高活力木聚糖酶菌株的筛选、酶学性质及酶解应用

实验分离鉴定了高产木聚糖酶曲霉菌株,研究其固态发酵产酶条件及酶学性质。经菌落形态观察、ITS基因序列分析菌株在系统分类中的地位。通过单因素固态发酵实验确定其最佳产酶条件。结果表明,高产木聚糖酶曲霉菌株鉴定为黑曲霉（Aspergillus niger）。其最佳产酶条件为：玉米芯与麸皮比例为1∶3、氮源为尿素、初始pH为3.5、料水比为1∶3.5和接种量为10.0%。在此条件下发酵120 h,木聚糖酶酶活最高可达10 446.92 IU/g。酶学性质研究表明,在pH为5.0、温度为45℃条件下木聚糖酶处于最优条件。糠醛（23.0 g /L）和5-羟甲基糠醛（25.7 g/L）对木聚糖酶的激活率分别达到15.9%和18.4%。Aspergillus niger SM751可以作为木聚糖酶潜在的生产菌株用于木质纤维素的酶解领域。     

优化产酶培养基用于稻草发酵产乳酸的研究

对利用稻草为碳源发酵产纤维酶的过程从菌种选择、培养基的选择、碳源的结构、含水率进行了优化。以黑曲霉及里氏木霉的混合菌株为产酶菌种,在稻草粉和麦麸以3:1的比例混合作碳源,培养基中含水率为70%时发酵产出的纤维素酶酶活达到最高,最高的Cx酶活为1568.47U/g,滤纸酶活为489.3U/g。以该条件下产的纤维素酶分别用短乳杆菌和米根霉进行乳酸发酵实验,产乳酸结果为:短乳杆菌:10.8g/L;米根霉:9.2g/L。     

基于响应曲面法的厨余发酵产氢条件优化研究

利用响应曲面法优化厨余发酵产氢条件。以初始pH、物料比和碳氮比为自变量,培养7天的产氢量为因变量,采用Box-Behnken(BB)设计,研究各自变量及其交互作用对厨余发酵产氢效果的影响。以模拟得到的二次多项式回归方程的预测模型为基础,得到物料比为10%、50%和90%,最佳反应条件下的产氢量(VS)分别为35.49,49.12,48.39 ml/g,远高于单因素实验的最高值34.21,46.36,45.21 ml/g,试验结果为厨余发酵产氢技术应用提供了技术参数。     

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。     

绿色木霉的选育及固态发酵产纤维素酶的研究

从纤维素酶产生菌绿色木霉TY-2出发,通过紫外诱变技术选育出1株遗传稳定性良好的高产菌株H-28,其产纤维素酶的滤纸酶活力稳定在2.67 U/g左右,较出发菌株提高58.08%。以麸皮和蔗渣为主要原料对变异株H-28进行固态发酵研究,单因素优化了培养基、培养条件和表面活性剂对菌株H-28发酵产纤维素酶的影响,最后选取影响产酶较大的4个因素:发酵时间、Mandels营养盐液、蛋白胨、吐温-80做4因素3水平正交试验。最终优化后突变菌株H-28的产酶能力最高值为6.79 U/g,是出发菌株的4.07倍。     

牛粪、鸡粪发酵产氢潜力的研究

采用恒温厌氧发酵工艺,用乳酸调控发酵pH值,进行了牛粪和鸡粪发酵产氢的实验研究。实验结果表明,pH为4.7～5.5时,牛粪的产氢潜力为32.33ml/g(TS)和41.39ml/g(VS);鸡粪的产氢潜力为33.58ml/g(TS)和50.88ml/g(VS)。     

肉座菌Z28产纤维素酶工艺研究

采用液体发酵法,对肉座菌(Hypocrea sp.)Z28的最适产纤维素酶条件进行了研究.结果表明:在以稻草粉为碳源,硫酸铵为氮源,接种量2%,发酵时间6d,温度为30℃,pH值6.0,转速为180r/min的条件下,纤维素酶活力最高,其CMCase活力为66.35U/mL,FPA活力为13.75U/mL,β-葡萄糖苷酶活力为14.26U/mL.     

