Utilization of by-products derived from bioethanol production process for cost-effective production of lactic acid

The by-products of bioethanol production such as thin stillage (TS) and condensed distillers solubles (CDS) were used as a potential nitrogen source for economical production of lactic acid. The effect of those by-products and their concentrations on lactic acid fermentation were investigated using Lactobacillus paracasei CHB2121. Approximately, 6.7 g/L of yeast extract at a carbon source to nitrogen source ratio of 15 was required to produce 90 g/L of lactic acid in the medium containing 100 g/L of glucose. Batch fermentation of TS medium resulted in 90 g/L of lactic acid after 48 h, and the medium containing 10 % CDS resulted in 95 g/L of lactic acid after 44 h. Therefore, TS and CDS could be considered as potential alternative fermentation medium for the economical production of lactic acid. Furthermore, lactic acid fermentation was performed using only cassava and CDS for commercial production of lactic acid. The volumetric productivity of lactic acid [2.94 g/(L·h)] was 37 % higher than the productivity obtained from the medium with glucose and CDS.     

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)。     

大肠杆菌AFP111菌体回收连续转化生产丁二酸

在利用大肠杆菌AFP111厌氧发酵生产丁二酸过程中,随着产物丁二酸的不断积累,菌体活力和产酸能力逐渐降低,而通过回收菌体在新鲜培养基中重复发酵,可延长厌氧发酵时间,但是丁二酸生产效率较低。为了提高菌体回收丁二酸的转化效率,通过在回收菌体时有氧诱导 3 h,以纯水为培养基,进行丁二酸转化发酵。在连续进行 3 批次的发酵后,丁二酸的总产量和最终收率分别为 56.50 g/L和90%,生产速率达到了 0.81 g/(L·h),比未诱导情况下的生产速率提高了13%。     

Effective conversion of industrial starch waste to L-Lactic acid by Lactococcus lactis in a dialysis sac bioreactor

We describe here a simple technological process based on the direct fermentation of potato starch waste (PSW), an inexpensive agro-processing industrial waste, by a potential probiotic strain, Lactococcus lactis subsp. lactis, for enhancing L-lactic acid production. To maximize bioconversion and increase cell stability, we designed and tested a novel dialysis sac-based bioreactor. Shake flask fermentation (SFF) and fed batch fermentation in the dialysis sac bioreactor were compared for L-lactic acid production efficiency. The results showed that the starch (20 g/L) in the PSW-containing medium was completely consumed within 24 h in the dialysis sac bioreactor, compared with 48 h in the SFF. The maximum lactic acid concentration (18.9 g/L) and lactic acid productivity (0.79 g/L·h) obtained was 1.2- and 2.4-fold higher in the bioreactor than by SFF, respectively. Simultaneous saccharification and fermentation was effected at pH 5.5 and 30 °C. L. lactis cells were viable for up to four cycles in the fed batch fermentation compared to only one cycle in the SFF.     

Optimization of nutritional supplements for enhanced lactic acid production utilizing sugar refinery by-products

The present study investigated the synergistic effect of nutritional supplements (amino acid and Tween 80) on lactic acid production by Lactobacillus delbruckii utilizing a sugar refinery by product (cane molasses) in a submerged fermentation process. Initially, the effect of individual factors on lactic acid yield was studied by supplementing amino acids and their combinations, Tween 80 and cane molasses at varying concentrations in production medium. A combination of l-phenylalanine and l-lysine gave a maximum lactic acid yield of 47.89?±?0.1 g/L on a dry cell weight basis at individual factor level. Similarly, maximum lactic acid yield was obtained by supplementing the production medium with 40.0 g/L and 2.0 g/L Tween 80 and cane molasses, respectively, at individual factor level. In order to further improve the lactic acid yield, nutritional supplements were optimized by central composite rotatable design (CCRD) using Minitab 15 software. Shake flask cultivation under optimized conditions, i.e., cane molasses (32.40 g/L), Tween 80 (2.0 g/L) and l-phenylalanine and l-lysine (34.0 mg/L) gave a lactic acid yield of 64.86?±?0.2 g/L, corresponding to 95.0 % of the predicted yield of 67.78?±?0.3 g/L. Batch cultivation performed in 7.5 L bioreactor (working volume: 3.0 L) under optimized conditions gave maximum lactic acid yield and productivity of 79.12?±?0.2 g/L and 3.40 g/L·h, which is higher than previous studies with reduced fermentation time. Screening of lactic acid producing bacteria and characterization of lactic acid was also done.     

