产纤维素酶黏细菌在生物转化的作用

目的:预处理对木质纤维素降解的影响.方法:从土壤中分离筛选到高纤维素酶活的黏细菌菌株So ce sh1008.该菌具有CMC酶活(CMCase)及微晶纤维素酶活性.研究NaOH联合黏细菌降解盐蒿、稻草、棉花秸秆和甘蔗渣四种木质纤维素的情况.结果:碱(2％ NaOH) -黏细菌处理的方法优于黏细菌-碱的方法,其中降解棉花秸秆降解效果最明显,以5.0g木质纤维素为原料,其最终干重损失达2.1g,溶液中总糖含量和还原糖含量均值分别为12.8 mg/mL和0.93 mg/mL.酵母菌发酵产乙醇的研究结果表明,最佳发酵时间为47h,碱-黏细菌甘蔗渣降解液发酵效果最好,乙醇产出达6.0％.结论:黏细菌联合2％ NaOH能有效降解甘蔗渣,提高乙醇产量.     

碱液湿磨预处理稻草的酶解性能

预处理是提高酶法木质纤维素糖化效率的重要加工过程.本论文对稻草进行碱液湿磨预处理和酶解,探讨了预处理碱液浓度对稻草的成分、结构和酶解的影响,同时利用米氏方程对稻草酶解反应进行动力学分析,求出了米氏常数Km 和最大反应速率rmax.实验结果表明,碱液湿磨预处理明显改善了稻草的酶解性能.未处理稻草酶解的还原糖收率为13.4%、Km 为66.8 mg/mL、rmax 为312.5μmol/(min · mL).采用1%NaOH 溶液对稻草进行预处理1h 后,还原糖收率提高到41.4%,Km 减小到15.9 mg/mL, rmax 提高到666.7μmol/(min · mL).预处理过程中木质素去除、纤维素晶体结构消除、底物可及度增加是酶解中还原糖收率和反应速率上升的主要原因.     

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％.     

不同纤维素原料超临界水解的研究

分别以稻草秸秆、经预处理的稻草秸秆、脱脂棉、微晶纤维素和定性滤纸为原材料,利用间歇式的超临界反应设备,在400℃的盐浴中进行木质纤维素的超临界水解,采用3,5-二硝基水杨酸(DNS)法对产物中的还原糖进行测定,研究反应时间对不同纤维素原料水解产糖的影响。结果表明:在超临界条件下,不同原料在较短的时间内还原糖含量均出现峰值,随着反应时间的延长还原糖产量呈现下降的趋势;稻秆、预处理后的稻秆、脱脂棉、微晶纤维素和定性滤纸的最大产糖量分别为7.42、9.05、12.55、18.01和14.24 g/L;与此对应的最佳反应时间分别为3.5、4、3、3、4 min;对应的最大还原糖产率分别为14.84%、18.10%、25.10%、36.02%、28.48%。     

玉米秸秆分批补料获得高还原糖浓度酶解液的条件优化

木质纤维素高浓度还原糖水解液的获得是纤维乙醇产业化发展的方向。在发酵工业领域,分批补料法是实现这一目标的重要研究途径。本研究采用分批补料法对获得高浓度玉米秸秆酶解还原糖的条件进行了优化。以稀硫酸预处理的玉米秸秆为原料,考察了液固比、补加量与补加时间对分批补料糖化的影响。结果表明,秸秆高浓度酶解液条件的初始物料为20％ (重量/体积),木聚糖酶220 U/g (底物),纤维素酶6 FPU/g (底物),果胶酶50 U/g (底物),在24 h、48 h后分批补加8％预处理后的物料,同时添加与补料量相应的木聚糖酶20 U/g (底物),纤维素酶2 FPU/g (底物),72 h后,最终糖化结果与非补料法相比,还原糖浓度从48.5 g/L提高到138.5 g/L,原料的酶解率最终达到理论值的62.5％。试验结果表明补料法可以显著提高秸秆水解液还原糖浓度。     

60Co-γ射线辐照协同氢氧化钠预处理对油菜秸杆酶解产糖的影响

研究了辐照协同氢氧化钠预处理油菜秸秆对酶解产还原糖的影响。利用响应面法对氢氧化钠反应条件进行了优化,得出最优条件为氢氧化钠浓度为2．38％,反应温度为100℃,反应时间为0．5h。这一条件预期还原糖产量为524．93mg／g,通过实验验证,实际还原糖含量（528．51mg／g）能够很好地与预期相吻合。扫描电镜观察表明,辐照协同氢氧化钠预处理后秸秆表面积明显增大,出现很多蜂窝状孔洞结构,能够有效增大酶解可及表面积,从而提高酶解效率。     

