FED-BATCH SIMULTANEOUS SACCHARIFICATION AND FERMENTATION OF MICROWAVE/ACID/ALKALI/H2O2 PRETREATED RICE STRAW FOR PRODUCTION OF ETHANOL

The batch simultaneous saccharification and fermentation (SSF) of microwave/acid/alkali/H₂O₂ pretreated rice straw to ethanol was optimized using cellulase from Trichoderma reesei and Saccharomyces cerevisiae YC-097 cells prior to the fed-batch SSF studies. The batch SSF optima were 10% w/v substrate, 40°C, 15 mg cellulase/g substrate, initial pH 5.3, and 72 hours. Under the optimum conditions the ethanol concentration and its yield were 29.1 g/L and 61.3% respectively. Based on the optimal batch SSF, the fed-batch SSF was investigated and its operation parameters were optimized. Under its optimal conditions the ethanol concentration reached 57.3 g/L, while its productivity and yield were only slightly less than those in the batch SSF. This suggests that fed-batch SSF is a potential operation mode for effective ethanol production from microwave/acid/alkali/H₂O₂ pretreated rice straw.     

FED-BATCH SIMULTANEOUS SACCHARIFICATION AND FERMENTATION OF MICROWAVE/ACID/ALKALI/H2O2 PRETREATED RICE STRAW FOR PRODUCTION OF ETHANOL

The batch simultaneous saccharification and fermentation (SSF) of microwave/acid/alkali/H₂O₂ pretreated rice straw to ethanol was optimized using cellulase from Trichoderma reesei and Saccharomyces cerevisiae YC-097 cells prior to the fed-batch SSF studies. The batch SSF optima were 10% w/v substrate, 40°C, 15 mg cellulase/g substrate, initial pH 5.3, and 72 hours. Under the optimum conditions the ethanol concentration and its yield were 29.1 g/L and 61.3% respectively. Based on the optimal batch SSF, the fed-batch SSF was investigated and its operation parameters were optimized. Under its optimal conditions the ethanol concentration reached 57.3 g/L, while its productivity and yield were only slightly less than those in the batch SSF. This suggests that fed-batch SSF is a potential operation mode for effective ethanol production from microwave/acid/alkali/H₂O₂ pretreated rice straw.     

A comprehensive ligninolytic pre-treatment approach from lignocellulose green biotechnology to produce bio-ethanol

In the present study, we developed a novel ligninolytic enzymes based pre-treatment method for lignocellulosic wheat straw to depolymerize lignin and expose the cellulose polymers to produce bio-ethanol. Wheat straw was pre-treated with ligninolytic enzymes extract produced from Ganoderma lucidum under optimum solid state fermentation conditions. The pre-treated biomass was further subjected to the enzymatic hydrolysis by the crude unprocessed cellulases (β-1,4 endoglucanase, 53.5 ± 1.24 U/mL; β-1,4 exoglucanase, 41.3 ± 1.31 U/mL; β-1,4 glucosidase, 46.8 ± 1.43 U/mL; and xylanase 39 ± 2.2 U/mL) produced by Trichoderma harzaianum. Under optimal conditions for enzymatic saccharification, 10% (w/v) of pre-treated biomass was hydrolyzed completely and converted to 72.5 and 2.4 g/L of glucose and xylose, respectively. Initial time screening Sequential Saccharification and Fermentation (SSF) of the concentrated enzymatic hydrolyzate (10%, w/v) by Saccharomyces cerevisiae produced 22.6 g/L ethanol in a fermented medium after 72 h of temperature controlled incubation at 37 °C. For maximum ethanol production, different physical and nutritional parameters like pH, temperature, substrate level and inoculum sizes were optimized. Under optimal conditions ethanol production of 33.5 g/L was obtained from ligninolytic treated residual (wheat straw) biomass.     

