嵌合纤维小体酵母菌群同步糖化发酵木质纤维素产乙醇的研究

为探究CBP酿酒酵母菌群表面展示嵌合纤维小体的协同作用,该文通过组装纤维素酶和半纤维素酶进行木质纤维素乙醇发酵的性能,构建由分别可表面展示纤维素酶嵌合纤维小体(由支架蛋白ScafI,内切葡聚糖酶、外切葡聚糖酶和β-葡糖苷酶构成)和半纤维素酶嵌合纤维小体(由支架蛋白ScafII,内切木聚糖酶和外切木糖苷酶构成)的重组酿酒酵母菌EBY100株组成的CBP菌群。通过蛋白质的SDS-PAGE、Western Bolt验证、酶活性测定、细胞表面定位和纤维小体组装效率分析,证明了融合蛋白能够以活性形式组装成2种形式的嵌合纤维小体。以蒸汽爆破玉米秸秆为底物进行同步糖化发酵产乙醇,96 h时最大乙醇产量达到0.673 g/L,消耗每克碳水化合物的乙醇产量0.255 g,相当于乙醇产率理论值的50.1%,协同因子为1.06。结果证明两株酵母细胞表面的不同类型嵌合纤维小体在水解木质纤维素过程中存在明显的协同促进作用。     

自组装嵌合纤维小体酿酒酵母菌群的乙醇发酵研究

在可共代谢葡萄糖木糖的二倍体酿酒酵母Y6细胞表面,通过组成型强启动子(PGK)控制的a凝集素表面展示系统,构建一个可共代谢纤维素半纤维素的双支架纤维小体。通过免疫荧光、流式细胞术及酶催化活性的测定,证明纤维小体结构能正确组装并以活性形式固定在细胞表面。以纤维素和半纤维素为底物进行乙醇发酵实验,60 h时最大乙醇产量达1.98 g/L,乙醇产率为0.33 g/g,相当于理论值的65%。结果证明,表面展示嵌合纤维小体的酿酒酵母可实现共降解纤维素和半纤维素生产乙醇的目标。     

玉米秸秆同步糖化发酵产乙醇优化实验研究

以耐高温酿酒酵母为对象,研究高温酿酒酵母利用玉米秸秆为原料同步糖化产氢乙醇的影响因素,采用Plackett-Burman(PB)法对影响乙醇产率的5个因素进行筛选,采用CCD模型及响应面分析对影响因素进行优化设计,确定高温酿酒酵母利用玉米秸秆为原料同步糖化产氢乙醇的最佳工艺条件。研究结果表明:高温酿酒酵母利用玉米秸秆酶解液同步糖化产乙醇过程中接种量、温度、酶浓度和发酵时间4个因素对乙醇产量的影响最为明显,温度和发酵时间的交互作用最为显著。利用优化设计得到的最佳产乙醇工艺为:接种量7.4%、温度34.2℃、初始p H值5.0、酶浓度49.36 U/g。在此条件下,发酵105.12 h后乙醇产率可达59.88%,实验结果与响应面拟合方程的预测值(60.85%)吻合良好。     

稀硫酸-亚硫酸盐法预处理枯死松木的原位纤维素乙醇生产

报道了稀硫酸-亚硫酸盐法预处理软木的纤维素乙醇生产效率。通过采用一株耐受代谢抑制剂的酿酒酵母菌株Y5,对预处理后的固体酶解物及水解液的混合物进行批式及补料批式同步糖化发酵的研究。结果表明:预处理软木混合物中的所有可发酵糖能够在72h内被Y5全部利用,最后的乙醇浓度达到22.2g/L,相应的乙醇发酵产率为0.44g/g。批式与补料批式SSF,除了补料批式发酵24h内的乙醇生产速率高于批式发酵外,并没有其他明显区别。     