不同品种水稻秸秆的理化特性及酶解产糖的研究

本研究以不同品种水稻秸秆为原料,测定分析了三大主要组分纤维素、半纤维素和木质素含量及理化特性的差异,并分析对比了经碱预处理后酶解及直接酶解的产糖率差异。利用X-射线衍射仪测定分析了结晶度和晶粒尺寸的异同,红外光谱仪定性评估了特定基团的差异,扫描电镜观察分析了表面形态结构的差异。综合考虑理化特性和酶解产糖率结果,与a(常规水稻黄华占)相比,5种不同品种的超级杂交水稻秸秆中,b(杂交水稻Y两优1)、c(杂交水稻隆两优2010)和f(杂交水稻Y两优143)三个品种的秸秆材料比较适合作为酶解产糖的原材料。     

中和剂对稀酸蒸爆玉米秸秆酶解效果的影响

以稀酸蒸爆的玉米秸秆为研究对象,考察直接水洗、Ca(OH)2、NaOH、氨水中和物料至pH 5,在固液比1∶10、酶添加量为每克纤维素14 U(滤纸酶活)的酶解条件下对纤维素转化率的影响。结果表明:水洗、Ca(OH)2、NaOH、氨水中和物料酶解72 h后,纤维素转化率分别为91.7%、80.7%、83.1%及81.7%。同时对影响纤维素酶解效率的各种因素进行了探讨。从综合成本及后续发酵过程考虑,用氨水中和稀酸蒸爆物料更适合于工业化生产。     

沙柳原料高浓度底物酶解发酵乙醇工艺

为了提高沙柳生物转化过程的经济可行性,考察了沙柳原料经过蒸爆、超微粉碎+稀酸、超微粉碎+稀碱预处理后高浓度底物补料酶解的效果,并对其高浓度水解糖液进行了乙醇发酵。结果表明:蒸爆处理法水解效果最好,通过补料酶解,底物质量分数可以达到30%,酶解液中总糖质量浓度达到132 g/L,葡萄糖质量浓度105 g/L;超微粉碎+稀酸预处理原料底物质量分数可以达到22%,酶解液中总糖质量浓度达到123 g/L,葡萄糖质量浓度73 g/L;超微粉碎+稀碱预处理原料底物质量分数可以达到22%,酶解液中总糖质量浓度133 g/L,葡萄糖质量浓度77 g/L。3种预处理使沙柳原料的酶解糖液都可以较好地被酿酒酵母利用发酵产乙醇,蒸爆处理原料的酶解糖液乙醇发酵效果最好,乙醇质量浓度达到47 g/L。     

响应曲面优化秸秆稀酸水解工艺用于发酵产壳聚糖

为实现利用秸秆水解产生的五碳糖发酵产壳聚糖,以米根霉为研究对象,研究水解温度、水解时间、酸浓度等不同预处理方式获得的半纤维素水解液对米根霉发酵产壳聚糖的影响。结果表明:水解温度、水解时间对水解液中木糖含量以及甲酸、乙酸、糠醛等抑制剂浓度具有显著影响,并进一步影响后续发酵产壳聚糖的生成量。利用响应曲面对稀酸水解预处理条件进行优化,获得最佳工艺条件:H_2SO_413.6 g/L,99.5℃,水解时间1.91 h,在此条件下预测壳聚糖发酵产量为0.79 g/L,实验验证产量为0.82 g/L,占菌体生物量的15%~18%。研究结果为秸秆资源的高效利用及发酵生产壳聚糖提供新思路。     

Production of xylanase under solid-state fermentation by <Emphasis Type="Italic">Aspergillus tubingensis</Emphasis> JP-1 and its application