蒸汽爆破麦草同步糖化发酵转化乙醇的研究

近年来对木质生物资源同步糖化发酵转化乙醇的研究较多,但是,麦草同步糖化发酵转化乙醇的最佳工艺条件还未确定。文中采用正交试验设计的方法,对在混合酶(纤维素酶Celluclast 1.5 1,β-葡萄糖苷酶Novozym 188)与酿酒酵母菌作用下,稀硫酸催化的蒸汽爆破麦草原料同步糖化发酵转化乙醇的工艺条件进行研究,详细讨论了反应温度、底物质量浓度、发酵液pH值、纤维素酶浓度对乙醇质量浓度和得率的影响。结果表明,工艺条件对乙醇质量浓度和得率的影响程度由高到低依次为:底物质量浓度、纤维素酶浓度、发酵液pH值、反应温度。最佳工艺条件为反应温度35℃,底物质量浓度100 g/L,发酵液pH值5.0,纤维素酶浓度30 FPU/g。在此条件下,随着反应时间的延长,乙醇质量浓度持续上升。反应72 h后,乙醇质量浓度和得率分别达到22.7 g/L和65.8%。     

耐热酿酒酵母FE-B的分离筛选及应用研究

从米酒酒曲中筛选出来一株乙醇发酵菌株,命名为FE-B,经鉴定为酿酒酵母,CGMCC保藏号为2735。FE-B在28,36和40℃都能生长,28 h左右达到稳定期时培养物的菌体浓度分别为4.24,3.95和3.23 g(DCW)/L。FE-B在28,36和42℃发酵24 h,发酵液中的乙醇浓度分别为8.0%,8.1%和3.3%。FE-B在温度≤41℃,酶加入量25 IU/g(秸秆干基),固含量10%条件下进行同步糖化与发酵,乙醇产生速率随温度上升而递增,当温度为41℃时,72 h后体系中乙醇浓度可达2.11%,纤维素水解率87.6%,具有很好的应用前景。     

Comparison of bioethanol production of simultaneous saccharification & fermentation and separation hydrolysis & fermentation from cellulose-rich barley straw

Cellulose rich barley straw, which has a glucan content of 62.5%, followed by dilute acid pretreatment, was converted to bioethanol by simultaneous saccharification and fermentation (SSF). The optimum fractionation conditions for barley straw were an acid concentration of 1% (w/v), a reaction temperature of 158 °C and a reaction time of 15 min. The maximum saccharification of glucan in the fractionated barley straw was 70.8% in 72 h at 60 FPU/gglucan, while the maximum digestibility of the untreated straw was only 18.9%. With 6% content WIS (water insoluble solid) for the fractionated barley straw, 70.5 and 83.2% of the saccharification yield were in SHF and SSF (representing with glucose equivalent), respectively, and a final ethanol concentration of 18.46 g/L was obtained under the optimized SSF conditions: 34 °C with 15 FPU/g-glucan enzyme loading and 1 g dry yeast cells/L. The results demonstrate that the SSF process is more effective than SHF for bioethanol production by around 18%.     

表面活性剂对麦草同步糖化发酵转化乙醇的影响

研究了5种非离子型表面活性剂(BSA, Tween-20, Tween-80, PEG-4000, PEG-6000)促进麦草同步糖化发酵的效果. 结果表明,5种表面活性剂均能促进麦草同步糖化发酵,以Tween-20效果最为显著. 反应体系中添加Tween-20可降低酶用量而保持乙醇浓度基本相同. 在pH 5.0、温度37℃、底物浓度50 g/L及Celluclast 1.5 l用量25 FPU/g、Novozym 188用量15 IU/g的反应体系中,添加0.03 g/g Tween-20,反应72 h,乙醇浓度达到18.7 g/L,比未添加表面活性剂的体系提高了14.0%,反应时间缩短了12 h.     

Bioethanol production from wheat straw by the thermotolerant yeast Kluyveromyces marxianus CECT 10875 in a simultaneous saccharification and fermentation fed-batch process

Solid content in the simultaneous saccharification and fermentation (SSF) broth should be as high as possible in order to reach higher ethanol concentration. In this work, several feeding strategies for ethanol production from steam-exploded wheat straw by Kluyveromyces marxianus CECT 10875 have been studied with the aim of obtaining higher ethanol concentrations. Previous fermentability tests as well as SSF processes showed the difficulty of using the slurry for ethanol production under the studied conditions. Notwithstanding, fed-batch SSF processes with water-insoluble solids (WIS) fraction resulted in better configuration, reaching the highest ethanol concentration (36.2 g/L) with an initial WIS content of 10% (w/v) and 4% (w/v) of substrate addition at 12 h, which meant 20% more ethanol when compared with batch SSF.     