生物统合加工嵌合纤维小体组装模块反应机制探究

为探究生物统合加工嵌合纤维小体中重组蛋白粘连模块与对接模块的分子相互作用及其对嵌合纤维小体组装效率的影响,采用酿酒酵母细胞分泌表达支架蛋白和酶分子催化模块,在胞外通过非变性蛋白凝胶电泳和等温滴定量热法测定分析二级支架蛋白、纤维素酶分别与一级支架蛋白结合时的相互作用反应。结果显示,分别连接二级支架蛋白和纤维素酶蛋白的对接模块与粘连模块的亲和力常数增大,亲和力减小,组装反应为焓变驱动的放热反应并产生氢键,证明这些蛋白分子间亲和力减小是导致二级支架蛋白与一级支架蛋白组装效率较低的主要影响因素。     

汽爆玉米秸秆产乙醇的补料同步糖化发酵优化

为提高反应系统中的底物浓度,减少酶用量,以蒸汽爆破预处理的玉米秸秆及高性能的酵母菌株Y5进行补料同步糖化发酵研究。通过同步糖化发酵策略的优化,经两次补料使反应体系底物浓度提高至25.7%,同步糖化发酵72h,即可获得41.7g/L的乙醇浓度,综合转化率64%。同时,纤维素酶及β-葡萄糖苷酶用量分别可降至7FPU/(g纤维素)及7IU/(g纤维素)。     

混合固态发酵降解甘蔗渣产糖产乙醇研究

以碱处理甘蔗渣为原料,比较里氏木霉(Trichoderma reesei CICC40359)和斜卧青霉(Penicillium decumbens LSM-1)单菌和混菌固态发酵及转化乙醇效果,研究发酵过程菌体生长、产酶产糖和乙醇转化情况。结果表明:混菌发酵效果优于单菌,在接种量8%,发酵温度30℃,混菌固态发酵(SSF)72h后总糖和还原糖产量最大值为20.21 g/L和12.47 g/L;β-葡萄糖苷酶活力和菌体生物量在144 h后分别达到0.48 IU/m L和0.21 g/g DM;对发酵3 d后底物(包括生成糖、合成酶及未降解基质)接种酵母进行乙醇同步糖化发酵,乙醇浓度在发酵24 h时达到5.83 g/L,发酵效率达到理论值的40.84%。利用多菌混合固态发酵转化底物产乙醇能避免传统乙醇生产过程高成本纤维素酶的应用,为纤维乙醇生产提供一条经济有效的新途径。     

双菌比较优化稻草“液化”发酵产乙醇的研究

利用稻草液化产物为底物,分别采用酿酒酵母和休哈塔假丝酵母发酵生产乙醇,对影响发酵阶段的各因素进行优化,选取最佳菌种完成秸秆到乙醇的转化。结果表明,液化产物经酶解后葡萄糖浓度可达69.5mg/mL,是发酵制备乙醇的良好底物。优化发酵后,酿酒酵母更适合做液化产物的发酵菌种。适宜的发酵条件:初始葡萄糖浓度60~65 mg/mL,温度30℃,pH=6.0,装液量80 mL,接种量10%,发酵时间36 h,在此条件下乙醇得率可达49.3%,能达到理论得率的96.1%,转化率最高为0.27 g/g(乙醇/液化产物)。     

玉米秸秆酶解糖化液乙醇发酵的工艺优化

利用从4组混合乙醇酵母中筛选出的优势混合酵母,对玉米秸秆酶解糖化液的乙醇发酵工艺过程进行了优化试验。试验结果表明,管囊酵母和酿酒酵母组成的混合酵母具有较高的乙醇发酵能力,经60 h发酵,乙醇浓度最高可达12.55 g/L,乙醇产率为最大理论值的68.63%。根据对糖化液乙醇发酵的二次回归正交组合优化试验,当发酵温度为28.0℃,初始pH为5.2,接种量为8.1%时,实际乙醇浓度最高可达13.03g/L,乙醇产率为0.36 g/g,为最大理论值的70.59%,与所得乙醇发酵回归方程预测值基本相符。     

