Lactic acid production from pretreated corn stover with recycled streams

In order to overcome bottlenecks of the high amount of cellulase consumption in lignocellulosic l-Lactic acid (LA) production, a non-sterilized fed-batch simultaneous saccharification and fermentation (SSF) -membrane separation integration process was established in this current work. During the process, residual cellulase that remaining in the waste aqueous solution and solid residuals of corn stover (CS) were recycled and reused in subsequent fermentations. A total 6 rounds of operation were performed. Averagely, LA yield of 0.389 g g⁻¹ (pretreated CS) was achieved, which was 1.20 times higher than that of the conventional process without waste stream recycling. Moreover, the wastewater discharge and the cost of nutrients for fermentation can also hugely decrease. Results indicated that cellulase, wastewater discharge and nutrients consumption of the process reduced by 47.4 %, 73.7 % and 86.1 %, respectively. This study opens a promising way for the reduction of second-generation LA production cost, which could significantly change the economic feasibility of the LA biorefineries.     

Lactic acid production from whey permeate by immobilized Lactobacillus casei

Two matrices have been assessed for their ability to immobilize Lactobacillus casei cells for lactic acid fermentation in whey permeate medium. Agar at 2% concentration was found to be a better gel than polyacrylamide in its effectiveness to entrap the bacterial cells to carry out batch fermentation up to three repeat runs. Of the various physiological parameters studied, temperature and pH were observed to have no significant influence on the fermentation ability of the immobilized organism. A temperature range of 40–50°C and a pH range of 4.5–6.0 rather than specific values, were found to be optimum when fermentation was carried out under stationary conditions. In batch fermentation ～90% conversion of the substrate (lactose) was achieved in 48 h using immobilized cell gel cubes of 4 × 2 × 2 mm size, containing 400 mg dry bacterial cells per flask and 4.5% w/v (initial) whey lactose content as substrate. However, further increase in substrate levels tested (>4.5% w/v) did not improve the process efficiency. Supplementation of Mg²⁺ (1 mM) and agricultural by-products (mustard oil cake, 6%) in the whey permeate medium further improved the acid production ability of the immobilized cells under study.     

Direct lactic acid fermentation: Focus on simultaneous saccharification and lactic acid production

In the recent decades biotechnological production of lactic acid has gained a prime position in the industries as it is cost effective and eco-friendly. Lactic acid is a versatile chemical having a wide range of applications in food, pharmaceutical, leather and textile industries and as chemical feedstock for so many other chemicals. It also functions as the monomer for the biodegradable plastic. Biotechnological production is advantageous over chemical synthesis in that we can utilize cheap raw materials such as agro-industrial byproducts and can selectively produce the stereo isomers in an economic way. Simultaneous saccharification and fermentation can replace the classical double step fermentation by the saccharification of starchy or cellulosic biomass and conversion to lactic acid concurrently by adding inoculum along with the substrate degrading enzymes. It not only reduces the cost of production by avoiding high energy consuming biomass saccharification, but also provides the higher productivity than the single step conversion by the providing adequate sugar release.     

Effects of intermittent addition of cellulase for production of L-lactic acid from wastewater sludge by simultaneous saccharification and fermentation

An attempt was made to create L-lactic acid, a precursor of poly-lactic acid, which is a biodegradable plastic, from wastewater sludge from the paper-manufacturing industry. The sludge contained a high percentage of cellulose and needed to be hydrolyzed to glucose by the action of the cellulase before being treating with lactic acid bacteria. Therefore, a method involving simultaneous saccharification and fermentation (SSF) was carried out. The optimum pH of the SSF for production of the lactic acid by the newly isolated lactic acid bacterium with a high selectively of L-lactic acid was found out to be around pH = 5.0, and the optimum temperature to be approximately 40 degrees C. On the basis of the measurement of the cell density changes in the lactic acid bacteria, it was ascertained that the bacterial activity could continue at a high level for a relatively long period of time, and that the L-lactic acid productivity was diminished by the rapid deactivation of the cellulase. With the intermittent addition of cellulase once daily for the sake of compensating for the cellulase deactivation, the L-lactic acid attained a maximum concentration of 16.9 g/L, i.e., a 72.2% yield based on the potential glucose contained in the sludge under optimum pH and temperature conditions.     

