Fe3O4纳米颗粒固定化纤维素酶的酶学特性研究

试验研究了超顺磁性纳米颗粒直接固定纤维素酶的酶学特性.以化学共沉淀法合成出的纳米Fe3O4颗粒为载体,利用水溶性碳化二亚胺(EDC)活化将纤维素酶固定,探讨了不同酶量、交联剂、pH值等因素对固定化纤维素酶性能的影响,得出固定纤维素酶的最佳条件:酶浓度为9.0 mg/mL,EDC浓度为3.0 mg/mL,pH值为4.0.试验还研究了在不同温度和pH值条件下的固定化酶与游离酶的活性,酶固定后最佳水解温度为60℃,最适pH值向碱性迁移且范围有所增加.     

响应面法优化蔗渣酶解工艺研究

利用Minitab软件对甘蔗渣酶解工艺条件进行优化研究。采用Plackett-Burman设计,从温度、pH值、加酶量、液固比和转速5个影响因素中,筛选出具有显著效应的温度、pH值和加酶量3个主要影响因素。然后采用响应面法对3个主要影响因素做进一步优化研究。优化结果表明:温度47.6℃,p H=4.76和加酶量37.76 Fpu/g时,综纤维素转化率可达74.5%。该条件下,实际试验值为74.44%,与预测值吻合较好。     

酶法酯交换棉籽毛油合成生物柴油

以戊二醛交联壳聚糖固定A.niger Li-38脂肪酶,固定条件为0.015%的戊二醛,交联50min。以该固定化酶催化棉籽毛油合成生物柴油,反应条件为:以叔丁醇为反应介质,醇油摩尔比1:1,水分含量0.6%,反应温度55℃,酶载量为8%,分批补加甲醇,反应28h后最终转化率达到89.1%。该固定化酶的贮藏稳定性较好,室温放置12d,酶活保持80%以上,30d时酶活为46.8%。固定化酶在30～70℃,pH值5.5～6.5之间较稳定,其热稳定性和pH稳定性较游离酶有所提高。固定化酶可重复使用7次,转化率保持在80%以上。     

膨化玉米秸秆酶解糖化条件优化的研究

采用二次回归正交组合试验设计,以温度、pH值、底物浓度、加酶量和酶解糖化时间5个因素为变量,研究各因素在不同试验水平下对膨化玉米秸秆酶解糖化效率的影响,并对试验条件进行优化组合。结果表明,温度为50℃,pH值为4.8,底物浓度为95g/L,加酶量为40U/g底物,酶解糖化时间为60h,膨化玉米秸秆酶解效果最佳,该条件下还原糖含量达到28.61%。     

中性脂肪酶在凹凸棒石表面固定条件优化及其活性

以凹凸棒石为载体,采用吸附法,对脂肪酶进行固定化,研究了固定化条件对固定化脂肪酶催化活性的影响,得到最佳固定化条件:给酶量为25900U/g,固定化温度为25℃,pH值为7.5,吸附时间为5h,此时固定化酶的活力约为2800U/g载体。固定化酶的最适反应温度从游离酶的40℃下降至30℃,固定化酶的最适pH从游离酶的7.0上升到7.5。固定化酶的热稳定性和pH稳定性较游离酶有了较大改善,其在60℃以下能保持75%以上的酶活,而游离酶60℃残余酶活仅为20%。在pH为5～9的范围内,固定化酶的酶活能保持70%以上,而游离酶只能保持约20%。用固定化的中性脂肪酶催化不同的油品,即大豆油、菜籽油及泔水油生产生物柴油,菜籽油的酯化率最高。     

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。     

酸碱预处理后玉米秸秆的酶解工艺优化

对玉米秸秆进行酸碱预处理,在单因素实验基础上,以还原糖转化率为影响值设计正交实验,研究温度、pH值、液固比、酶浓度及酶解时间5因素对纤维素酶解过程的影响。得出玉米秸秆酶解最佳工艺条件为:温度48℃、pH值4.6、液固比20、酶浓度55 U/g;酶解时间44 h。在此工艺条件下还原糖转化率达到80.97%。结合红外光谱对秸秆中各组分特征基团分析表明,膨化后的玉米秸秆酶解的纤维素基团特征峰变化更为明显。     