缺失PHO13基因与进化工程构建高效利用木糖酵母

过表达了木糖还原酶、木糖醇脱氢酶和木酮糖激酶基因的重组工业酿酒酵母菌株KAM-6X内缺失了编码对硝基苯磷酸盐磷酸化酶的PHO13基因,接着通过EMS诱变和进化工程筛选,获得了一株高效利用木糖的菌株,命名为PE。有氧条件下,在含50 g/L木糖和100 g/L木糖的YPX中最大比生长速率分别为0.299和0.282 h^-1,分别比出发菌提高了95.43%和102.87%,同时PE菌株能在前24 h内耗掉36.12 g/L木糖,48 h内耗掉70.25 g/L木糖。微好氧条件下副产物产量降低,糖醇转化率最高达到0.382 g/g,证明PE是一株高效利用木糖发酵的工业酵母菌株。     

Simultaneous saccharification and fermentation of alkaline-pretreated corn stover to ethanol using a recombinant yeast strain

Bio-ethanol converted from cheap and abundant lignocellulosic materials is a potential renewable resource to replace depleting fossil fuels. Simultaneous saccharification and fermentation (SSF) of alkaline-pretreated corn stover for the production of ethanol was investigated using a recombinant yeast strain Saccharomyces cerevisiae ZU-10. Low cellobiase activity in Trichoderma reesei cellulase resulted in cellobiose accumulation. Supplementing the simultaneous saccharification and fermentation system with cellobiase greatly reduced feedback inhibition caused by cellobiose to the cellulase reaction, thereby increased the ethanol yield. 12 h of enzymatic prehydrolysis at 50 °C prior to simultaneous saccharification and fermentation was found to have a negative effect on the overall ethanol yield. Glucose and xylose produced from alkaline-pretreated corn stover could be co-fermented to ethanol effectively by S. cerevisiae ZU-10. An ethanol concentration of 27.8 g/L and the corresponding ethanol yield on carbohydrate in substrate of 0.350 g/g were achieved within 72 h at 33 °C with 80 g/L of substrate and enzyme loadings of 20 filter paper activity units (FPU)/g substrate and 10 cellobiase units (CBU)/g substrate. The results are meaningful in co-conversion of cellulose and hemicellulose fraction of lignocellulosic materials to fuel ethanol.     

木质纤维生物质同步糖化发酵(SSF)生产乙醇的研究进展

综述了有关木质纤维生物质原料同步糖化发酵生产乙醇的最新研究进展和未来发展方向:同步糖化发酵是一种用于从木质纤维原料生产乙醇的工艺过程,此工艺的优点是酶水解与发酵同时进行,可以减少最终产物对酶水解的抑制作用,并减少投资成本,是最具发展潜力和优势的工艺之一。近年来在优化预处理工艺、降低纤维素酶成本以及己糖戊糖协同发酵等方面的研究都取得了长足的进步,其中以小麦秸秆为原料进行同步糖化发酵所得到的乙醇浓度接近40g/L。     

Simultaneous saccharification and fermentation of wheat bran flour into ethanol using coculture of amylotic Aspergillus niger and thermotolerant Kluyveromyces marxianus

Studies on simultaneous saccharification and fermentation (SSF) of wheat bran flour, a grain milling residue as the substrate using coculture method were carried out with strains of starch digesting Aspergillus niger and nonstarch digesting and sugar fermenting Kluyveromyces marxianus in batch fermentation. Experiments based on central composite design (CCD) were conducted to maximize the glucose yield and to study the effects of substrate concentration, pH, temperature, and enzyme concentration on percentage conversion of wheat bran flour starch to glucose by treatment with fungal α-amylase and the above parameters were optimized using response surface methodology (RSM). The optimum values of substrate concentration, pH, temperature, and enzyme concentration were found to be 200 g/L, 5.5, 65°C and 7.5 IU, respectively, in the starch saccharification step. The effects of pH, temperature and substrate concentration on ethanol concentration, biomass and reducing sugar concentration were also investigated. The optimum temperature and pH were found to be 30°C and 5.5, respectively. The wheat bran flour solution equivalent to 6% (w/V) initial starch concentration gave the highest ethanol concentration of 23.1 g/L after 48 h of fermentation at optimum conditions of pH and temperature. The growth kinetics was modeled using Monod model and Logistic model and product formation kinetics using Leudeking-Piret model. Simultaneous saccharificiation and fermentation of liquefied wheat bran starch to bioethanol was studied using coculture of amylolytic fungus A. niger and nonamylolytic sugar fermenting K. marxianus.     