发酵条件对发酵产氢细菌B49产氢的影响

采用间歇发酵实验，研究了葡萄糖浓度、接种量、温度、氮源、不同有机底物对发酵产氢产酸细菌新菌种IM9(AF481148 in EMBL)生物产氢的影响。结果表明，接种量影响IM9的产氢；IM9生长和产氢适宜温度均为35℃；IM9不能利用无机氮源，而有机氮是IM9生长、产氢的适宜氮源；葡萄糖是IM9发酵产氢的最适宜底物，当浓度为10g／L时，IM9的葡萄糖利用率为100％，氢气得率为1．69molH2／mol glucose；此外，IM9可利用小麦、大豆、玉米、土豆及糖蜜废水和啤酒废水产氢，其中利用糖蜜废水、啤酒废水产氢分别为137．9ml H2／g COD和49．9ml H2／g COD。     

猪粪发酵产氢潜力的研究

采用批量发酵工艺，以解猪粪为原料，进行了厌氧发酵产氢的研究，发酵料液pH值控制在5．0左右，实验结果表明，鲜猪粪的产氢潜力为127ml/g(TS)和158ml/g(VS).     

餐厨废弃物糖化发酵炼制燃料乙醇工艺优化

利用高效液化酶(Liquozyme)和糖化酶(AMG)对餐厨废弃物进行液化糖化,优化了酶组成、加酶量、pH、操作温度、操作时间、酵母接种量等参数,建立了由餐厨废弃物炼制燃料乙醇的最佳工艺。结果表明:通过液化酶和糖化酶复配,可降低原料粘度、提高传质效率,使淀粉类多糖快速转化为可发酵性单糖;液化酶最优作用条件为85℃,pH值5.1~5.2,加酶量为0.75 U/g(干基),液化时间为40 min;糖化酶最优作用条件为45℃,pH值5.0,加酶量0.5 U/g(干基),糖化时间为30 min;最佳的发酵条件是酵母接种量3 g/L,发酵时间20 h,所得乙醇浓度为54 g/L,相当于0.438 g/g(乙醇/葡萄糖),达到理论产量的86%。     

Hydrolysis of pretreated rice straw by an enzyme cocktail comprising acidic xylanase from Aspergillus sp. for bioethanol production

The most crucial enzyme involved in xylan hydrolysis is endoxylanase which cleaves the internal glycosidic bonds of xylan. The aim of this work was to study the production of extracellular xylanase by a locally isolated strain of Aspergillus sp. under solid-state fermentation (SSF) and to evaluate the potential of the enzyme in enzymatic hydrolysis of pretreated rice straw. Xylanase production reached maximum with incubation period (96 h), moisture level (80%), inoculum size (3 × 10⁶ spores/mL), pH (4.8), temperature (25 °C), carbon source (wheat bran) and nitrogen source (yeast extract). Under optimized conditions, xylanase production reached to 5059 IU/gds. Crude xylanase was used for supplementing the enzyme cocktail comprising cellulases (Zytex, India), β-glucosidase (In-house) and xylanase (In-house) for the saccharification of alkali-pretreated rice straw to get the maximum reducing sugar production. The cocktail containing the three enzymes resulted a maximum of 574.8 mg/g of total reducing sugars in comparison to 430.2 mg/g sugars by the cocktail without xylanase. These results proved that the crude xylanase preparation from Aspergillus sp. could be a potent candidate for the enzyme cocktail preparation for biomass hydrolysis in lignocellulosic bioethanol program.     

纤维素乙醇木聚糖酶的固体发酵工艺研究

该研究立足于河南天冠企业集团纤维素乙醇项目,以酶解糖化工艺对木聚糖酶的需求为出发点,利用黑曲霉X06作为产酶菌株,采用固体发酵工艺,通过正交设计试验,优化了培养基配方和发酵控制工艺,最优方案:麸皮∶玉米芯=6∶4、硝酸铵4%、尿素1%、磷酸二氢钾0.4%、硫酸镁0.2%、初始含水量65%、初始pH=4.0、温度28℃、环境相对湿度70%、72 h酶活达到10 096.74 IU/g。这一方案在生产中得到进一步放大和优化,所生产的固体木聚糖酶应用在秸秆酶解工艺中,酶解液中木糖含量提高了66.9%。     

One-factor-at-a-time (OFAT) optimization of hemicellulases production from Fusarium moniliforme in submerged fermentation