The production of extracellular xylanase by a locally isolated strain of Aspergillus tubingensis JP-1 was studied under solid-state fermentation. Among the various agro residues used wheat straw was found to be the best for high yield of xylanase with poor cellulase production. The influence of various parameters such as initial pH, moisture, moistening agents, nitrogen sources, additives, surfactants and pretreatment of substrates were investigated. The production of the xylanase reached a peak in 8 days using untreated wheat straw with modified MS medium, pH 6.0 at 1:5 moisture level at 30 °C. Under optimized conditions yield as high as 6,887 ± 16 U/g of untreated wheat straw was achieved. Crude xylanase was used for enzymatic saccharification of agro-residues like wheat straw, rice bran, wheat bran, sugarcane bagasse and industrial paper pulp. Dilute alkali (1 N NaOH) and acid (1 N H₂SO₄) pretreatment were found to be beneficial for the efficient enzymatic hydrolysis of wheat straw. Dilute alkali and acid-pretreated wheat straw yielded 688 and 543 mg/g reducing sugar, respectively. Yield of 726 mg/g reducing sugar was obtained from paper pulp after 48 h of incubation.     

Effect of physicochemical pre-treatment of rice straw on its digestibility by <Emphasis Type="Italic">Clostridium cellulolyticum</Emphasis>

Rice straw (RS) may serve as a low-cost biomass for the production of biofuels and biochemicals, but its native structure is resistant to enzymatic and microbial deconstruction. Therefore, an efficient pre-treatment method is required to modify crystalline cellulose to a more reactive amorphous form. This work investigated pre-treatments of rice straw involving size reduction (S) followed by either sodium hydroxide (NaOH) or diluted sulfuric acid (H₂SO₄) and liquid hot water (LHW). The shrinkage of the vascular bundles in the rice straw structure pre-treated with NaOH–LHW–S was higher than that with LHW–S and H₂SO₄–LHW–S pre-treatments. The highest levels of total fermentative products and residual sugars were obtained at the concentrations of 7.8 ± 0.2 and 2.1 ± 0.3 g/L, respectively, after fermentation by Clostridium cellulolyticum for NaOH–LHW–S pre-treated rice straw at 121 °C for 120 min. Overall, the combined physicochemical pre-treatment of RS led to improved microbial hydrolysis during cellulose degradation at the percentage of 85.5 ± 0.5.     

Simultaneous saccharification and fermentation (SSF) of <Emphasis Type="Italic">Jatropha curcas</Emphasis> shells: utilization of co-products from the biodiesel production process

Jatropha curcas has great potential as an oil crop for use in biodiesel applications, and the outer shell is rich in lignocellulose that may be converted to ethanol, giving rise to the concept of a biorefinery. In this study, two dilute pretreatments of 0.5% H₂SO₄ and 1.0% NaOH were performed on Jatropha shells with subsequent simultaneous saccharification and fermentation (SSF) of the pretreated water-insoluble solids (WIS) to evaluate the effect of inhibitors in the pretreatment slurry. A cellulase loading of 15 FPU/g WIS, complimented with an excess of cellobiase (19.25 U/g), was used for SSF of either the washed WIS or the original slurry to determine the effect of inhibitors. Ethanol and glucose were monitored during SSF of 20 g of pretreated biomass. The unwashed slurry showed to have a positive effect on SSF efficiency for the NaOH-pretreated biomass. Maximum efficiencies of glucan conversion to ethanol in the WIS were 40.43% and 41.03% for the H₂SO₄- and NaOH-pretreated biomasses, respectively.     

Bioconversion of wheat straw to ethanol: Chemical modification,enzymatic hydrolysis,and fermentation

Native wheat straw (WS) was pretreated with various concentrations of H₂SO₄ and NaOH followed by secondary treatments with ethylene diamine (EDA) and NH₄OH prior to enzymatic saccharification. Conversion of the cellulosic component to sugar varied with the chemical modification steps. Treatment solely with alkali yield 51–75% conversion, depending on temperature. Acid treatment at elevated tempeatures showed a substantial decrease in the hemicellulose component, whereas EDA-treated WS (acid pretreated) showed a 69–75% decrease in the lignin component. Acid-pretreated EDA-treated straw yielded a 98% conversion rate, followed by 83% for alkali–NH₄OH treated straws. In other experiments, WS was pretreated with varying concentration of H₂SO₄ or NaOh followed by NH₄OH treatment prior to enzymatic hydrolysis. Pretreatment of straw with 2% NaOH for 4 h coupled to enzymatic hydrolysis yield a 76% conversion of the cellulosic component. Acid–base combination pretreatment yielded only 43% conversions. A reactor column was subsequently used to measure modification–saccharification–fermentation for wheat straw conversion on a larger scale. Thirty percent conversions of wheat straw cellulosics to sugar were observed with subsequent fermentation to alcohol. The crude cellulase preparation yielded considerable quantities of xylose in addition to the glucose. Saccharified materials were fermented directly with actively proliferating proliferating yeast cells without concentration of the sugars.     