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.     

木薯粉同步糖化发酵(SSF)产丁二酸

【目的】通过优化产琥珀酸放线杆菌GXAS137同步糖化发酵木薯粉产丁二酸的发酵培养基,提高丁二酸产量,降低生产成本。【方法】在单因素试验的基础上,先利用Plackett-Burman试验设计筛选出影响丁二酸发酵的重要参数,再采用正交试验确定重要参数的最佳水平。【结果】价格低廉玉米浆可用作氮源,影响丁二酸产量的重要参数是木薯粉、玉米浆、碱式碳酸镁和糖化酶浓度。最佳条件为(g/L):木薯粉100,玉米浆14,糖化酶2.0 AGU/g底物,碱式碳酸镁75。优化后丁二酸产量达到69.31 g/L,丁二酸得率为90.01%,生产强度为1.44 g/(L·h)。与初始条件(52.34 g/L)相比,丁二酸浓度提高了32.42%。并利用1.3 L发酵罐对SSF与SHF两种发酵工艺进行了比较,SSF丁二酸产量(72.21 g/L)远高于SHF(56.86 g/L)。【结论】产琥珀酸放线杆菌同步糖化发酵木薯粉丁二酸产量高,生产成本低,具有较好的工业化应用前景。     

过量表达苹果酸脱氢酶对大肠杆菌NZN111产丁二酸的影响

大肠杆菌NZN111是敲除了乳酸脱氢酶的编码基因 (ldhA) 和丙酮酸-甲酸裂解酶的编码基因 (pflB) 的工程菌,厌氧条件下由于辅酶NAD(H) 的不平衡导致其丧失了代谢葡萄糖的能力。构建了苹果酸脱氢酶的重组菌大肠杆菌NZN111/pTrc99a-mdh,在厌氧摇瓶发酵过程中通过0.3 mmol/L的IPTG诱导后重组菌的苹果酸脱氢酶 (Malate dehydrogenase,MDH) 酶活较出发菌株提高了14.8倍,NADH/NAD+的比例从0.64下降到0.26,同时NAD+和NADH浓度分别     

发酵产丁二酸过程中废弃细胞的循环利用

对厌氧发酵产丁二酸后的废弃细胞进行破壁处理,考察了以细胞水解液作为有机氮源重新用于丁二酸发酵的可行性。比较了超声破碎、盐溶、酶解3种方法破碎细胞获得的水解液作为氮源发酵产丁二酸的效果,结果表明酶解制得的细胞水解液效果最佳。以总氮含量为1.11g/L的酶解液(相当于10g/L酵母膏)作为氮源发酵,丁二酸产量可达42.0g/L,继续增大酶解液用量对耗糖、产酸能力没有显著提高。将细胞酶解液与5g/L酵母膏联用发酵36h后,丁二酸产量达75.5g/L,且丁二酸生产强度为2.10g/(L·h),比使用10g/L酵母膏时提高了66.7%。因此,厌氧发酵产丁二酸结束后的废弃细胞酶解液可以替代原培养基中50%的酵母膏用于发酵。     

Effects of culture redox potential on succinic acid production by Corynebacterium crenatum under anaerobic conditions

During mixed-acid fermentation by Corynebacterium crenatum under anaerobic conditions, two moles of NADH are required to synthesize 1 mol of succinic acid. In this work, four controlled culture redox potentials and different carbon sources with different oxidation states were used to investigate the possibility of enhancing the succinic acid production by increasing the availability of NADH. When the culture redox potential was ?300 mV, the yield of succinic acid was 0.31 g/g, representing a 72% increase compared with the yield when the culture redox potential was ?40 mV. Meanwhile, the molar ratio of succinic acid/lactic acid increased from 0.27 to 0.48. When 0.1% neutral red was added to the acid production medium, the yield of succinic acid was 0.25 g/g, and the molar ratio of succinic acid/lactic acid was 0.38. Both values were higher than those obtained from glucose only (0.19 g/g, 0.26) or gluconate (0.05 g/g, 0.18). A higher NADH/NAD⁺ ratio and increased enzymatic activity could be achieved to enhance the succinic acid production by manipulating the culture to a more reductive environment.     

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.     

