Production ofcis,cis-muconic acid from benzoic acidvia microbial transformation

For the production ofcis,cis-muconic acidvia biocatalytic conversion reactions from a toxic cosubstrate, benzoic acid, a fed-batch process using computer-controlled DO-stat feeding was developed. The mutant strain ofPseudomonas putida BM014 producedcis,cis-muconic acid from benzoic acid with high conversion yield. More than 32 g/L ofcis,cis-muconic acid was accumulated in 42h and a productivity of 1.4 g/(L·h) was achieved.     

Production of succinic acid and lactic acid by Corynebacterium crenatum under anaerobic conditions

A new succinic acid and lactic acid production bioprocess by Corynebacterium crenatum was investigated in mineral medium under anaerobic conditions. Corynebacterium crenatum cells with sustained acid production ability and high acid volumetric productivity harvested from the glutamic acid fermentation broth were used to produce succinic acid and lactic acid. Compared with the first cycle, succinic acid production in the third cycle increased 120% and reached 43.4 g/L in 10 h during cell-recycling repeated fermentations. The volumetric productivities of succinic acid and lactic acid could maintain above 4.2 g/(L·h) and 3.1 g/(L·h), respectively, for at least 100 h. Moreover, wheat bran hydrolysates could be used for succinic acid and lactic acid production by the recycled C. crenatum cells. The final succinic acid concentration reached 43.6 g/L with a volumetric productivity of 4.36 g/(L·h); at the same time, 32 g/L lactic acid was produced.     

Process development of itaconic acid production by a natural wild type strain of <Emphasis Type="Italic">Aspergillus terreus</Emphasis> to reach industrially relevant final titers

Itaconic acid is a promising organic acid and is commercially produced by submerged fermentation of Aspergillus terreus. The cultivation process of the sensitive filamentous fungus has been studied intensively since 1932, with respect to fermentation media components, oxygen supply, shearing rate, pH value, or culture method. Whereas increased final titers were achieved over the years, the productivity has so far remained quite low. In this study, the impact of the pH on the itaconic acid production was investigated in detail. The pH during the growth and production phase had a significant influence on the final itaconic acid concentration and pellet diameter. The highest itaconic acid concentration of 160 g/L was achieved at a 1.5-L scale within 6.7 days by raising and controlling the pH value to pH 3.4 in the production phase. An ammonia solution and an increased phosphate concentration were used with an itaconic acid yield of 0.46 (w/w) and an overall productivity of 0.99 g/L/h in a fed-batch mode. A cultivation with a lower phosphate concentration resulted in an equal final concentration with an increased yield of 0.58 (w/w) after 11.8 days and an overall productivity of 0.57 g/L/h. This optimized process was successfully transferred from a 1.5-L scale to a 15-L scale. After 9.7 days, comparable pellet morphology and a final concentration of 150 g/L itaconic acid was reached. This paper provides a process strategy to yield a final titer of itaconic acid from a wild-type strain of A. terreus which is in the same range as the well-known citric acid production.     

Production of mannitol by Lactobacillus intermedius NRRL B-3693 in fed-batch and continuous cell-recycle fermentations

Improved fermentation processes were developed for the production of mannitol by a heterofermentative lactic acid bacterium (Lactobacillus intermedius NRRL B-3693). A fed-batch fermentation protocol overcame limitations caused by high substrate concentrations. The process was developed using corn steep liquor and glucose as inexpensive industrial nutrient sources, supplemented with a small amount of soy peptone and manganese. The fed-batch process resulted in a concentration of 176 ± 0.5 g mannitol from 184 ± 0 g fructose and 92 ± 0.1 g glucose per L of final fermentation broth in 30 h with a volumetric productivity of 5.9 g/(L h). Further increases in volumetric productivity of mannitol were obtained in a continuous cell-recycle fermentation process that reached more than 40 g/(L h), despite reduced mannitol levels of 78–98 g/L and residual substrate of 10–20 g/L. This is the first report of such a high volumetric productivity of mannitol by a heterofermentative lactic acid bacterium.     

