菠萝渣纤维素降解菌的筛选及鉴定

为了加快菠萝渣快速发酵,通过利用多种选择性培养基,从自然发酵的菠萝渣中分离到多种纤维素分解菌,经过初筛和复筛,获得了降解菠萝渣纤维素的菌株c3b1-3,其最适合的培养基为蛋白纤维素培养基;通过形态、生理生化特征和分子综合鉴定得出c3b1-3为金黄杆菌属（Chryseobacterium sp.）。     

利用木糖-葡萄糖为原料产乙醇酵母的筛选、鉴定及发酵试验

建立筛选利用木糖为碳源产乙醇酵母模型,获得一株适合利用木质纤维素为原料产乙醇的酵母菌株。样品经麦芽汁培养基培养后,以木糖为唯一碳源的筛选培养基初筛,再以重铬酸钾显色法复筛。通过生理生化和26D1/D2区对筛选得到的菌株进行分析和鉴定,该菌初步鉴定为Pichia caribbica。经过筛选得到的菌株Y2-3以木糖（40g/L）为唯一碳源发酵时：生物量为23.5g/L,木糖利用率为94.7 %,乙醇终产量为4.57 g/L;以混合糖（葡萄糖40 g/L,木糖20 g/L）发酵时：生物量为28.6 g/L,木糖利用率为94.2 %,葡萄糖利用率为95.6%,乙醇终产量为20.6 g/L。Pichia caribbica是可以转化木糖及木糖-葡萄糖混合糖为乙醇的酵母菌株,为利用木质纤维素发酵乙醇的进一步研究奠定了基础。     

利用基本培养基初步筛选牛瘤胃中纤维素降解菌

目的初步筛选牛瘤胃中纤维素降解菌。方法分别采用基本培养基（牛肉膏蛋白胨培养基、马丁培养基）,利用好氧、兼性和厌氧3种不同的培养方法进行初选,初步分离牛瘤胃中的细菌与真菌,再通过复选培养基（加入微晶纤维素）,筛选降解纤维素的菌种。结果筛选分离出降解纤维素的1株厌氧细菌和1株厌氧真菌。结论此实验方法简单易行,能够有效地从牛瘤胃中筛选出生长良好的纤维素降解菌。     

贯叶马兜铃的组织培养与快速繁殖

1植物名称贯叶马兜铃（Aristolochia delavayiFranch）。2材料类别腋芽。3培养条件基本培养基为MS。启动培养基：（1）MS＋NAA 1 mg·L-（-1）（单位下同）＋6-BA 0.5;增殖培养基：（2）MS＋NAA 0.5＋6-BA 2;生根培养基：（3）1/2MS＋IBA 0.5。以上培养基中均加入30 g·L-（-1）蔗糖、7 g·L-（-1）琼脂,pH 5.8。培养温度（25±3）℃,光照时间12 h·d-（-1）,光照强度40～50μmol·m-（-2）·s-（-1）。     

岩生报春的组织培养与快速繁殖

1植物名称岩生报春（Primula saxatilis Kom.）。2材料类别种子。3培养条件（1）种子萌发培养基：1/2MS;（2）无菌苗增殖培养基：MS;（3）丛生芽诱导和增殖培养基：MS＋6-BA 2.5 mg·L-（-1）（单位下同）＋NAA 1.0;（4）生根培养基：MS＋NAA 0.1。上述各培养基均添加7 g·L-（-1）琼脂和30 g·L-（-1）蔗糖,pH 5.8～6.0。培养温度为20～23℃,光照强度为20～25μmol·m-（-2）·s-（-1）,光照时间为14 h·d-（-1）。     

辽东栎的组织培养和植株再生

1植物名称辽东栎（Quercus liaotungensis Koidz.）。2材料类别成熟合子胚。3培养条件以MS为基本培养基。（1）不定芽诱导培养基：MS＋6-BA 1.0 mg·L-（-1）（单位下同）＋2,4-D 0.5;（2）不定芽增殖培养基：MS＋6-BA 1.0＋NAA 0.5＋GA_30.5;（3）生根培养基：1/2MS＋NAA 0.5。以上培养基均加入30 g·L-（-1）蔗糖和6.5 g·L-（-1）琼脂,pH 5.8。培养温度为（25±2）℃,光照强度为40～50μmol·m-（-2）·s-（-1）,光照时间为16 h·d-（-1）。     

卡佩指甲草的组织培养与快速繁殖

1植物名称卡佩指甲草[Paronychia kapela（Hacq.）Kerner]。2材料类别带芽茎段。3培养条件（1）启动培养基：MS＋6-BA 0.5 mg·L-（-1）（单位下同）＋NAA 0.2;（2）丛生芽诱导和增殖培养基：MS＋6-BA 0.5＋NAA 0.1;（3）生根培养基：1/2MS＋NAA0.1。以上培养基中均加入30 g·L-（-1）蔗糖和7 g·L-（-1）琼脂,pH为5.8～6.0。培养温度为（25±2）℃,光照强度40～60μmol·m-（-2）·s-（-1）,日光灯补光,光照时间16h.d-（-1）。     