Effect of chemical treatments on the degradability of cotton straw by rumen microorganisms and by fungal cellulase

Three different chemical treatments—sulfur dioxide, ozone, and sodium hydroxide—were applied on cotton straw, and the effect on cell-wall degradability was assessed by using rumen microorganism and Trichoderma reesei cellulase. Sulfur dioxide (applied at 70°C for 72 h) did not change the lignin content of cotton straw but reduced the concentration of hemicellulose by 48%. Ozone exerted a dual effect, both on lignin (a 40% reduction) and hemicellulose (a 54% decrease). The treatment with NaOH did not solublize cell-wall components. The in vitro organic matter digestibility with rumen fluid of cotton straw was increased significantly by ozone and SO₂ treatments, by 120% and 50%, respectively, but not by NaOH. T. reesei cellulase was applied on the chemically pretreated cotton straw at a low level (6 filter paper U/g straw, organic matter), and the release of reducing sugars was determined. The highest level of reducing sugars (30.6 g/100 g organic matter) was obtained with the O₃-cellulase combination, which solubilized 64% of the cellulose and 88% of the hemicellulose. the SO₂- and the NaOH-pretreated cotton straw were hydrolyzed by T. reesei cellulase to the same extent (21 g reducing sugars/100 g organic matter). The rumen fluid digestibility of the enzymatic ally hydrolyzed straw was not increased further over the effect already obtained with the chemical pretreatments. However, the fermentability of the combined treatments was increased markedly. In the O₃-cellulase-treated cotton straw, 83% of the rumen fluid digestible material consisted of highly fermentable components. Although ozone proved to be the most potent pretreatment for enzymic saccharification in this study, the absolute result was modest. The limited effect of the combined O₃-cellulase treatment was probably associated with the pretreatment limitations, but not with the enzyme level. Based on the differential response of the chemically treated cotton straw to attack by rumen microorganisms on the one hand, and by T. reesei cellulase on the other hand, a hypothesis has been suggested as to the location of lignin and hemicellulose in the cellwall unit of cotton straw.     

Biohydrogen production from cellulosic hydrolysate produced via temperature-shift-enhanced bacterial cellulose hydrolysis

A “temperature-shift” strategy was developed to improve reducing sugar production from bacterial hydrolysis of cellulosic materials. In this strategy, production of cellulolytic enzymes with Cellulomonas uda E3-01 was promoted at a preferable temperature (35 °C), while more efficient enzymatic cellulose hydrolysis was achieved under an elevated culture temperature (45 °C), at which cell growth was inhibited to avoid consumption of reducing sugar. This temperature-shift strategy was shown to markedly increase the reducing sugar (especially, monosaccharide and disaccharide) concentration in the hydrolysate while hydrolyzing pure (carboxymethyl-cellulose, xylan, avicel and cellobiose) and natural (rice husk, rice straw, bagasse and Napier-grass) cellulosic materials. The cellulosic hydrolysates from CMC and xylan were successfully converted to H₂ via dark fermentation with Clostridium butyricum CGS5, attaining a maximum hydrogen yield of 4.79 mmol H₂/g reducing sugar.     

Effect of dilute acid pretreatment on the saccharification and fermentation of rye straw

This research shows the effect of dilute acid pretreatment with various sulfuric acid concentrations (0.5–2.0% [wt/vol]) on enzymatic saccharification and fermentation yield of rye straw. After pretreatment, solids of rye straw were suspended in Na citrate buffer or post-pretreatment liquids (prehydrolysates) containing sugars liberated after hemicellulose hydrolysis. Saccharification was conducted using enzymes dosage of 15 or 25 FPU/g cellulose. Cellulose saccharification rate after rye straw pretreatment was enhanced by performing enzymatic hydrolysis in sodium citrate buffer in comparison with hemicellulose prehydrolysate. The maximum cellulose saccharification rate (69%) was reached in sodium citrate buffer (biomass pretreated with 2.0% [wt/vol] H₂SO₄). Lignocellulosic complex of rye straw after pretreatment was subjected to separate hydrolysis and fermentation (SHF) or separate hydrolysis and co-fermentation (SHCF). The SHF processes conducted in the sodium citrate buffer using monoculture of Saccharomyces cerevisiae (Ethanol Red) were more efficient compared to hemicellulose prehydrolysate in respect with ethanol yields. Maximum fermentation efficiency of SHF processes obtained after rye straw pretreatment at 1.5% [wt/vol] H₂SO₄ and saccharification using enzymes dosage of 25 FPU/g in sodium citrate buffer, achieving 40.6% of theoretical yield. However, SHCF process using cocultures of pentose-fermenting yeast, after pretreatment of raw material at 1.5% [wt/vol] H₂SO₄ and hydrolysis using enzymes dosage of 25 FPU/g, resulted in the highest ethanol yield among studied methods, achieving 9.4 g/L of ethanol, corresponding to 55% of theoretical yield.     