Integrated production of lactic acid and biomass on distillery stillage

The possibilities of parallel lactic acid and biomass production in batch and fed-batch fermentation on distillery stillage from bioethanol production were studied. The highest lactic acid yield and productivity of 92.3 % and 1.49 g L^?1 h^?1 were achieved in batch fermentation with initial sugar concentration of 55 g L^?1. A significant improvement of the process was achieved in fed-batch fermentation where the concentration of lactic acid was increased to 47.6 % and volumetric productivity for 21 % over the batch process. A high number of Lactobacillus rhamnosus ATCC 7469 viable cells of 10⁹ CFU ml^?1 was attained at the end of fed-batch fermentation. The survival of 92.9 % of L. rhamnosus cells after 3 h of incubation at pH 2.5 validated that the fermentation media remained after lactic acid removal could be used as a biomass-enriched animal feed thus making an additional value to the process.     

谷氨酸棒状杆菌厌氧产丁二酸的发酵条件

考察谷氨酸棒状杆菌ATCC13032Δldh厌氧产丁二酸的发酵条件。结果发现:补加NaHCO3的效果最好,并且考察了NaHCO3浓度对葡萄糖转化速率及丁二酸生成速率的影响。运用代谢流分析方法分析了乳酸脱氢酶基因敲除对谷氨酸棒状杆菌厌氧代谢的影响,发现乳酸脱氢酶基因敲除导致磷酸烯醇式丙酮酸生成丁二酸的流量提高了214.3%,流向乳酸的流量变为0;分批厌氧转化36 h生成41.2 g/L丁二酸,产率45.0%。     

Enhancing polyhydroxybutyrate production from high cell density fed-batch fermentation of Bacillus megaterium BA-019

This study demonstrated the improved polyhydroxybutyrate (PHB) production via high cell density cultivation of Bacillus megaterium BA-019 with balanced initial total sugar concentration and carbon to nitrogen (C/N) weight ratio. In the 10 L stirred fermentor operated at 30 °C, pH 7.0, 600 rpm, and 1.0 vvm air, with the initial total sugar concentration of 60 g/L and urea at the C/N weight ratio of 10:1, 32.48 g/L cell biomass with the corresponding PHB weight content of 26.94 % and volumetric productivity of 0.73 g/L h were obtained from batch cultivation. Continuing cultivation by intermittent feeding of the sugarcane molasses along with urea at the C/N weight ratio of 12.5:1 gave much improved biomass and PHB production (90.71 g/L biomass with 45.84 % PHB content and 1.73 g/L h PHB productivity). Similar biomass and PHB yields were obtained in the 90 L stirred fermentor when using the impeller tip speed as the scale-up criterion.     

Novel fermentation process strengthening strategy for production of propionic acid and vitamin B12 by Propionibacterium freudenreichii

An efficient fermentation-strengthening approach was developed to improve the anaerobic production of propionic acid and vitamin B12 by co-fermentation with Propionibacterium freudenreichii. Vitamin B12 production from glucose resulted in relatively high productivity (0.35 mg/L h) but a low propionic acid yield (0.55 g/g). By contrast, glycerol gave a high propionic acid yield (0.63 g/g) but low productivity (0.16 g/L h). Co-fermentation of glycerol and glucose with a gradual addition strategy gave high yields (propionic acid: 0.71 g/g; vitamin B12: 0.72 mg/g) and productivities (propionic acid: 0.36 g/L h; vitamin B12: 0.36 mg/L h). Finally, the integrated feedstock and fermentation system strengthening strategy was demonstrated as an efficient method for the economic production of bio-based propionic acid and vitamin B12.     

Efficient production of l-lactic acid by newly isolated thermophilic Bacillus coagulans WCP10-4 with high glucose tolerance

A thermophilic Bacillus coagulans WCP10-4 with tolerance to high concentration of glucose was isolated from soil and used to produce optically pure l-lactic acid from glucose and starch. In batch fermentation at pH?6.0, 240 g/L of glucose was completely consumed giving 210 g/L of l-lactic acid with a yield of 95 % and a productivity of 3.5 g/L/h. In simultaneous saccharification and fermentation at 50 °C without sterilizing the medium, 200 g/L of corn starch was completely consumed producing 202.0 g/L of l-lactic acid. To the best of our knowledge, this strain shows the highest osmotic tolerance to glucose among the strains ever reported for lactic acid production. This is the first report of simultaneous saccharification and fermentation of starch for lactic acid production under a non-sterilized condition.     