肿节石斛的无菌播种和快速繁殖

1植物名称肿节石斛（Dendrobium pendulumRoxb.）。2材料类别成熟种子。3培养条件以N6和MS为基本培养基。种子萌发培养基：（1）N6;（2）MS;（3）1/2MS;（4）VW（Vacin-Went培养基）;（5）MS＋椰乳100 mg·L-（-1）（单位下同）。增殖与分化成苗培养基：（6）MS＋6-BA 0.2＋NAA 0.2。生根壮苗培养基：（7）MS＋NAA 0.5;（8）MS＋土豆泥100。上述培养基均附加30 g·L-（-1）蔗糖和6 g·L-（-1）琼脂,pH 5.5～5.8。培养温度为（24±2）℃;光照强度20～40μmol·m-（-2）·s-（-1）,光照时间12 h·d-（-1）。     

浙皖粗筒苣苔的组织培养和快速繁殖

1植物名称浙皖粗筒苣苔（Briggsia chienii Chun）。2材料类别种子及无菌苗叶片。3培养条件MS为基本培养基。诱导种子发芽培养基：（1）MS＋6-BA 1.0 mg·L-（-1）（单位下同）＋NAA 0.5;诱导叶片分化培养基：（2）MS＋6-BA 2.0＋NAA 0.5;增殖继代培养基：同（1）;壮苗培养基：（3）MS＋6-BA0.1＋NAA 0.05;生根培养基：（4）1/2MS＋0.1%活性炭。以上培养基均含30 g·L-（-1）蔗糖和7.0 g·L-（-1）琼脂,pH 5.8。培养温度为（25±2）℃;     

闽赣长蒴苣苔的组织培养和快速繁殖

1植物名称闽赣长蒴苣苔（Didymocarpus heucherifoliusHand.-Mazz.）。2材料类别幼叶。3培养条件MS为基本培养基。诱导不定芽分化培养基：（1）MS＋6-BA0.1 mg·L-（-1）（单位下同）＋NAA0.1;（2）MS＋6-BA 1.0＋NAA 0.5;（3）MS＋6-BA 2.0＋NAA0.5。增殖继代培养基：（2）;（3）;（4）MS＋6-BA 2.0＋NAA 1.0;（5）MS＋6-BA 0.05＋NAA 0.05。生根培养基：（6）1/2MS＋NAA 0.5;（7）1/2MS＋0.5%活性炭;（8）1/2MS＋0.1%活性炭。以上培养基均含30 g·L-（-1）蔗糖和7.0 g·L-（-1）琼脂,pH 5.8。培养温度为（25±2）℃,光照强度约为30μmol·m-（-2）·s-（-1）,光照时间为16h.d-（-1）。     

甘蔗渣纤维素降解菌株的筛选与鉴定

甘蔗渣是制糖工业的主要副产物。筛选甘蔗渣纤维素降解菌株对甘蔗渣乙醇产业具有重要的意义。以甘蔗渣为原料,通过分离和纯化得到14株菌株,对其进行纤维素刚果红平板染色实验和滤纸崩解实验,最终获得3株可以生产纤维素酶的菌株02-2-2、21-1-2和40-1-1。酶活性测定结果表明,菌株40-1-1的酶活力在培养3 d后达到最高,为27.26 U/mg。通过形态学和分子生物学鉴定,菌株02-2-2为枝顶孢属(Acremonium sp.),菌株21-1-2和40-1-1为光滑短梗霉属(Acrophialophora sp.)。研究筛选的菌株将为开展甘蔗渣纤维素降解利用提供参考。     

Enhanced enzymatic hydrolysis of sugarcane bagasse by N-methylmorpholine-N-oxide pretreatment

The cellulose dissolution solvent used in Lyocell process for cellulose fiber preparation, N-methylmorpholine-N-oxide (NMMO) monohydrate, was demonstrated to be an effective agent for sugarcane bagasse pretreatment. Bagasse of 20wt% was readily dissolved in NMMO monohydrate at 130 degrees C within 1h. After dissolution, bagasse could be regenerated by rapid precipitation with water as a porous and amorphous mixture of its original components. The regenerated bagasse exhibited a significant enhancement on enzymatic hydrolysis kinetic. Not only the reducing sugars releasing rate but also hydrolysis yield was enhanced at least twofold as compared with that of untreated bagasse. The cellulose fraction of regenerated bagasse was nearly hydrolyzed to glucose after 72h hydrolysis with Cellulase AP3. The recycled NMMO demonstrated the same performance as the fresh one on bagasse pretreatment for hydrolysis enhancement. The regenerated bagasse was directly used in simultaneous saccharification and fermentation (SSF) for ethanol production by Zymomonas mobilis. No negative effect on ethanol fermentation was observed and ethanol yield approximately 0.15 g ethanol/g baggasse was achieved.     