Continuous conversion of rice starch hydrolysate to 2-keto-D-gluconic acid by Arthrobacter globiformis C224

A continuous conversion process of rice starch hydrolysate to 2-keto-D-gluconic acid (2KGA) by Arthrobacter globiformis C224 was developed. Its feasibility for industrial application was also evaluated. Results showed that the initial cell concentration exceeding 1.25 g/L met the continuous 2KGA production at a stable dilution rate and media composition, while the dilution rate and feeding glucose concentration had a significant effect on 2KGA production performance. The optimal operating parameters were obtained as: 0.090/h of dilution rate and 171.0 g/L of feeding glucose concentration. Under these conditions, the steady state had a produced 2KGA concentration of 124.74 g/L, average volumetric productivity of 11.23 g/L/h, and yield of 0.97 g/g. In conclusion, continuous 2KGA production by the A. globiformis C224 strain would be a superior industrial process for the production of 2KGA in terms of its high 2KGA productivity and yield.     

Optimization of Repeated-Batch Fermentation of a Recombinant Strain of the Yeast <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> for Succinic Acid Production at Low pH

The capacity of a recombinant Yarrowia lipolytica yeast strain VKPM Y3753 for succinic acid biosynthesis in a laboratory bioreactor at low pH has been studied. The batch and repeated-batch modes of fermentation of the strain were compared. The optimal conditions for repeated-batch fermentation were selected; they resulted in the accumulation of 55.3 g/L of succinic acid and the maximal productivity for this compound, 2.6 g/(L h), while lowering the pH of the broth culture to 3.65 at the end of the biosynthesis process.     

Itaconic Acid Production by Filamentous Fungi in Starch-Rich Industrial Residues

Several fungi and starch-rich industrial residues were screened for itaconic acid (IA) production. Out of 15 strains, only three fungal strains were found to produce IA, which was confirmed by HPLC and GC–MS analysis. These strains were identified as Aspergillus terreus strains C1 and C2, and Ustilago maydis strain C3 by sequencing of 18S rRNA gene and internal transcribed spacer regions. Cis-aconitate decarboxylase (cad) gene, which encodes a key enzyme in IA production in A. terreus, was characterized from strains C1 and C2. C1 and C2 cad gene sequences showed about 96% similarity to the only available GenBank sequence of A. terreus cad gene. 3-D structure and cis-aconitic acid binding pocket of Cad enzyme were predicted by structural modeling. Rice, corn and potato starch wastes were screened for IA production. These materials were enzymatically hydrolyzed under experimentally optimized conditions resulting in the highest glucose production of 230 mg/mL from 20% potato waste. On comparing the production potential of selected strains with different wastes, the best IA production was achieved with strain C1 (255.7 mg/L) using potato waste. Elemental composition as well as batch-to-batch variation in waste substrates were analyzed. The difference in IA production from two different batches of potato waste was found to inversely correlate with their phosphorus content, which indicated that A. terreus produced IA under phosphate limiting condition. The potato waste hydrolysate was deionized to remove inhibitory ions like phosphate, resulting in improved IA production of 4.1 g/L by C1 strain, which is commercially competitive.     

循环利用重组大肠杆菌细胞转化合成丁二酸

研究了回收丁二酸发酵液中的大肠杆菌进行细胞转化的可行性,以转化率和生产效率为指标,考察了不同菌体浓度、底物浓度、pH调节剂对细胞转化的影响。发酵结果表明大肠杆菌可以在仅含有葡萄糖和pH调节剂的水环境中转化生产丁二酸,并确定了最佳的转化条件为:细胞浓度(OD600)50,底物浓度40g/L,缓冲盐为MgCO3。基于优化好的条件,在7L发酵罐中进行重复批次转化,第1次转化的转化率和生产效率分别达到91%和3.22g/(L·h),第2次转化的生产效率和转化率达到了86%和2.04g/(L·h),第3次转化的转化率和生产效率分别达到了83%和1.82g/(L·h)。     

Emerging biotechnologies for production of itaconic acid and its applications as a platform chemical