未脱毒汽爆玉米秸秆高底物浓度的同步糖化和乙醇发酵

为取消蒸汽爆破预处理玉米秸秆水洗脱毒步骤和提高乙醇发酵的乙醇浓度,利用专利菌株酿酒酵母Y5,对蒸汽爆破预处理玉米秸秆不经脱毒处理,直接进行同步糖化和发酵(SSF)。蒸汽爆破玉米秸秆的浓度30%,同步糖化和发酵的时间96 h,100 m L、3000 m L反应器和5L发酵罐的乙醇浓度分别达到50.0、47.8、47.5 g/L。研究结果表明,菌株Y5在纤维素乙醇生产中对简化生产工艺、降低设备投资、减少水消耗、降低生产成本具有重要的应用前景。     

Kinetics of batch production of ethanol from cheese whey

A kinetic model for ethanol fermentation was developed. The model accounted for substrate limitation, substrate inhibition, ethanol inhibition and cell death and performed satisfactorily for predicting the transient responses of cell growth, ethanol production and substrate utilization during the batch fermentation process of cheese whey (R² = 0.96 to 0.99). The maximum specific growth rate (μ_m), the saturation constant (K₅), the ethanol inhibition constant and the substrate inhibition constant were found to be 0.051 h⁻¹, 1.9 g/l, 20.65 g/l and 112.51 g/l, respectively. The maximum ethanol concentration above which Candida pseudotropicalis does not grow was found to be 100 g/l. The maximum ethanol production occurred at about 150 g/l initial substrate concentration after about 62 h. High initial substrate concentrations reduced both the specific growth rate and the substrate utilization rate due to the substrate inhibition phenomenon.     

改进的柳枝稷预处理方法及乙醇发酵研究

为了提高柳枝稷中纤维素和半纤维素糖的转化率,降低水解液中抑制剂的浓度,首先,用稀酸在温和条件下对柳枝稷进行水解,然后用碱对酸水解后的固体物进行预处理,接着用纤维素酶酶解并分别对稀酸水解液和酶解液进行乙醇发酵.结果表明:纤维素转化率达到94.26%,半纤维素转化率为60.93%,稀酸水解液乙醇发酵的乙醇产率为0.441g乙醇/g糖,达到最高理论值的86.47%.酶解液乙醇发酵的乙醇产率为0.486g乙醇/g葡萄糖,达到最高理论值的95.29%.     

Fermentative biohydrogen production by a new chemoheterotrophic bacterium Citrobacter sp. Y19

A newly isolated Citrobacter sp. Y19 for CO-dependent H₂ production was studied for its capability of fermentative H₂ production in batch cultivation. When glucose was used as carbon source, the pH of the culture medium significantly decreased as fermentation proceeded and H₂ production was seriously inhibited. The use of fortified phosphate at 60–180 mM alleviated this inhibition. By increasing culture temperatures (25–36°C), faster cell growth and higher initial H₂ production rates were observed but final H₂ production and yield were almost constant irrespective of temperature. Optimal specific H₂ production activity was observed at 36°C and pH 6–7. The increase of glucose concentration (1–20 g/l) in the culture medium resulted in higher H₂ production, but the yield of H₂ production (mol H₂/mol glucose) gradually decreased with increasing glucose concentration. Carbon mass balance showed that, in addition to cell mass, ethanol, acetate and CO₂ were the major fermentation products and comprised more than 70% of the carbon consumed. The maximal H₂ yield and H₂ production rate were estimated to be 2.49 molH₂/mol glucose and 32.3 mmolH₂/gcellh, respectively. The overall performance of Y19 in fermentative H₂ production is quite similar to that of most H₂-producing bacteria previously studied, especially to that of Rhodopseudomonas palustris P4, and this indicates that the attempt to find an outstanding bacterial strain for fermentative H₂ production might be very difficult if not impossible.     