响应面法优化海带酸水解预处理工艺

采用响应面法优化酸水解海带预处理的工艺条件。在对硫酸浓度、水解时间、底物浓度、水解温度4个因素进行单因素试验基础上,以还原糖得率为响应值,以Box-Behnken中心组合设计方法,建立预处理工艺参数的回归模型,利用软件Design-Expert 7.1.6作响应曲面分析,得出海带预处理理论最佳工艺参数;对理论参数进行了平行试验验证及校正,得到最优工艺参数:酸浓度为0.48 mol/L,底物浓度为8%,水解时间为43 min,水解温度为120℃,在此条件下还原糖得率为25.47%。通过验证可知,建立的数学模型能够较好地预测试验结果。     

核桃内生细菌Bacillus subtilis HB1310发酵棉秆水解糖液产油工艺优化

为实现核桃内生产油细菌Bacillus subtilis HB1310发酵棉秆水解糖液高效生产微生物油脂,检测了糖液浓度、氮源浓度、发酵起始p H值以及发酵时间4个主要因素对该菌株发酵棉秆水解糖液产油率的影响。通过单因素试验确定了响应面试验中各因素的水平中心点。通过响应面试验确定了菌株发酵棉秆水解糖液产油的优化工艺参数为糖液浓度7%(w/v)、氮源浓度5 g/L、发酵起始p H值6.0、发酵时间2 d,在此优化工艺条件下进行验证试验,获得的产油率可达30.26%±1.05%(w/w),与模型的理论预测值较接近,说明建立的模型是切实可行的。     

小麦秸秆转化为可发酵糖的研究

对小麦秸秆水解转化为可发酵糖进行了研究，考察了小麦秸秆预处理方法以及温度、pH值、酶用量、底物浓度和反应时间等因素对小麦秸秆酶水解的影响。试验结果表明，汽蒸加蒽醌方法是较好的预处理方法。酶解最佳工艺为：温度48℃，pH值5．2，酶解时间24h，酶用量与底物的最佳配比0．160：1；底物浓度≥1％，以1．5％～2．5％为宜，此时还原糖得率达32．4％。     

Production and optimization of high grade cellulase from waste date seeds by Cellulomonas uda NCIM 2353 for biohydrogen production

Production of high grade cellulolytic enzymes from waste agricultural biomass would valorise these wastes to valuable products as well as avoid the pollution problems associated with landfilling of the biomass. In the present study, waste date palm (Phoenix dactylifera) seeds were valorised for cellulase production from Cellulomonas uda NCIM 2353 and for its subsequent usage in biohydrogen production. Optimization of key operational parameters such as date seed concentration, xylose, casein and initial media pH were performed using central composite design to obtain the maximum enzyme yield. The optimum values obtained were (g/L): date seed concentration 30.65, xylose concentration 0.55, casein 7.00 and pH 7.40 for a determination coefficient of 0.999. The results demonstrated a higher prediction accuracy of response surface methodology as the cellulase activity increased six fold (175.96 IU/mL) after optimization. The optimum pH and temperature of purified cellulase was 7 and 50 °C respectively where the enzyme retained nearly 80% of activity upto 180 min. Enzymatic hydrolysis studies showed that a high saccharification efficiency of 60.5% was obtained for acid pretreated sugarcane bagasse by the indigenous cellulase, equivalent to the performance of commercial cellulase. Further, the as-obtained reducing sugars were decomposed by Clostridium thermocellum to produce biohydrogen of maximum concentration 187.44 mmol/L at end of 24 h of fermentation. Results show that date seed substrate based cellulase protein can be employed for industrial processes of biohydrogen production.     

Immobilization of Saccharomyces cerevisiae to modified bagasse for ethanol production

Ethanol production was carried out by growing yeast cells immobilized on bagasse carriers. The effects of cellulase hydrolysis of carriers on cell immobilization and ethanol production were investigated. The electrical conductivity of carriers after three days cellulase hydrolysis was 3.7 times higher than that of basic bagasse carrier and the immobilized cell concentration of modified carriers was 1.46 times higher than that of unmodified ones. The scanning electron microscopy (SEM) of the carriers indicated that cellulase hydrolysis disrupted the sleek surface into rough and porous, which promoted the mass transfer and resulted in high specific ethanol production rate. Cellulase hydrolysis of carriers has positive effects on both of cell immobilization and ethanol productivity.     