稀碱预处理棕榈残渣制备纤维乙醇

以棕榈残渣(Empty fruit bunch,EFB)为原料,通过预处理、酶解、发酵等过程制备纤维乙醇.首先对比了碱、碱/过氧化氢等预处理条件对棕榈残渣组成及酶解的影响,结果表明稀碱预处理效果较好.适宜的稀碱预处理条件为:NaOH浓度为1％,固液比为1∶10,在40℃浸泡24 h后于121℃下保温30 min,在该条件下,EFB的固体回收率为74.09％,纤维素、半纤维素和木质素的含量分别为44.08％、25.74％和13.89％.对该条件下预处理后的固体样品,以底物浓度5％、酶载量30 FPU/g底物酶解72 h,纤维素和半纤维素的酶解率分别达到84.44％和89.28％.进一步考察了酶载量和底物浓度对酶解的影响以及乙醇批式同步糖化发酵,当酶载量为30 FPU/g底物,底物浓度由5％增加至25％时,利用酿酒酵母Saccharomyces cerevisiae(接种量为5％,VIV)发酵72 h后乙醇的浓度分别为9.76 g/L和35.25 g/L,可分别达到理论得率的79.09％和56.96％.     

马铃薯淀粉同步糖化发酵制备L-乳酸条件的统计学优化

以马铃薯淀粉为原料,采用同步糖化发酵方法制备乳酸。通过Plackett-Burman实验设计对影响乳酸产量的7个因子进行筛选,结果表明淀粉质量浓度、糖化酶用量和发酵温度3个因素对乳酸产量影响显著。利用最陡爬坡试验逼近最大响应区,采用中心复合实验设计及响应面分析法进行回归分析,建立影响乳酸产量的二次模型。模型求解得出最优淀粉质量浓度为271.89g/L,糖化酶用量为265.09U/g,发酵温度为39.05℃,最大理论乳酸产量为196.99g/L。3批验证实验结果平均值与预测值接近,表明该模型与实际情况拟合良好,实际最大乳酸产量为193.6g/L,较优化前提高了13.9%,L-乳酸的平均纯度达到95.2%。     

Production of lactic acid from wastepaper as a cellulosic feedstock

Lactic acid promises to be an important commodity chemical in the future as a monomer for the production of biodegradable polylactic acid (PLA). As the demand for lactic acid increases, the need to explore alternative feedstock sources and process options that are inexpensive and efficient is bound to gain importance. This paper reports the results of a study of the production of lactic acid from wastepaper as a representative cellulosic feedstock, using a batch, bench-scale simultaneous saccharification and fermentation (SSF) process. The effect on process performance of operating parameters such as pH, temperature, enzyme loading, solids concentration, and enzyme preparation has been examined. A lactic acid product yield of 84% of theoretical was achieved at a solids loading of 5%, using 25 filter paper units (FPU) of cellulase per gram of cellulose, at 45°C and pH 5.0. The pH and temperature of operation have been selected to achieve good performance of both the cellulase and the microoganism in the SSF process. Our studies show that a feedstock such as wastepaper offers considerable promise and opportunity in the future for development of a biomass-based process for lactic acid production. Received 09 January 1996/ Accepted in revised form 22 August 1996     

H2O2 feeding enables LPMO-assisted cellulose saccharification during simultaneous fermentative production of lactic acid

Simultaneous saccharification and fermentation (SSF) is a well-known strategy for valorization of lignocellulosic biomass. Because the fermentation process typically is anaerobic, oxidative enzymes found in modern commercial cellulase cocktails, such as lytic polysaccharide monooxygenases (LPMOs), may be inhibited, limiting the overall efficiency of the enzymatic saccharification. Recent discoveries, however, have shown that LPMOs are active under anoxic conditions if they are provided with H₂O₂ at low concentrations. In this study, we build on this concept and investigate the potential of using externally added H₂O₂ to sustain oxidative cellulose depolymerization by LPMOs during an SSF process for lactic acid production. The results of bioreactor experiments with 100 g/L cellulose clearly show that continuous addition of small amounts of H₂O₂ (at a rate of 80 µM/h) during SSF enables LPMO activity and improves lactic acid production. While further process optimization is needed, the present proof-of-concept results show that modern LPMO-containing cellulase cocktails such as Cellic CTec2 can be used in SSF setups, without sacrificing the LPMO activity in these cocktails.     

Ellagic acid production by <Emphasis Type="Italic">Aspergillus niger</Emphasis> in solid state fermentation of pomegranate residues

Two Aspergillus niger strains (GH1 and PSH) previously isolated from a semiarid region of Mexico were characterized for their effectiveness in converting pomegranate ellagitannins (ET) into ellagic acid (EA) in a solid state fermentation (SSF). Pomegranate seeds and husk were used as support for the SSF. Released EA was evaluated by liquid chromatography. Yields of 6.3 and 4.6 mg of EA per gram of dried pomegranate husk were obtained with A. niger GH1 and PSH, respectively. Total hydrolyzable polyphenols of pomegranate husk were degraded during the first 72 h of culture (71 and 61%, by GH1 and PSH strains, respectively). Tannin acyl hydrolase activity was not clearly associated with EA production. EA that accumulated in cultures of A. niger GH1 was remarkably pure after a simple extraction process. Pomegranate husk is a good support, and at the same time an excellent substrate in the production of high commercial interest metabolites like EA due the degradation of its ET content.     