Pretreatment and hydrolysis of cellulosic agricultural wastes with a cellulase-producing Streptomyces for bioethanol production

Production of reducing sugar by hydrolysis of corncob material with Streptomyces sp. cellulase and ethanol fermentation of cellulosic hydrolysate was investigated. Cultures of Streptomyces sp. T3-1 improved reducing sugar yields with the production of CMCase, Avicelase and ??-glucosidase activity of 3.8, 3.9 and 3.8 IU/ml, respectively. CMCase, Avicelase, and ??-glucosidase produced by the Streptomyces sp. T3-1 favored the conversion of cellulose to glucose. It was recognized that the synergistic interaction of endoglucanase, exoglucanase and ??-glucosidase resulted in efficient hydrolysis of cellulosic substrate. After 5 d of incubation, the overall reducing sugar yield reached 53.1 g/100 g dried substrate. Further fermentation of cellulosic hydrolysate containing 40.5 g/l glucose was performed using Saccharomyces cerevisiae BCRC 21812, 14.6 g/l biomass and 24.6 g/l ethanol was obtained within 3 d. The results have significant implications and future applications regarding to production of fuel ethanol from agricultural cellulosic waste.     

Pretreatment of cassava stems and peelings by thermohydrolysis to enhance hydrolysis yield of cellulose in bioethanol production process

The potential of wastes obtained from the cultivation of Manihot esculenta Crantz as raw material for bioethanol production was studied. The objective was to determine the optimal conditions of hemicellulose thermohydrolysis of cassava stems and peelings and evaluate their impact on the enzymatic hydrolysis yield of cellulose. An experimental design was conducted to model the influence of factors on the pentose, reducing sugar and phenolic compound contents. Residues obtained from the optimal pretreatment conditions were hydrolysed with cellulase (filter paper activity 40 FPU/g). The hydrolysates from pretreatment and enzymatic hydrolysis were fermented respectively using Rhyzopus spp. and Sacharomyces cerevisiae. The yield of enzymatic hydrolysis obtained under the optimal conditions were respectively 73.1% and 86.6% for stems and peelings resulting in an increase of 39.84% and 55.40% respectively as compared to the non-treated substrates. The ethanol concentrations obtained after fermentation of enzymatic hydrolysates were 1.3 and 1.2 g/L respectively for the stem and peeling hydrolysates. The pentose and phenolic compound concentrations obtained from the multi-response optimization were 10.2 g/L; 0.8 g/L and 10.1 g/L; 1.3 g/L respectively for stems and peelings. The hydrolysates of stems and peelings under these optimal conditions respectively gave ethanol concentrations of 5.27 g/100 g for cassava stems and 2.6 g/100 g for cassava peelings.     

Formation of ethanol from carbon monoxide via a new microbial catalyst

A recently discovered clostridial bacteria converts components of synthesis gas (CO, CO₂, H₂) into liquid products such as ethanol, butanol and acetic acid. Isolated from an agricultural lagoon, the stability and productivity characteristics of the bacteria were studied in a continuous 4.5 l bubble column bioreactor at 37°C using artificial blends of CO, CO₂, and N₂. Preliminary results on the rates of cell growth, substrate utilization, product formation, and yields of products and cells from CO are discussed. At steady state, apparent yields (mole C in products per mole CO consumed) of ethanol, butanol, and acetic acid were 0.15, 0.075 and 0.025, respectively, and the cell yield was 0.25 g/mol CO. The theoretical yield of ethanol is 0.33 if CO is only utilized for the production of ethanol. The experimental yield of CO₂ from CO was approximately 60% compared to the theoretical yield of 67% with ethanol as the sole product. As a comparison with another ethanol-producing bacteria, the results of a similar fermentation study using batch-grown Clostridium ljungdahlii showed yields of 0.062 for ethanol and 0.094 for acetic acid and a cell yield of 1.378 g/mol.     