Optimization of key variables for the enhanced production of hydrogen by Ethanoligenens harbinense W1 using response surface methodology

The optimization of process conditions for the production of hydrogen by Ethanoligenens harbinense W1 was investigated using response surface methodology (RSM). Three parameters namely inoculum to substrate ratio, initial pH value and temperature were chosen as variables. The adequately high R² value (99.4%) indicated the statistical significance of the model. The optimum process conditions for hydrogen production rate were determined by analyzing the response surface three-dimension surface plot and contour plot and by solving the regression model equation with Design Expert software. The central composite design (CCD) was used to optimize the process conditions, which showed that an inoculum to substrate ratio of 14%, initial pH value of 4.32 and the experimental temperature of 34.97 °C were the best conditions. Under the optimized conditions, the maximum specific hydrogen production rate (SHPR) was 35.74 mL/g-CDW.h based on cell dry weight. The results were further verified by triplicate experiments. The batch reactors were operated under an optimized condition of the inoculum to substrate ratio of 14%, initial pH value of 4.3 and the experimental temperature of 35 °C. The maximum SHPR was estimated at 35.57 mL/g-CDW.h, which further verified the practicability of this optimum strategy.     

Biodiesel production from Calophyllum inophyllum oil using lipase producing Rhizopus oryzae cells immobilized within reticulated foams

Biodiesel production from non-edible Calophyllum inophyllum linn oil with high levels of Free Fatty Acid (FFA) (acid value −6.732 mg KOH/g of oil) was investigated using whole-cell biocatalysts. Rhizopus oryzae cells immobilized within reticulated polyurethane foams were used as biocatalysts for biodiesel production. The effects of reaction parameters such as methanol-to-oil molar ratio, water content, and temperature for the production of biodiesel through methanolysis in a packed-bed reactor (PBR) were studied. Molar ratio of methanol-to-oil – 12:1, water content – 15%v/v, cell concentration – 20% and temperature 35 °C were found to be the optimum. The yield of biodiesel obtained in batch methanolysis from C. inophyllum oil under optimized condition was 92%. Long-term stability of immobilized cells for methanolysis was verified using re-usability studies.     

Covalent immobilization of enzymes and yeast: Towards a continuous simultaneous saccharification and fermentation process for cellulosic ethanol

The immobilization of enzymes and yeast cells is a key factor for establishing a continuous process of cellulosic ethanol production, which can combine the benefits of a separated hydrolysis and fermentation process and a simultaneous saccharification and fermentation process. This paper investigates the use of cellulase enzyme and yeast cell immobilization under a flow regime of ethanol production from soluble substrates such as cellobiose and carboxymethyl cellulose. The immobilization was achieved by incubating enzymes and yeast cells on polystyrene surfaces which had been treated by nitrogen ion implantation. The saccharification by immobilized enzymes and the fermentation by immobilized yeast cells were conducted in two separate vessels connected by a pump. During the experiments, glucose concentrations were always maintained at low levels which potentially reduce product inhibition effects on the enzymes. Covalent immobilization of enzymes and yeast cells on the plasma treated polymer reduces loss by shear flow induced detachment. The potential for continuous flow production of ethanol and the influence of daughter yeast cells in the circulating flow on the immobilized enzyme activity are discussed.     

Effect of doping pretreated corn stover conditions on yield of bioethanol in immobilized cell systems

The surface characteristics of immobilized yeast before and after adding CO₂-laser pretreated corn stover (LPCS) substrates were investigated using bioethanol production. Response surface methodology (RSM), based on the Box–Behnken design (BBD) for experiments, was used to optimize the doping condition. An optimum experimental condition was obtained at pH 4.5, 2.08% yeast concentration, and 0.20% LPCS substrates. Under this condition, doping LPCS increased the yield of bioethanol from 53% to 84%, which matched the predicted value. After doping LPCS, the results of inverted microscope (IM) and atomic force microscopy (AFM) illustrated that the immobilized gel beads changed from rod-like in shape with a smooth surface to a larger rod-like ultrastructure with a rougher surface. The yield was relatively stable within 28 d, with a downward trend subsequently appearing.     

