hydrolyzate and corn steep liquor

Lactic acid production was investigated for batch and repeated batch cultures of Enterococcus faecalis RKY1, using wood hydrolyzate and corn steep liquor. When wood hydrolyzate (equivalent to 50 g l⁻¹ glucose) supplemented with 15–60 g l⁻¹ corn steep liquor was used as a raw material for fermentation, up to 48.6 g l⁻¹ of lactic acid was produced with, volumetric productivities ranging between 0.8 and 1.4 g l⁻¹ h⁻¹. When a medium containing wood hydrolyzate and 15 g l⁻¹ corn steep liquor was supplemented with 1.5 g l⁻¹ yeast extract, we observed 1.9-fold and 1.6-fold increases in lactic acid productivity and cell growth, respectively. In this case, the nitrogen source cost for producing 1 kg lactic acid can be reduced to 23% of that for fermentation from wood hydrolyzate using 15 g l⁻¹ yeast extract as a single nitrogen source. In addition, lactic acid productivity could be maximized by conducting a cell-recycle repeated batch culture of E. faecalis RKY1. The maximum productivity for this process was determined to be 4.0 g l⁻¹ h⁻¹.     

Utilisation of fruits waste for citric acid production by solid state fermentation

A solid state fermentation method was used to utilise pineapple, mixed fruit and maosmi waste as substrates for citric acid production using Aspergillus niger DS 1. Experiments were carried out in the presence and absence of methanol at different moisture levels. In the absence of methanol the maximum citric acid was obtained at 60% moisture level whereas in the presence of methanol the maximum citric acid was obtained at 70% moisture level. The stimulating effect of methanol was less at lower moisture level. The inhibitory effect of metal ions was also not observed and maximum citric acid yield of 51.4, 46.5 and 50% (based on sugar consumed) was obtained from pineapple, mixed fruit and maosmi residues, respectively.     

Biotechnological conversions of bio‐diesel‐derived crude glycerol by Yarrowia lipolytica strains

In the present report, crude glycerol, waste discharged from bio‐diesel production, was used as carbon substrate for three natural Yarrowia lipolytica strains (LFMB 19, LFMB 20 and ACA‐YC 5033) during growth in nitrogen‐limited submerged shake‐flask experiments. In media with initial glycerol concentration of 30 g/L, all strains presented satisfactory microbial growth and complete glycerol uptake. Although culture conditions favored the secretion of citric acid (and potentially the accumulation of storage lipid), for the strains LFMB 19 and LFMB 20, polyol mannitol was the principal metabolic product synthesized (maximum quantity 6.0 g/L, yield 0.20–0.26 g per g of glycerol consumed). The above strains produced small quantities of lipids and citric acid. In contrast, Y. lipolytica ACA‐YC 5033 produced simultaneously higher quantities of lipid and citric acid and was further grown on crude glycerol in nitrogen‐limited experiments, with constant nitrogen and increasing glycerol concentrations (70–120 g/L). Citric acid and lipid concentrations increased with increment of glycerol; maximum total citric acid 50.1 g/L was produced (yield 0.44 g per g of glycerol) while simultaneously 2.0 g/L of fat were accumulated inside the cells (0.31 g of lipid per g of dry weight). Cellular lipids were mainly composed of neutral fraction, the concentration of which substantially increased with time. Moreover, in any case, the phospholipid fraction was more unsaturated compared with total and neutral lipids, while at the early growth step, microbial lipid was more rich in saturated fatty acids (e.g. C16:0 and C18:0) compared with the stationary phase.     

Succinic acid production from n‐alkanes

A novel process of production of succinic acid (SA) has been developed, which includes the synthesis of alpha‐ketoglutaric acid by a thiamine‐auxotrophic yeast strain Yarrowia lipolytica VKM Y‐2412 from n‐alkanes and subsequent oxidation of the acid by hydrogen peroxide to SA. The concentration of SA in the culture broth and its yield were found to be 38.8 g/L and 82.45% of n‐alkane consumed, respectively. The isolation procedure involved the extraction of the residual alkanes with the mixture of ethyl acetate and hexane, the decomposition of H₂O₂ in the filtrate followed by filtrate bleaching and acidification with a mineral acid; the evaporation of filtrate and the ethanol extraction of SA from lyophilized residue. The purity of the SA isolated from the culture liquid filtrate reached 99.5%.     

A novel recycling process using the treated citric acid wastewater as ingredients water for citric acid production

In this study, an integrated process coupling citric acid and methane fermentations was proposed to solve severe wastewater pollution problem in cassava-based citric acid production. The accumulation patterns of the potential and major inhibitors in this process, including organic compounds, volatile fatty acids (VFAs), total ions and pigments were investigated. Both simulation and experimental results indicated that these inhibitors could reach their equilibrium levels after 3–7 fermentation runs when reutilizing the treated citric acid wastewater. As a result, the proposed citric acid fermentation process by recycling the wastewater treated in methane fermentation could be stably operated for more than 15 runs, which could save a large amount of fresh water and relieve the severe wastewater pollution in citric acid production potentially.     

Efficient docosahexaenoic acid production by Schizochytrium sp. via a two-phase pH control strategy using ammonia and citric acid as pH regulators

The cell growth and docosahexaenoic acid (DHA) synthesis of Schizochytrium sp. are closely related to the culture pH. A two-phase pH control strategy based on nitrogen consumption was developed in which pH 7.0 was used for biomass accumulation and pH 5.0 for DHA synthesis. Using this strategy, the cell dry weight and DHA content reached 98.07 and 25.85 g/L, respectively. Furthermore, ammonia and citric acid were used as pH regulators. Application of citric acid further resulted in 7.88 and 4.87% improvements of total lipids and the ratio of DHA to total fatty acids, respectively. Ammonia, as a suitable nitrogen source, promoted non-lipid biomass accumulation. Using this method, a maximum DHA yield of 32.75 g/L was obtained with non-lipid biomass (58.01 g/L) and the ratio of DHA to total fatty acids (52.36%). This study provides an easy strategy for large-scale industrial production of DHA via high-cell-density fermentation of Schizochytrium sp.