Utilization of excess corn kernels for hydrogen gas biofuel production

Corn kernels are good candidates for production of various value-added products such as gas biofuel, hydrogen due to the carbohydrate-rich composition. In this study, widely grown corn, field corn kernels were dissolved in subcritical water at different temperatures to determine optimal thermal hydrolysis condition. Organic-rich hydrolysate obtained from hydrolysis process was gasified by aqueous-phase reforming (APR) for hydrogen gas production.Since hydrolysis at 200 °C resulted in significantly more total organic carbon release than other temperatures and the lowest amount of insolubilized solid residue. Different concentrations of this hydrolysate (diluted with water at different ratios) were evaluated for high yielding hydrogen gas production. Gasification performance of corn kernels was also compared with lignocellulosic biomass using corn stover as a representative biomass material.The hydrolysate with 2486 mg/L TOC concentration showed the best performance for hydrogen gas production (130 mL H₂/g corn) and left less amount of ungasified solid residue. Corn kernels produced 2.3 times more hydrogen gas compared to corn stover biomass. Thus, corn kernels are promising feed materials for APR process, and excess production of corn can be utilized for hydrogen gas production in higher yield and richer composition.     

Ethanol production process from banana fruit and its lignocellulosic residues: Energy analysis

Tropical countries, such as Brazil and Colombia, have the possibility of using agricultural lands for growing biomass to produce bio-fuels such as biodiesel and ethanol. This study applies an energy analysis to the production process of anhydrous ethanol obtained from the hydrolysis of starch and cellulosic and hemicellulosic material present in the banana fruit and its residual biomass. Four different production routes were analyzed: acid hydrolysis of amylaceous material (banana pulp and banana fruit) and enzymatic hydrolysis of lignocellulosic material (flower stalk and banana skin). The analysis considered banana plant cultivation, feedstock transport, hydrolysis, fermentation, distillation, dehydration, residue treatment and utility plant. The best indexes were obtained for amylaceous material for which mass performance varied from 346.5 L/t to 388.7 L/t, Net Energy Value (NEV) ranged from 9.86 MJ/L to 9.94 MJ/L and the energy ratio was 1.9 MJ/MJ. For lignocellulosic materials, the figures were less favorable; mass performance varied from 86.1 to 123.5 L/t, NEV from 5.24 to 8.79 MJ/L and energy ratio from 1.3 to 1.6 MJ/MJ. The analysis showed, however, that both processes can be considered energetically feasible.     

A limited LCA comparing large- and small-scale production of rape methyl ester (RME) under Swedish conditions

Production of rape methyl ester (RME) can be carried out with different systems solutions, in which the choice of system is usually related to the scale of the production. The purpose of this study was to analyse whether the use of a small-scale RME production system reduced the environmental load in comparison to a medium- and a large-scale system. To fulfil this purpose, a limited LCA, including air-emissions and energy requirements, was carried out for the three plant sizes. For small plants and physical allocation, the global warming potential was 40.3 g CO₂-eq/MJ_fuel, the acidification potential 236 mg SO₂-eq/MJ_fuel, the eutrophication potential 39.1 mg PO₄³⁻-eq/MJ_fuel, the photochemical oxidant creation potential 3.29 mg C₂H₄-eq/MJ_fuel and the energy requirement 295 kJ/MJ_fuel. It was shown that the differences in environmental impact and energy requirement between small-, medium- and large-scale systems were small or even negligible. The higher oil extraction efficiency and the more efficient use of machinery and buildings in the large-scale system were, to a certain degree, outweighed by the longer transport distances. The dominating production step was the cultivation, in which production of fertilisers, soil emissions and tractive power made major contributions to the environmental load. The results were, however, largely dependent on the method used for allocation of the environmental burden between the RME and the by-products meal and glycerine. This indicates that when different biofuels or production strategies are to be compared, it is important that the results are calculated with the same allocation strategies and system limitations.