Synergism of cellulase,pectinase and xylanase on hydrolyzing differently pretreated sweet potato residues

Abstract

The synergism of cellulase (C), pectinase (P), and xylanase (X) for the saccharification of sweet potato residues (SPR) was investigated. The removal of starch from SPR was easily achieved by using amylase, but the cellulose conversion of de-starched SPR was relatively low, thus dilute H₂SO₄, NaOH, and H₂O₂ pretreatment was conducted to improve the enzymatic digestibility. The lignin content of NaOH pretreated SPR was the lowest, whereas H₂SO₄ pretreatment resulted in the lowest contents of hemicellulose and pectin. The combination of C, P, and X exhibited different sugar production patterns, C–P displayed synergistic action on glucose and galactose production from each type of SPR, C–X also exhibited synergistic effect on glucose production except when H₂SO₄ pretreated SPR was used, whereas no synergism between P–X on monosaccharide production was observed. The presence of synergism between cellulase and mixed accessory enzymes [C–(PX)] on glucose formation was determined by C–X, and the degree of synergism between C–P and C–(PX) on glucose production had a positive relationship with pectin content. The highest cellulose conversion of 96.2% was obtained from NaOH pretreated SPR using mixed enzymes comprising C, P, and X with the ratio of 8:1:1.     

Utilization of agro-industrial waste for xylanase production by Aspergillus foetidus MTCC 4898 under solid state fermentation and its application in saccharification

Xylanase production by Aspergillus foetidus MTCC 4898 was carried out under solid state fermentation using wheat bran and anaerobically treated distillery spent wash. Response surface methodology involving Box–Behnken design was employed for optimizing xylanase production. The interactions among various fermentation parameters viz. moisture to substrate ratio, inoculum size, initial pH, effluent concentration and incubation time were investigated and modeled. The predicted xylanase activity under optimized parameters was 8200–8400 U/g and validated xylanase activity was 8450 U/g with very poor cellulase activity. Crude xylanase was used for enzymatic saccharification of agroresidues like wheat straw, rice straw and corncobs. Dilute NaOH and ammonia pretreatments were found to be beneficial for the efficient enzymatic hydrolysis of all the three substrates. Dilute NaOH pretreated wheat straw, rice straw and corncobs yielded 4, 4.2, 4.6 g/l reducing sugars, respectively whereas ammonia treated wheat straw, rice straw and corncobs yielded 4.9, 4.7, 4.6 g/l reducing sugars, respectively. The hydrolyzates were analysed by HPTLC. Xylose was found to be the major end product with traces of glucose in the enzymatic hydrolyzates of all the substrates.     

Mild alkaline pretreatment can achieve high hydrolytic and fermentation efficiencies for rice straw conversion to bioethanol

Abstract

Mild alkaline pretreatment was evaluated as a strategy for effective lignin removal and hydrolysis of rice straw. The pretreatment efficiency of different NaOH concentrations (0.5, 1.0, 1.5 or 2.0% w/w) was assessed. Rice straw (RS) pretreated with 1.5% NaOH achieved better sugar yield compared to other concentrations used. A cellulose conversion efficiency of 91% (45.84?mg/ml glucose release) was attained from 1.5% NaOH pretreated rice straw (PRS), whereas 1% NaOH pretreated rice straw yielded 35.10?mg/ml of glucose corresponding to a cellulose conversion efficiency of 73.81%. The ethanol production from 1% and 1.5% NaOH pretreated RS hydrolysates was similar at ～3.3% (w/v), corresponding to a fermentation efficiency of 86%. The non-detoxified hydrolysate was fermented using the novel yeast strain Saccharomyces cerevisiae RPP-03O without any additional supplementation of nutrients.