Hemicellulose degrading enzymes play an important role in bioconversion of lignocellulosic and agro-industrial wastes. In this study, production of hemicellulase by six fungal isolates was determined under submerged culture using corn cobs xylan as a carbon source and enzyme inducer at different incubation periods. The results indicated that Alternaria tenuis showed the lowest enzyme productivity (156.95 ± 2.07U/l) while the highest enzyme production (2,594.44 ± 62.25 U/l) was obtained by Fusarium moniliforme. One-factor-at-a-time revealed maximum enzyme productivity of 10,950.11 ± 98.45 U/l at corn cobs xylan (6 g/l), yeast extract (4 g/l), inorganic salts (1.5 g/l KH₂PO₄, 1.0 g/l MgSO₄.7H₂O, 0.2 g/l CaCl₂, 0.4 g/l FeSO₄.7H₂O, and 0.3 g/l MnSO₄.5H₂O), initial pH (5), initial inoculum size (4%), 150 rpm, and temperature (30°C) in a submerged fermentation process.     

耐酒精半纤维素降解菌的筛选、鉴定及产酶分析

从石油污染严重地区的贫瘠土壤中筛选得到的一株高产木聚糖酶且能够耐受一定浓度酒精的细菌,经生理生化实验及16SrDNA鉴定为短小芽孢杆菌(Bacillus pumilus),命名为DT83。对其产木聚糖酶的发酵条件进行优化,结果表明:最适碳源为木聚糖,最适氮源为蛋白胨,产酶最适温度为28℃,最佳初始pH值为8.5。SDS-PAGE分析表明,木聚糖酶分子量约为23kD。DT83和E.coli BL21的酒精耐受实验结果比较表明,DT83具有较高酒精耐受度,可达到8%～10%。     

Effect of carbon sources on cellulase production by clostridium cellulolyticum

The effect of varying carbon source on cellulase activity in C. cellulolyticum has been investigated using a basal culture medium supplemented with various compounds, including Avicel which gave the best results indicating that both the production of cellulase components (FPAse, CMCase, PNPGase and xylanase) and the bacterial hydrolysis yield increase with the degree of crystallinity. Glucose and cellobiose alternative products of cellulolysis, had contrasting effects. At concentrations from 0.5 to 5 mM when mixed with Avicel glucose enhances, while cellobiose inhibits, the production of cellulase. In contrast, in the absence of Avicel, cellobiose has a greater effect in enhancing cellulase production than glucose. At 7.5 g.l⁻¹ other sugars (fructose, xylose and arabinose) are also more effective than cellobiose in promoting cellulase production. Glucose, in contrast to cellobiose, has a greater effect on derepression of Avicelase and inhibits neither Avicelase activity in solution nor bacterial cellulolysis in solid culture media. Cultures grown in a fermenter, using either sugar, show that growth inhibition can be lowered and production greatly improved. The best results were obtained under conditions of cellobiose limitation using ultrafiltration to the growth inhibiting metabolites. Culture carboxymethylcellulose (CMC), a soluble cellulose derivative, causes both immediate growth and a rapid production of CMCase, PNGase and xylanase activities whereas the capacity to hydrolyse cellulose of filter paper (FPase) is absent. The sugars released during cellulolysis inhibit both cell growth and cellulase production indicating that C. cellulolyticum produces a “true cellulase system” subject to end-product regulation.     

Parthenium sp. as a plant biomass for the production of alkalitolerant xylanase from mutant Penicillium oxalicum SAUE-3.510 in submerged fermentation

The use of congress grass (Parthenium sp.) and water hyacinth (Eichhornia crassipes) as low cost raw materials for xylanase production from mutant Penicillium oxalicum SAU_E-3.510 in submerged fermentation was investigated. For development of mutant from wild type P. oxalicum SA-8 ITCC 6024, a strategy of mixed mutagenesis was followed using UV-irradiation and ethidium bromide, which had resulted into 1.87 fold increases in the activity of the enzyme. For enzyme production, the fungus was cultivated in mineral medium containing congress grass as carbon source. Considerably higher levels of production (475.2 ± 6.0 IU ml^?1) were achieved in media containing congress grass, although it was slightly less than that was obtained (488.5 ± 6.5 IU ml^?1) in presence of commercial oat spelt xylan. This fact confirms the feasibility of using this low cost non-food resource as an alternative carbon source to save costs of the enzyme production process. Maximum xylanase activity was reported at 55 °C with its stability at 80 °C for 2 h. The highest activity of xylanase at pH 9.0 and its stability at similar pH for 24 h denote the alkalitolerant nature of enzyme.     