Enhancement and modeling of microparticle-added <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> lactic acid production

Lactic acid has a wide industrial application area and can be produced by fungal strains. However, excessive bulk growth form of fungi during the fermentations is a major problem, which limits the fermentation performance. Microparticles are excellent tools to prevent bulk fungal growth and provide homogenized fermentation broth to increase uniformity and the prediction performance of the models. Therefore, in this study, addition of aluminum oxide and talcum microparticles into fermentations was evaluated to enhance the production of lactic acid by Rhizopus oryzae. The results showed that the bulk fungal growth was prevented and the lactic acid concentration increased from 6.02 to 13.88 and 24.01 g/L, when 15 g/L of aluminum oxide or 10 g/L of talcum was used, respectively, in the shake-flask fermentations. Additionally, substrate concentration, pH, and agitation were optimized in the bioreactors using response surface methodology, and optimum values were determined as 126 g/L of glucose, 6.22 pH, and 387 rpm, respectively. Under these conditions, lactic acid production further increased to 75.1 ± 1.5 g/L with 10 g/L of talcum addition. Also, lactic acid production and glucose consumption in the batch fermentation were successfully modeled with modified Gompertz model and modified logistic model. RMSE and MAE values for lactic acid production were calculated as 2.279 and 1.498 for the modified Gompertz model; 3.6 and 4.056 for the modified logistic model. Additionally, modified logistic model predicted glucose consumption with ?2.088 MAE and 2.868 RMSE, whereas these values were calculated as 2.035 and 3.946 for the modified Gompertz model.     

Enhanced succinic acid production in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> with increasing the available NADH supply and glucose consumption rate by decreasing H<Superscript>+</Superscript>-ATPase activity

Objectives

To enhance succinic acid production in Corynebacterium glutamicum by increasing the supply of NADH and the rate of glucose consumption by decreasing H⁺-ATPase activity.

Results

A mutant of C. glutamicum NC-3-1 with decreased H⁺-ATPase activity was constructed. This increased the rate of glycolysis and the supply of NADH. Fermentation of C. glutamicum NC-3-1 gave 39 % higher succinic acid production (113 and 81 g/l), a 29 % higher succinic acid yield (0.94 and 0.73 g succinic acid/g glucose) and decreased by-products formation compared to that of C. glutamicum NC-3 in 5 l bioreactor.

Conclusion

The point mutation in C. glutamicum NC-3-1 increased the rate of glycolysis and resulted in higher succinic acid production, higher succinic acid yield and significantly decreased formation of by-products.

     

Ultrasonic pretreatment and acid hydrolysis of sugarcane bagasse for succinic acid production using Actinobacillus succinogenes

Immense interest has been devoted to the production of bulk chemicals from lignocellulose biomass. Diluted sulfuric acid treatment is currently one of the main pretreatment methods. However, the low total sugar concentration obtained via such pretreatment limits industrial fermentation systems that use lignocellulosic hydrolysate. Sugarcane bagasse hemicellulose hydrolysate is used as the carbon and nitrogen sources to achieve a green and economical production of succinic acid in this study. Sugarcane bagasse was ultrasonically pretreated for 40 min, with 43.9 g/L total sugar obtained after dilute acid hydrolysis. The total sugar concentration increased by 29.5 %. In a 3-L fermentor, using 30 g/L non-detoxified total sugar as the carbon source, succinic acid production increased to 23.7 g/L with a succinic acid yield of 79.0 % and a productivity of 0.99 g/L/h, and 60 % yeast extract in the medium could be reduced. Compared with the detoxified sugar preparation method, succinic acid production and yield were improved by 20.9 and 20.2 %, respectively.     

过量表达异柠檬酸裂解酶对ldh-1谷氨酸棒状杆菌产丁二酸的影响

谷氨酸棒状杆菌SA001是缺失了乳酸脱氢酶基因 (ldhA) 的菌株。为了增加厌氧条件下经异柠檬酸到丁二酸的代谢通量,以提高丁二酸的产量。将来自大肠杆菌Escherichia coli K12的异柠檬酸裂解酶基因导入谷氨酸棒状杆菌SA001 (SA001/pXMJ19-aceA) 中。该菌经0.8 mmol/L的IPTG有氧诱导12 h后,转入厌氧发酵16 h,丁二酸的产量为10.38 g/L,丁二酸的生产强度为0.83 g/(L·h)。与出发菌株比较,异柠檬酸裂解酶的酶活提高了5.8倍,丁二酸的产量提高了48%。结果表明过量表达异柠檬酸裂解酶可以增加由乙醛酸途径流向丁二酸的代谢流。