产纤维素酶菌株Trametes sp.LYW-1的分离鉴定及产酶条件分析

为寻找新纤维素酶和纤维素酶基因资源,以羧甲基纤维素钠作为唯一碳源,从湿润木屑中分离和筛选获得1株高产纤维素酶目的菌株LYW-1。对该菌保守序列ITS进行扩增和测序后,提交NCBI并与相近物种ITS序列进行比对和分析,基于亲缘关系构建系统发育树。将ITS rDNA保守序列比较分析结果与形态学观察结果相结合,初步鉴定该菌株属于栓菌属(Trametes)。以羧甲基纤维素钠溶液为底物,采用3’5’-二硝基水杨酸(DNS)法测定纤维素酶活力,分析pH、温度及培养基中同碳源、氮源对该菌产纤维素酶活力的影响。结果表明,菌株LYW-1在pH 7.0、37℃、淀粉为碳源和乙酸铵为氮源时为最适培养条件,总酶活力达到最大。     

秸秆饲料化全细胞纤维素酶的构建及应用

为提升秸秆饲料的品质，通过构建全细胞酿酒酵母纤维素酶，探究全细胞纤维素酶用于秸秆饲料化的应用前景。通过与其他青贮菌剂对比用于秸秆饲料化发酵，比较分析全细胞酿酒酵母纤维素酶在秸秆饲料化中的应用效果。试验发酵条件设置为：发酵温度28℃，发酵时间14 d，真空厌氧发酵。结果表明：与乳酸菌发酵相比，纤维素酶与乳酸菌混合发酵的玉米秸秆铵态氮/总氮比没有明显上升（P>0.05），因此不会造成饲料中的蛋白过量分解；纤维素含量显著下降（P<0.01），相比未发酵秸秆下降15.03个百分点，比乳酸菌发酵效果提高一倍以上，与商用青贮剂发酵效果相当；半纤维素含量相比未发酵秸秆下降2.70个百分点。研究表明酿酒酵母全细胞纤维素酶与乳酸菌混合处理秸秆，可提高纤维素酶酶解效率，有效减低秸秆中纤维素、半纤维素含量，酶解产物可加速乳酸菌发酵进程，全细胞酶可增加秸秆单细胞蛋白含量，实现玉米秸秆发酵效果、营养价值和利用效率的同步提升。     

高温秸秆降解菌的筛选及其纤维素酶活性研究

筛选能在高温（55~75℃）好氧堆肥中高效降解纤维素的菌株，并评估其降解秸秆的能力与产纤维素酶的热稳定性。从高温期堆肥中采样，以水稻秸秆粉为唯一碳源，通过55、65℃和75℃连续高温传代驯化并分离筛选耐高温菌，结合水解圈和水稻秸秆崩解试验筛选不同高温下高效降解秸秆的目标菌株，采用16S rRNA测序和系统发育分析鉴定目标菌株分类地位，通过分析目标菌株纤维素降解相关酶在50~90℃之间的热稳定性，解析其高温适应性机制，评价其在实际生产中的应用潜力。高温驯化分离得到13株耐高温降解菌，其中B-5、B-6、B-7和B-11的纤维素和秸秆降解能力较强，而只有B-7和B-11在55~65℃和75℃具有高效降解水稻秸秆的能力，将其认定为目标菌株。系统发育分析表明B-7和B-11菌株与芽孢杆菌科高度相似，分别命名为短小芽孢杆菌B-7和嗜热脂肪芽孢杆菌B-11。酶活热稳定性分析发现B-7和B-11各纤维素酶活性在50~90℃之间先升高后降低，其最适温度范围不同，分别为55~65℃和70~80℃，其中B-11在85℃时的相对酶活仍高于60%。研究表明，菌株B-7和B-11是耐高温高效秸秆降解菌，其具有不同高温偏好性，纤维素酶热稳定性强，在秸秆高温好氧堆肥中具有潜在的应用前景。     