Lactic acid and animal feeds production from Sophora flavescens residues by Rhizopus oryzae fermentation

In this study, the feasibility of producing lactic acid and animal feeds from Sophora flavescens residues (SFR) by Rhizopus oryzae was explored. Results showed that the simultaneous saccharification and fermentation (SSF) is the optimal fermentation mode, which was simple and high-efficiency. When the inoculation volume of R. oryzae was 10 % and the pH value was adjusted by adding CaCO₃ in stages during SSF, the maximum concentration of lactic acid was 46.78 g/L, and the maximum lactic acid productivity reached 0.97 g/L/h. Results also showed that the protein content of the solid residues after fermentation of R. oryzae reached 12.15 %. This content was 46 times higher than that by the original SFRs and nearly 4.3 times the protein content of the solid residues after fermentation by Enterococcus faecium. In addition, the solid residues after fermentation rich in Fe and Zn could be used as animal feeds or feed additives. Thus, it is expected that this study may provide a novel approach for Chinese medicine residues treatment towards full resource recovery.     

米根霉发酵生产L-乳酸

报道了Ｌ－乳酸菌株的分离与筛选,探讨了不同碳源、氮源、通气量、温度等发酵条件对产Ｌ－乳酸的影响,从７８株米根霉中筛选出１３株产Ｌ－乳酸较高的菌株,其中米根霉（Ｒｈｉｚｏｐｕｓ ｏｒｙｚａｅ）Ｒｓ９２８产Ｌ－乳酸最高,产酸最稳定。试验结果表明,该菌株最适发酵培养组成（％）：淀粉水解糖１６,ＭｇＳＯ４ ０．０８,ＫＨ２ＰＯ４ ０．０５,ＺｎＳＯ４ ０．０１,ＣａＣＯ３ ７,ｐＨ自然。在６０ｔ发酵罐中,     

Product inhibition in simultaneous saccharification and fermentation of cellulose into lactic acid

The product, lactic acid, strongly inhibited microbial activity in lactic acid fermentation. The volumetric productivity declined from 1.19 g/l.h with zero lactic acid (control) to only 0.18 g/l.h when lactic acid reached 65 g/l. Lactic acid also inhibited cellulase activity but less severely than the inhibition on microbial activity as lactic acid above 90 g/l was needed for 50% inhibition. A gradual deterioration of the Simultaneous Saccharification and Fermentation (SSF) process occurred with the build-up of lactic acid and the rate-controlling step in SSF shifted from hydrolysis to fermentation as the bioprocess proceeded.     

Lactic acid production by Lactobacillus delbrueckii in a dual reactor system using packed bed biofilm reactor

Aims:  An integrated dual reactor system for continuous production of lactic acid by Lactobacillus delbrueckii using biofilms developed on reticulated polyurethane foam (PUF) is demonstrated.
Methods and Results:  Lactobacillus delbrueckii was immobilized on PUF, packed in a bioreactor and used in lactic acid fermentation. The rate of lactic acid production was significantly high with a volumetric productivity of 5 g l⁻¹ h⁻¹ over extended period of time. When coupled to a bioreactor, the system could be operated as dual reactor for over 1000 h continuously without augmentation of inoculum and no compromise on productivity.
Conclusions:  Polyurethane foams offer an excellent support for biofilm formation.
Significance and Impact of the Study:  The system was very robust and could be operated for prolonged period at a volumetric productivity of 4–6 g l⁻¹ h⁻¹.     