Recently, itaconic acid (IA), an unsaturated C5-dicarboxylic acid, has attracted much attention as a biobased building block chemical. It is produced industrially (>80 g L^?1) from glucose by fermentation with Aspergillus terreus. The titer is low compared with citric acid production (>200 g L^?1). This review summarizes the latest progress on enhancing the yield and productivity of IA production. IA biosynthesis involves the decarboxylation of the TCA cycle intermediate cis-aconitate through the action of cis-aconitate decarboxylase (CAD) enzyme encoded by the CadA gene in A. terreus. A number of recombinant microorganisms have been developed in an effort to overproduce it. IA is used as a monomer for production of superabsorbent polymer, resins, plastics, paints, and synthetic fibers. Its applications as a platform chemical are highlighted. It has a strong potential to replace petroleum-based methylacrylic acid in industry which will create a huge market for IA.     

Process development of succinic acid production by Escherichia coli NZN111 using acetate as an aerobic carbon source

Escherichia coli strain NZN111 could convert glucose to succinic acid efficiently in anaerobic conditions after the induction of gluconeogenic carbon sources in aerobic conditions. Acetate shows a strong effect on both yield and productivity of succinic acid. In this study, the fed-batch process of succinic acid production by NZN111 using acetate in a chemically defined medium in the aerobic stage was investigated and developed. Increasing cell density could increase succinic acid with a productivity of 3.97 g/(L h) in the first 8 h of the anaerobic phase with an overall yield of 1.42 mol/mol glucose in a 5 L fermentor. However, there was strong repression from succinic acid in the later anaerobic stage. When succinic acid exceeded 30 g/L, the glucose consumption rate began to drop sharply along with the succinic acid production rate. Supplementation with glucose from 30 to 70 g/L in the anaerobic stage showed little effect on succinic acid production. Acetic acid and pyruvic acid accumulated had no effect on succinic acid formation because of their low concentration. With acetate as the sole carbon source for aerobic cultivation in the following scale-up, 60.09 g/L of succinic acid was produced with a yield of 1.37 mol/mol in a 50 L bioreactor.     

High-level production of 1,3-propanediol from crude glycerol by <Emphasis Type="Italic">Clostridium butyricum</Emphasis> AKR102a

The aim of this study was to optimize a biotechnological process for the production of 1,3-propanediol (1,3-PD) based on low-quality crude glycerol derived from biodiesel production. Clostridium butyricum AKR102a was used in fed-batch fermentations in 1-L and 200-L scale. The newly discovered strain is characterized by rapid growth, high product tolerance, and the ability to use crude glycerol at the lowest purity directly gained from a biodiesel plant side stream. Using pure glycerol, the strain AKR102 reached 93.7 g/L 1,3-PD with an overall productivity of 3.3 g/(L*h). With crude glycerol under the same conditions, 76.2 g/L 1,3-PD was produced with a productivity of 2.3 g/(L*h). These are among the best results published so far for natural producers. The scale up to 200 L was possible. Due to the simpler process design, only 61.5 g/L 1,3-PD could be reached with a productivity of 2.1 g/(L*h).     

Improved n-butanol production by a non-acetone producing Clostridium pasteurianum DSMZ 525 in mixed substrate fermentation

The kinetics of growth, acid and solvent production in batch culture of Clostridium pasteurianum DSMZ 525 were examined in mixed or mono-substrate fermentations. In pH-uncontrolled batch cultures, the addition of butyric acid or glucose significantly enhanced n-butanol production and the ratio of butanol/1,3-propanediol. In pH-controlled batch culture at pH?=?6, butyric acid addition had a negative effect on growth and did not lead to a higher n-butanol productivity. On the other hand, mixed substrate fermentation using glucose and glycerol enhanced the growth and acid production significantly. Glucose limitation in the mixed substrate fermentation led to the reduction or inhibition of the glycerol consumption by the growing bacteria. Therefore, for the optimal growth and n-butanol production by C. pasteurianum, a limitation of either substrate should be avoided. Under optimized batch conditions, n-butanol concentration and maximum productivity achieved were 21 g/L, and 0.96 g/L?×?h, respectively. In comparison, mixed substrate fermentation using biomass hydrolysate and glycerol gave a n-butanol concentration of 17 g/L with a maximum productivity of 1.1 g/L?×?h. In terms of productivity and final n-butanol concentration, the results demonstrated that C. pasteurianum DSMZ 525 is well suitable for n-butanol production from mixed substrates of biomass hydrolysate and glycerol and represents an alternative promising production strain.     