响应面法优化无花果果汁酶解提取工艺研究

【目的】研究双酶法提取无花果汁的最佳工艺参数,为制备无花果汁、酿造无花果酒奠定基础。【方法】以无花果为材料,以出汁率为考察指标,通过单因素试验和响应面试验,研究纤维素酶和果胶酶添加量、酶解温度、酶解时间4个因素及其交互作用对无花果岀汁率的影响,利用响应面试验结果建立回归方程,并对回归方程进行显著性和方差分析,得到无花果果汁酶解提取最佳工艺参数并进行试验验证。【结果】通过单因素试验得到的无花果果汁最佳酶解提取条件为:纤维素酶添加量1.5%(质量分数,下同),果胶酶添加量0.3%,酶解温度55℃,酶解时间90min。根据单因素试验结果进行响应面试验分析得出,无花果果汁的最佳酶解提取工艺条件为:纤维素酶添加量1.56%,果胶酶添加量0.28%,酶解温度53℃,酶解时间90min;在此条件下无花果的岀汁率为72.15%,与理论值(73.99%)基本吻合,且比未处理无花果出汁率提高了75.46%。纤维素酶添加量与酶解温度和酶解时间、果胶酶添加量与酶解温度和酶解时间、酶解温度与酶解时间的交互作用均可在较大程度上影响无花果的岀汁率。【结论】通过响应面试验得到了双酶法提取无花果汁的最佳工艺参数,该工艺可以大幅提高无花果出汁率。     

Isolation and identification of effective cellulolytic bacteria in composting process from different sources

Cellulolytic bacteria have a bio-activating role in the composting process. A study was carried out to isolate and identify cellulolytic bacteria from various sources. The isolates were cultured in the carboxymethyl cellulose (CMC) agar medium and incubated at 30°C for 3–7 days. Based on morphological characteristics of the isolates, maximum diameter of a clear zone around the colony and maximum cellulolytic activity, eight isolate were selected for further studies regarding composting experiments.

Molecular tests based on PCR amplification and sequencing of 16S rRNA gene of isolates showed the closest phylogenetic similarity with the species of Stenotrophomonas rhizophila DSM14405 (99.8%), Brevibacterium halotolerans DSM8802 (99.6%), Achromobacter marplatensis B2 (99.8%), Bacillus methylotrophicus CBMB205 (100%), Pseudomonas azotoformans IAM 1603 (99.7%), Bacillus sonorensis NBRC 101234 (99.8%), Bacillus subtilis KCTC 13429 (100%) and Ochrobactrum thiophenivorans DSM 7216 (99.3%). The study of the isolates impact on the composting of palm wastes in a randomized complete block design with 11 treatments in 3 replications showed that strain IB (B. methylotrophicus) caused a significant decrease in C:N ratio (58%). The increasing of microbial respiration compared with control after 30 days incubation at 37°C showed that the B. methylotrophicus strain IB with cellulolytic characteristics can be applied to hydrolysis of cellulosic biomass in the composting processes.     

Effect of fish meal replacement by Chlorella meal with dietary cellulase addition on growth performance,digestive enzymatic activities,histology and myogenic genes’ expression for crucian carp Carassius auratus

This study was conducted to investigate the effects of fish meal (FM) replacement by Chlorella meal (CM) with dietary cellulase supplementation on growth performance, digestive enzymatic activities, histology and myogenic genes’ expression in crucian carp Carassius auratus (initial body weight: 2.90 ± 0.02 g, mean ± SEM). Six isonitrogenous diets were formulated at two cellulase levels (0 and 2 g kg^?1). At each cellulase level, CM was added at three levels of 0, 533.1 and 710.8 g kg^?1 to substitute 0, 75 and 100% of dietary FM respectively. Each experimental diet was randomly assigned to triplicate groups with 25 juvenile fish per fibreglass tank for 8 weeks. Dietary CM substitution significantly increased growth, feed utilization, amylase activity and the expression of Myod, Mrf4 and Myf5, but reduced the Myog expression. Dietary cellulase addition increased hepatosomatic and viscerosomatic index, lipase activity and the expression of Mrf4, but reduced trypsin activity and the expression of Myog and Myf5. Dietary CM substitution enlarged the cell size and also caused some karyopyknosis in liver. Our results showed that CM could totally replace FM in diets; dietary cellulase supplementation at the level of 2 g kg^?1 played a subtle role in improving growth and feed utilization for crucian carp.     