Recombinant Escherichia coli engineered for production of L-lactic acid from hexose and pentose sugars

Recombinant Escherichia coli have been constructed for the conversion of glucose as well as pentose sugars into L-lactic acid. The strains carry the lactate dehydrogenase gene from Streptococcus bovis on a low copy number plasmid for production of L-lactate. Three E. coli strains were transformed with the plasmid for producing L-lactic acid. Strains FBR9 and FBR11 were serially transferred 10 times in anaerobic cultures in sugar-limited medium containing glucose or xylose without selective antibiotic. An average of 96% of both FBR9 and FBR11 cells maintained pVALDH1 in anaerobic cultures. The fermentation performances of FBR9, FBR10, and FBR11 were compared in pH-controlled batch fermentations with medium containing 10% w/v glucose. Fermentation results were superior for FBR11, an E. coli B strain, compared to those observed for FBR9 or FBR10. FBR11 exhausted the glucose within 30 h, and the maximum lactic acid concentration (7.32% w/v) was 93% of the theoretical maximum. The other side-products detected were cell mass and succinic acid (0.5 g/l). Journal of Industrial Microbiology & Biotechnology (2001) 27, 259–264. Received 05 November 2000/ Accepted in revised form 03 July 2001     

Direct production of 4-hydroxybenzoic acid from cellulose using cellulase-displaying Pichia pastoris

4-hydroxybenzoic acid (4-HBA) is an industrially important aromatic compound, and there is an urgent need to establish a bioprocess to produce this compound in a sustainable and environmentally friendly manner from renewable feedstocks such as cellulosic biomass. Here, we developed a bioprocess to directly produce 4-HBA from cellulose using a recombinant Pichia pastoris strain that displays heterologous cellulolytic enzymes on its cell surface via the glycosylphosphatidylinositol (GPI)-anchoring system. β-glucosidase (BGL) from Aspergillus aculeatus, endoglucanase (EG) from Trichoderma reesei, and cellobiohydrolase (CBH) from Talaromyces emersonii were co-displayed on the cell surface of P. pastoris using an appropriate GPI-anchoring domain for each enzyme. The cell-surface cellulase activity was further enhanced using P. pastoris SPI1 promoter- and secretion signal sequences. The resulting strains efficiently hydrolyzed phosphoric acid swollen cellulose (PASC) to glucose. Then, we expressed a highly 4-HBA-resistant chorismate pyruvate-lyase (UbiC) from Providencia rustigianii in the cellulase-displaying strain. This strain produced 975 mg/L of 4-HBA from PASC, which corresponding to 36.8% of the theoretical maximum yield, after 96 h of batch fermentation without the addition of commercial cellulase. This 4-HBA yield was over two times higher than that obtained from glucose (12.3% of the theoretical maximum yield). To our knowledge, this is the first report on the direct production of an aromatic compound from cellulose using cellulase-displaying yeast.     

Lactic acid production from cellulosic waste by immobilized cells of Lactobacillus delbrueckii

Industrial waste corn cob residue (from xylose manufacturing) without pretreatment was hydrolyzed by cellulase and cellobiase. The cellulosic hydrolysate contained 52.4 g l⁻¹ of glucose and was used as carbon source for lactic acid fermentation by cells of Lactobacillus delbrueckii ZU-S2 immobilized in calcium alginate gel beads. The final concentration of lactic acid and the yield of lactic acid from glucose were 48.7 g l⁻¹ and 95.2%, respectively, which were comparative to the results of pure glucose fermentation. The immobilized cells were quite stable and reusable, and the average yield of lactic acid from glucose in the hydrolysate was 95.0% in 12 repeated batches of fermentation. The suitable dilution rate of continuous fermentation process was 0.13 h⁻¹, and the yield of lactic acid from glucose and the productivity were 92.4% and 5.746 g l⁻¹ h⁻¹, respectively. The production of lactic acid by simultaneous saccharification and fermentation (SSF) process was carried out in a coupling bioreactor, the final concentration of lactic acid was 55.6 g l⁻¹, the conversion efficiency of lactic acid from cellulose was 91.3% and the productivity was 0.927 g l⁻¹ h⁻¹. By using fed-batch technique in the SSF process, the final concentration of lactic acid and the productivity increased to 107.6 g l⁻¹ and 1.345 g l⁻¹ h⁻¹, respectively, while the dosage of cellulase per gram substrate decreased greatly. This research work should advance the bioconversion of renewable cellulosic resources and reduce environmental pollution.     

用非常规培养方法提高乳酸菌产酸率的研究

以德氏乳酸杆菌为研究对象,考察了八种有机溶剂分别加入培养基对细菌生长及产酸的影响。结果表明,采用油醇和三辛胺混合溶剂时,既能降低对细胞生长的毒性,又保持了较强的萃取能力。对悬浮细胞发酵和萃取整合的方法和固定化细胞发酵和萃取整合的方法进行了比较,表明这两种方法均较常规培养方法提高乳酸产率60％以上。     

工业乳酸发酵的近期进展

乳酸是一种重要的多用途有机酸。通过菌种改良和发酵工艺技术的改进,可以大大提升微生物发酵技术水平,降低成本。简要综述有关的研究进展。