Simultaneous saccharification and fermentation of wheat bran flour into ethanol using coculture of amylotic Aspergillus niger and thermotolerant Kluyveromyces marxianus

Studies on simultaneous saccharification and fermentation (SSF) of wheat bran flour, a grain milling residue as the substrate using coculture method were carried out with strains of starch digesting Aspergillus niger and nonstarch digesting and sugar fermenting Kluyveromyces marxianus in batch fermentation. Experiments based on central composite design (CCD) were conducted to maximize the glucose yield and to study the effects of substrate concentration, pH, temperature, and enzyme concentration on percentage conversion of wheat bran flour starch to glucose by treatment with fungal α-amylase and the above parameters were optimized using response surface methodology (RSM). The optimum values of substrate concentration, pH, temperature, and enzyme concentration were found to be 200 g/L, 5.5, 65°C and 7.5 IU, respectively, in the starch saccharification step. The effects of pH, temperature and substrate concentration on ethanol concentration, biomass and reducing sugar concentration were also investigated. The optimum temperature and pH were found to be 30°C and 5.5, respectively. The wheat bran flour solution equivalent to 6% (w/V) initial starch concentration gave the highest ethanol concentration of 23.1 g/L after 48 h of fermentation at optimum conditions of pH and temperature. The growth kinetics was modeled using Monod model and Logistic model and product formation kinetics using Leudeking-Piret model. Simultaneous saccharificiation and fermentation of liquefied wheat bran starch to bioethanol was studied using coculture of amylolytic fungus A. niger and nonamylolytic sugar fermenting K. marxianus.     

β-葡萄糖苷酶高温同步糖化发酵产乙醇应用研究

利用Trichoderma sp.W2所产的嗜温耐乙醇β-葡萄糖苷酶及耐高温酵母Kluyveromyces marxianus NCYC 587,以气爆秸秆为原料进行高温同步糖化发酵。研究结果表明:在42℃条件下,接种体积分数10%,底物质量分数15%,发酵pH值为4.8,β-葡萄糖苷酶添加量为30 U/g底物条件下发酵效果最好。NCYC 587能迅速利用预水解产生的葡萄糖发酵并积累乙醇,同时能利用部分木糖,但在发酵后期,葡萄糖利用完全后会代谢利用一定量的乙醇,致使发酵过程中乙醇质量浓度始终维持在一个相对较低的水平。乙醇最高质量浓度达到20.56 g/L,乙醇产率达80.64%。添加嗜温耐乙醇β-葡萄糖苷酶于高温同步糖化发酵能有效解决纤维素酶解发酵过程终端产物抑制的难题。     

Glucobacter oxydans全细胞直接催化秸秆稀酸预处理水解液产木糖酸

以长期驯化选育得到的耐抑制物氧化葡萄糖酸杆菌Glucobacter oxydans NL71为催化菌株,对全细胞直接催化木质纤维(麦秆)稀酸预处理水解液产木糖酸的工艺进行研究。结果表明:未脱毒水解液对催化反应生产木糖酸具有抑制作用,且随浓度提高,抑制作用增强。提高细胞浓度和提高供氧能力可以有效抵抗麦杆稀水解液的抑制效应。在摇瓶体系中以2 g/L细胞接种量直接催化未经脱毒的麦秆稀酸水解浓缩液,当初始木糖质量浓度超过100 g/L时,细胞的催化性能受到严重抑制,木糖酸得率均低于50%;当细胞接种量提升至8 g/L时,木糖酸得率可以达到85.7%;在机械搅拌式通氧加压反应体系中,采用密封加氧技术对100 g/L木糖浓度的麦秸稀酸水解浓缩液全细胞催化24 h的细胞接种量8 g/L,木糖酸终质量浓度达到103.1 g/L,木糖酸得率可达到93.1%,产生速率为摇瓶体系的1.6倍。     