绿色木霉Fn10-1纤维素酶的分离纯化及酶学特性测定

试验研究了绿色木霉Fn10-1纤维素酶的初步纯化及酶学性质开展研究。结果表明,该纤维素酶的最适催化温度为70℃,最适pH 7.6,在温度为4~50℃下均能保持较稳定的酶活,高于50℃以后,热稳定性变差,酶活力开始急剧下降。pH在5.6~7.6时该酶有较好的稳定性。Na+、Mn2+、Fe2+、Mg2+、Co2+、Ca2+、Ba2+、Al3+对纤维素酶具有一定的激活作用,Ag+、Fe3+、Cu2+、Zn2+、K+对该酶有一定的抑制作用,其中Cu2+的抑制作用尤为明显。     

Characterization of biochar and their influence on microbial activities and potassium availability in an acid soil

Application of biochar to soil has increased considerably during recent years because of its effectiveness as a soil amendment causing beneficial effects on soil health. However, the effects have been reported to vary and depend upon types of feedstock and pyrolysis conditions during biochar production. Therefore, characterization of biochar is extremely important for its efficient utilization as a soil amendment. In the present study, biochar was prepared from agro-industrial by-products (rice husk and sugarcane bagasse) and weeds (Parthenium and Lantana) under similar pyrolysis conditions. Lantana biochar (LBC) showed the highest pH (10.4) while the lowest value (8.5) being recorded in rice husk biochar (RHBC). The energy-dispersive X-ray spectroscopy (EDS) analysis indicated that LBC and Parthenium biochar (PBC) were superior with respect to potassium (K) content than sugarcane bagasse biochar (SBBC) and RHBC. The Fourier-Transform Infrared Spectroscopy (FTIR) study exhibited the existence of different functional groups in biochar. All the biochar treated soils showed significantly higher microbial activities with different degrees. Application of LBC and PBC at 4.50 g kg^?1 soil significantly increased K availability in soil. Lantana biochar and PBC amended the soil at 9 g kg^?1 significantly increased the soil pH thus makes these biochar as potential liming materials.     

紫外线诱变纤维素酶高产菌株的筛选及其酶活力

为筛选产纤维素酶酶活力高的菌株应用于秸秆饲料发酵,利用紫外线诱变对1株产纤维素酶绿色木霉菌进行了试验研究.结果表明:筛选出1株纤维素酶产酶量高且性状稳定的高产菌株ZJUV18,其 发酵72h纤维素酶滤纸酶活达68.07 U/g,较原始菌株提高4.45倍.     

纤维素产氢菌的分离及不同秸秆处理方法的研究

为了增强生物方法对秸秆的利用能力，将秸秆较好地转化成氢能，本试验利用改良的Hungate厌氧滚管法及滤纸崩解法，从已得菌系JYB中，筛选得到1株能较好地降解纤维素产氢的细菌，并利用水热法、汽爆法等6种方法对秸秆进行预处理，以增强秸秆的产氢效果。结果表明：筛选得到菌株JYB-13，该菌可利用大量碳源产氢，产氢的最适条件为35℃，pH值8.5。对秸秆进行预处理，由产氢效果得知，该菌对2%硫酸处理后的秸秆降解效果最好，秸秆的降解率为53.1%，氢气含量达到37.2%。分析发酵前后秸秆的成分，可知该菌可以较好地利用纤维素和半纤维素，对木质素利用较少。