进化代谢选育高渗透压耐受型产琥珀酸大肠杆菌

在以碳酸钠为酸中和剂的大肠杆菌两阶段发酵产琥珀酸的过程中,由于Na+的积累造成发酵体系中渗透压的提高,严重抑制了琥珀酸的产物浓度。为了增强大肠杆菌对渗透压的耐受性,考察了利用进化代谢方法筛选高渗透压耐受型高产琥珀酸大肠杆菌菌株的可行性。进化代谢系统作为一种菌株突变装置,可以使菌体在连续培养条件下以最大的生长速率生长。以NaCl为渗透压调节剂,通过在连续培养装置中逐步提高NaCl浓度使菌体在高渗透压条件下快速生长,最终得到了一株高渗透压耐受型琥珀酸生产菌株Escherichia coli XB4。以碳酸钠为酸中和剂,在7 L发酵罐中利用Escherichia coli XB4进行两阶段发酵,厌氧培养60 h后,琥珀酸产量达到了69.5 g/L,琥珀酸生产速率达到了1.81 g/(L.h),分别比出发菌株提高了18.6%和20%。     

Improvement of l-lactic acid production by osmotic-tolerant mutant of Lactobacillus casei at high temperature

l-Lactic acid production by Lactobacillus casei was used as a model to study the mechanism of substrate inhibition and the strategy for enhancing l-lactic acid production. It was found that the concentration of cell growth and l-lactate decreased with the increase of glucose concentration and fermentation temperature. To enhance the osmotic stress resistance of the strain at high temperature, a mutant G-03 was screened and selected with 360?g/L glucose at 45°C as the selective criterion. To further increase the cell growth for lactic acid production, 3?g/L of biotin was supplemented to the medium. As a result, l-lactate concentration by the mutant G-03 reached 198.2?g/L (productivity of 5.5?g?L^?1?h^?1) at 41°C in a 7-L fermentor with 210?g/L glucose as carbon source. l-Lactate concentration and productivity of mutant G-03 were 115.2% and 97.8% higher than those of the parent strain, respectively. The strategy for enhancing l-lactic acid production by increasing osmotic stress resistance at high temperature may provide an alternative approach to enhance organic acid production with other strains.     

Model-based metabolic engineering enables high yield itaconic acid production by Escherichia coli

Itaconic acid is a high potential platform chemical which is currently industrially produced by Aspergillus terreus. Heterologous production of itaconic acid with Escherichia coli could help to overcome limitations of A. terreus regarding slow growth and high sensitivity to oxygen supply. However, the performance achieved so far with E. coli strains is still low.We introduced a plasmid (pCadCS) carrying genes for itaconic acid production into E. coli and applied a model-based approach to construct a high yield production strain. Based on the concept of minimal cut sets, we identified intervention strategies that guarantee high itaconic acid yield while still allowing growth. One cut set was selected and the corresponding genes were iteratively knocked-out. As a conceptual novelty, we pursued an adaptive approach allowing changes in the model and initially calculated intervention strategy if a genetic modification induces changes in byproduct formation. Using this approach, we iteratively implemented five interventions leading to high yield itaconic acid production in minimal medium with glucose as substrate supplemented with small amounts of glutamic acid. The derived E. coli strain (ita23: MG1655 ∆aceA ∆sucCD ∆pykA ∆pykF ∆pta ∆Picd::cam_BBa_J23115 pCadCS) synthesized 2.27 g/l itaconic acid with an excellent yield of 0.77 mol/(mol glucose). In a fed-batch cultivation, this strain produced 32 g/l itaconic acid with an overall yield of 0.68 mol/(mol glucose) and a peak productivity of 0.45 g/l/h. These values are by far the highest that have ever been achieved for heterologous itaconic acid production and indicate that realistic applications come into reach.     