Alkali-stable cellulase from a halophilic isolate,Gracilibacillus sp. SK1 and its application in lignocellulosic saccharification for ethanol production

A halophilic strain SK1 showing cellulolytic activity was isolated from Yuncheng Salt Lake, and was identified as the genus of Gracilibacillus by 16S rRNA gene sequence analysis. Cellulase production was strongly influenced by the salinity of culture medium with maximal level in the presence of 10% NaCl. Substrate specificity test indicated the crude cellulase was a multi-component enzyme system, showing a combined activity of endoglucanase, exoglucanase and β-glucosidase. Zymogram analysis indicated six different endoglucanases were secreted by this strain. The crude enzyme was highly active and stable over broad ranges of temperature (40–70 °C), pH (6.0–10.0) and NaCl concentration (7.5–17.5%), with an optimum at 60 °C, pH 8.0 and 12.5% NaCl, which showed excellent thermostable, alkali-stable and halostable properties. Moreover, it displayed high stability in the presence of hydrophobic organic solvents. Saccharification of corn stover and rice straw by the cellulase resulted in respective yields of 0.678 and 0.502 g g⁻¹ dry substrate of reducing sugars. The enzymatic hydrolysates of corn stover were then used as the substrate for ethanol production by Saccharomyces cerevisiae. The yield of ethanol was 0.186 g g⁻¹ dry substrate, and the efficiency of reducing sugars conversion to ethanol was about 52.8%, which suggested the prospects of the crude enzyme from Gracilibacillus sp. SK1 in application for bio-ethanol production.     

The preparation and application of crude cellulase for cellulose-hydrogen production by anaerobic fermentation

Strategies were adopted to cost-efficiently produce cellulose-hydrogen by anaerobic fermentation in this paper. First, cellulase used for hydrolyzing cellulose was prepared by solid-state fermentation (SSF) on cheap biomass from Trichoderma viride. Several cultural conditions for cellulase production on cheap biomass such as moisture content, inoculum size and culture time were studied. And the components of solid-state medium were optimized using statistical methods to further improve cellulase capability. Second, the crude cellulase was applied to cellulose-hydrogen process directly. The maximal hydrogen yield of 122 ml/g-TVS was obtained at the substrate concentration of 20 g/L and cultured time of 53 h. The value was about 45-fold than that of raw corn stalk wastes. The hydrogen content in the biogas was 44–57%(v/v) and there was no significant methane gas observed.     

Enzymatic transesterification of soybean oil by using immobilized lipase on magnetic nano-particles

Lipase was covalently immobilized onto magnetic Fe₃O₄ nano-particles by using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) as an activating agent, and the bound lipase was used to catalyze the transesterification of vegetable oils with methanol to produce fatty acid methyl esters. The binding of lipase to magnetic particles was confirmed by enzyme assays, transmission electron microscopy (TEM) and Fourier transform infrared (FT-IR) spectra. It was determined that the immobilized lipase exhibited better resistance to temperature and pH inactivation in comparison to free lipase. Using the immobilized lipase, the major parameters affecting the transesterification reaction, such as the alcohol/oil molar ratio, enzyme loading and free fatty acid present in reactants were investigated to obtain the optimum reaction condition. The conversion of soybean oil to methyl esters reached over 90% in the three-step transesterification when 40% immobilized lipase was used. Moreover, the lipase catalyst could be used for 3 times without significant decrease of the activity.     

Enzymatic depolymerization of Ricinus communis, a potential lignocellulosic for improved saccharification

Residual lignocellulosics left to decay in fields and forest has a huge potential to serve as a low cost feedstock for production of bioethanol. In Indian subcontinent Ricinus communis is a major lignocellulosics growing in arid conditions containing 42% cellulose and 19.8% lignin. In the present study, Response Surface Methodology (RSM) based on Central Composite Design (CCD) has been used to explore the effects of pH, temperature, solid to liquid ratio (w/v), enzyme concentration and incubation time on enzymatic depolymerization of R. communis. The maximum delignification obtained was 85.69%. In case of lignified R. communis the optimum reducing sugar produced was about 288.83 mg/g dry substrate, whereas, in case of delignified R. communis the optimum reducing sugar produced was about 775.17 mg/g dry delignified substrate. After delignification reducing sugar yield was increased to about 2.68 fold.