Optimization of concomitant production of cellulase and xylanase from Rhizopus oryzae SN5 through EVOP-factorial design technique and application in Sorghum Stover based bioethanol production

Current study deals with the production of cellulases and xylanases from the Rhizopus oryzae SN5 isolated from composed soil of Himalayan pine forest, in order to meet the challenges of lignocellulosic biomass based biorefineries. Culture parameters for concomitant production of cellulase and xylanase were optimized through EVOP-factorial design technique under solid state fermentation. And maximum yield of cellulase and xylanase were obtained 437.54 U/gds and 273.83 U/gds, respectively at 30 °C and pH 6.0 after 5 days of incubation. On applying these enzymes for the saccharification of the dilute acid pretreated Sorghum Stover (SS), 0.407 g/g sugar was yielded. This hydrolysate on fermentation, yielded 0.411 g/g ehanol with Saccharomyces cerevisiae (NCIM 3288), which could be considered a good conversion. Therefore, Rhizopus oryzae SN5 was found as potent strain for the production of the cocktail of lignocellulosic biomasss hydrolytic enzymes and would be promising tool in the area of lignocellulose based bio-refineries.     

Physicochemical characterization and enzymatic digestibility of Chinese pennisetum pretreated with 1-ethyl-3-methylimidazolium acetate at moderate temperatures

1-ethyl-3-methylimidazolium acetate ([EMIM] AC) pretreatment at moderate temperatures (60 °C and 75 °C) was evaluated for improving hydrolysability of Chinese pennisetum, a leading candidate as an energy crop, for bioethanol production. The pretreatment caused slight carbohydrate and lignin loss but significantly changed the material physicochemical characters, such as crystallinity and surface structure. Both changes exhibited positive effects on improving the enzymatic digestibility of the Chinese pennisetum. It was observed that approximately 90% of the cellulose and 50% of the xylan in the Chinese pennisetum after pretreatment at 75 °C were converted to fermentable monosaccharides by the combined cellulases and endo-xylanase. The results suggested that Chinese pennisetum could be effectively pretreated with ([EMIM] AC) pretreatment at moderate temperatures, and the high hydrolysis yield of fermentable sugars from pretreated Chinese pennisetum could be achieved by the synergistic action of accessory xylanase in enzymatic hydrolysis by cellulases.     

Ash-forming elements in four Scandinavian wood species. Part 1: Summer harvest

Woody biomass in Finland and Sweden comprises mainly four wood species: spruce, pine, birch and aspen. To study the ash, which may cause problems for the combustion device, one tree of each species were cut down and prepared for comparisons with fuel samples. Well-defined samples of wood, bark and foliage were analyzed on 11 ash-forming elements: Si, Al, Fe, Ca, Mg, Mn, Na, K, P, S and Cl. The ash content in the wood tissues (0.2–0.7%) was low compared to the ash content in the bark tissues (1.9–6.4%) and the foliage (2.4–7.7%). The woods’ content of ash-forming elements was consequently low; the highest contents were of Ca (410–1340 ppm) and K (200–1310), followed by Mg (70–290), Mn (15–240) and P (0–350). Present in the wood was also Si (50–190), S (50–200) and Cl (30–110). The bark tissues showed much higher element contents; Ca (4800–19,100 ppm) and K (1600–6400) were the dominating elements, followed by Mg (210–2400), P (210–1200), Mn (110–1100) and S (310–750), but the Cl contents (40–330) were only moderately higher in the bark than in the wood. The young foliage (shoots and deciduous leaves) had the highest K (7100–25,000 ppm), P (1600–5300) and S (1100–2600) contents of all tissues, while the shoots of spruce had the highest Cl contents (820–1360) and its needles the highest Si content (5000–11,300). This paper presented a new approach in fuel characterization: the method excludes the presence of impurities, and focus on different categories of plant tissues. This made it possible to discuss the contents of ash element in a wide spectrum of fuel-types, which are of large importance for the energy production in Finland and Sweden.