Efficient production of butyric acid from Jerusalem artichoke by immobilized Clostridium tyrobutyricum in a fibrous-bed bioreactor

Butyric acid is an important specialty chemical with wide industrial applications. The feasible large-scale fermentation for the economical production of butyric acid requires low-cost substrate and efficient process. In the present study, butyric acid production by immobilized Clostridium tyrobutyricum was successfully performed in a fibrous-bed bioreactor using Jerusalem artichoke as the substrate. Repeated-batch fermentation was carried out to produce butyric acid with a high butyrate yield (0.44 g/g), high productivity (2.75 g/L/h) and a butyrate concentration of 27.5 g/L. Furthermore, fed-batch fermentation using sulfuric acid pretreated Jerusalem artichoke hydrolysate resulted in a high butyric acid concentration of 60.4 g/L, with the yield of 0.38 g/g and the selectivity of ∼85.1 (85.1 g butyric acid/g acetic acid). Thus, the production of butyric acid from Jerusalem artichoke on a commercial scale could be achieved based on the system developed in this work.     

Conversion of acid hydrolysate of oil palm empty fruit bunch to L-lactic acid by newly isolated Bacillus coagulans JI12

Cost-effective conversion of lignocellulose hydrolysate to optically pure lactic acid is commercially attractive but very challenging. Bacillus coagulans JI12 was isolated from natural environment and used to produce L-lactic acid (optical purity?>?99.5 %) from lignocellulose sugars and acid hydrolysate of oil palm empty fruit bunch (EFB) at 50 °C and pH 6.0 without sterilization of the medium. In fed-batch fermentation with 85 g/L initial xylose and 55 g/L xylose added after 7.5 h, 137.5 g/L lactic acid was produced with a yield of 98 % and a productivity of 4.4 g/L?h. In batch fermentation of a sugar mixture containing 8.5 % xylose, 1 % glucose, and 1 % L-arabinose, the lactic acid yield and productivity reached 98 % and 4.8 g/L?h, respectively. When EFB hydrolysate was used, 59.2 g/L of lactic acid was produced within 9.5 h at a yield of 97 % and a productivity of 6.2 g/L?h, which are the highest among those ever reported from lignocellulose hydrolysates. These results indicate that B. coagulans JI12 is a promising strain for industrial production of L-lactic acid from lignocellulose hydrolysate.     

Optimized production of 2,3-butanediol by a lactate dehydrogenase-deficient mutant of Klebsiella pneumoniae

To obtain high-yield production of 2,3-butanediol (2,3-BD) from glucose, we optimized the culture conditions for a lactate dehydrogenase-deficient mutant (ΔldhA) of Klebsiella pneumoniae using response surface methodology. 2,3-BD production was successfully improved by optimizing pH (5.6), aeration (3.50 vvm) and concentration of corn steep liquor (45.0 mL/L) as a nitrogen source, resulting in a maximum level of 2,3-BD production of 148.8 g/L and productivity of 2.48 g/L/h. 2,3-BD was also obtained with high concentration (76.24 g/L) and productivity (2.31 g/L/h) from the K. pneumoniae mutant strain using sugarcane molasses as a carbon source.     

Xylanase and itaconic acid production by Aspergillus terreus NRRL 1960 within a biorefinery concept

Production of two industrially important products, xylanase and itaconic acid (IA), by Aspergillus terreus NRRL 1960 from agricultural residues was investigated within a biorefinery concept. Biological pretreatment was applied to lignocellulosic materials by using A. terreus, which produced xylanase while growing on agricultural residues. For IA production, already grown cells were transferred into a new medium. The first step provided not only the pretreatment of lignocellulosic material in order to be used as feedstock but also production of xylanase. For this purpose, cotton stalk, sunflower stalk and corn cob were used as carbon sources as lignocellulosic material. Among them, the highest xylanase production was obtained on corn cob. By application of two-step fermentation, about 70 IU/mL xylanase and 18 g/L IA production levels were achieved. This study shows the stepwise usage potential of the microorganism as a tool in a biorefinery concept.