木薯酒精渣同步糖化发酵制乙醇的工艺研究

研究了利用木薯酒精厂废渣为原料发酵生产乙醇的方法,结果表明:经过简单的机械粉碎后,通过同步糖化发酵生产乙醇是可行的。发酵条件为:木薯酒精渣经粉碎后取粒径小于0.85mm的部分,初始料水比1∶8,纤维素酶添加量为每克木薯渣(干重)30FPU,发酵过程中在24h内分批将剩余木薯渣加入至总料水比达到1∶2.5,利用5L发酵罐进行同步糖化发酵,发酵液中乙醇质量浓度达到52g/L,木薯酒精渣到乙醇的收率达到13%。纤维素酶的添加量对发酵效果影响显著,当达到每克木薯渣(干重)50FPU时,发酵液中乙醇质量浓度可达65g/L,乙醇收率达到16%。     

Study of simultaneous saccharification and fermentation for steam exploded wheat straw to ethanol

Although simultaneous saccharification and fermentation (SSF) has been investigated extensively, the optimum condition for SSF of wheat straw has not yet been determined. Dilute sulfuric acid impregnated and steam explosion pretreated wheat straw was used as a substrate for the production of ethanol by SSF through orthogonal experiment design in this study. Cellulase mixture (Celluclast 1.5 l and ?-glucosidase Novozym 188) were adopted in combination with the yeast Saccharomyces cerevisiae AS2.1. The effects of reaction temperature, substrate concentration, initial fermentation liquid pH value and enzyme loading were evaluated and the SSF conditions were optimized. The ranking, from high to low, of influential extent of the SSF affecting factors to ethanol concentration and yield was substrate concentration, enzyme loading, initial fermentation liquid pH value and reaction temperature, respectively. The optimal SSF conditions were: reaction temperature, 35°C; substrate concentration, 100 g·L^-1; initial fermentation liquid pH, 5.0; enzyme loading, 30 FPU·g^-1. Under these conditions, the ethanol concentration increased with reaction time, and after 72 h, ethanol was obtained in 65.8% yield with a concentration of 22.7 g·L^-1.     

直接降解稻草菌种筛选及降解稻草过程优化

以稻草降解率为指标,研究了从稻草等筛选到的菌株对稻草降解的活性。对最优菌株进行形态和18S rDNA基因序列分析表明,该菌株为烟曲霉(Aspergillus fumigatus)。进一步优化了菌株XC6稻草降解的过程参数,结果表明,最优参数条件下,该菌株可以实现对稻草的完全降解[降解率为(100±0.2)%];同时生物量也高达(22.9±1.1)g/L。为今后这方面进一步发酵放大研究打下了基础。     

纤维素水解液中木糖发酵制丁醇

对实验室菌种进行筛选后,得到一株能利用纤维素水解液木糖发酵生产丁醇的菌株。研究发现,该菌株不仅能利用水解液中的葡萄糖,还可以利用水解液中的木糖。对菌种生长特性探索,批式发酵中碳源、氮源以及CaCO3等条件优化后,得到最佳种子培养时间为20～24 h,并确定了木糖浓度为20 g/L的纤维素水解液用于15 L发酵罐实验,在37 ℃静置培养84 h,丁醇产量10.95 g/L,总溶剂16.78 g/L（丙酮、乙醇、丁醇三者之和）,木糖利用率达到70%以上,总溶剂转化率为39.4%。解决了纤维素水解液中木糖不能被利用而造成的经济损失问题。     

乙酸预浸对玉米秸秆蒸汽爆破预处理的影响

对经乙酸预浸汽爆预处理的玉米秸秆进行了组分含量和抑制物分析,并研究了玉米秸秆预处理后的酶水解性和同步糖化发酵。与未经乙酸预浸相比,乙酸预浸玉米秸秆能在相对低温下进行汽爆预处理,在提高半纤维素水解程度的同时,并不会明显增加糠醛等发酵抑制物。酶水解实验表明,玉米秸秆经乙酸预浸,再以 200 ℃ 进行汽爆后的酶水解效果较好,每克原料可获得 284 mg 葡萄糖,提高了 10.2 %,为理论值的 76.8 %;乙酸预浸玉米秸秆经过 96 h 同步糖化发酵,获得了 22.5 g/L 的乙醇浓度,为理论值的 72 %;相比未经乙酸预浸的玉米秸秆,提高了11.9个百分点。