Saccharification and fermentation of Sugar Cane bagasse by Klebsiella oxytoca P2 containing chromosomally integrated genes encoding the Zymomonas mobilis ethanol pathway

Pretreatment of sugar cane bagasse is essential for a simultaneous saccharification and fermentation (SSF) process which uses recombinant Klebsiella oxytoca strain P2 and Genencor Spezyme CE. Strain P2 has been genetically engineered to express Zymomonas mobilis genes encoding the ethanol pathway and retains the native ability to transport and metabolize cellobiose (minimizing the need for extracellular cellobiase). In SSF studies with this organism, both the rate of ethanol production and ethanol yield were limited by saccharification at 10 and 20 filter papaer units (FPU) g(-1) acid-treated bagasse. Dilute slurries of biomass were converted to ethanol more efficiently (over 72% of theoretical yield) in simple batch fermentations than slurries containing high solids albeit with the production of lower levels of ethanol. With high solids (i.e., 160 g acid-treated bagasse L(-1)), a combination of 20 FPU cellulase g(-1) bagasse, preincubation under saccharification conditions, and additional grinding (to reduce particle size) were required to produce ca. 40 g ethanol L(-1). Alternatively, almost 40 g ethanol L(-1) was produced with 10 FPU cellulase g(-1) bagasse by incorporating a second saccharification step (no further enzyme addition) followed by a second inoculation and short fermentation. In this way, a theoretical ethanol yield of over 70% was achieved with the production of 20 g ethanol 800 FPU(-1) of commercial cellulase. (c) 1994 John Wiley & Sons, Inc.     

从海水环境分离筛选甘蔗渣纤维素降解菌

【目的】筛选海水环境高效甘蔗渣纤维素降解菌,并研究不同菌株间的混合发酵对甘蔗渣纤维素酶活力的影响,为纤维素降解菌在海水养殖中的应用提供理论基础。【方法】采用刚果红染色法进行菌株初筛,利用DNS法测定各菌株胞外纤维素酶活力及不同菌株间的混合酶液与混合发酵酶液的纤维素酶活力。【结果】筛选得到两株具有较强纤维素分解能力的细菌菌株Z4和S5,经16S rRNA基因序列分析,初步鉴定为地衣芽孢杆菌(Bacillus licheniformis)。菌株S5具有最高的全酶活和甘蔗渣纤维素酶活,分别为1.16 U/mL和2.80 U/mL。菌株Z4与S5间混合发酵能明显提高菌株的纤维素酶活力,比S5单独发酵时全酶活、甘蔗渣纤维素酶活分别提高40.60%、14.21%。同时菌株S5与芽孢杆菌BZ5混合发酵也能提高其纤维素酶活力,比S5单独发酵时全酶活、甘蔗渣纤维素酶活分别提高6.23%、25.92%。【结论】筛选得到两株酶系较全且酶活较高的纤维素降解菌Z4、S5,适宜的混合发酵可明显提高纤维素降解能力,在海水养殖中有较大的应用前景。     

Chemical characteristics and ethanol fermentation of the cellulose component in autohydrolyzed bagasse

The chemical characteristics, enzymatic saccharification, and ethanol fermentation of autohydrolyzed lignocellulosic material that was exposed to steam explosion were investigated using bagasse as the sample. The effects of the steam explosion on the change in pH, organic acids production, degrees of polymerization and crystallinity of the cellulose component, and the amount of extractive components in the autohydrolyzated bagasse were examined. The steam explosion decreased the degree of polymerzation up to about 700 but increased the degree of crystallinity and the micelle width of the cellulose component in the bagasse. The steam explosion, at a pressure of 2.55 MPa for 3 mins, was the most effective for the delignification of bagasse. 40 g/L of glucose and 20 g/L of xylose were produced from 100 g/L of the autohydrolyzed bagasse by the enzymatic saccharification using mixed cellulases, acucelase and meicelase. The maximum ethanol concentration, 20 g/L, was obtained from the enzymatic hydrolyzate of 100 g/L of the autohydrolyzed bagasse by the ethanol fermentation usingPichia stipitis CBS 5773; the ethanol yield from sugars was 0.33 g/g sugars.     

Effect of nutritional factors on cellulase enzyme and microbial protein production by Aspergillus terreus and its evaluation

The biomass yield, cellulolytic activity, and protein recovery using Aspergillus terreus GN1 with alkali-treated sugarcane bagasse was studied using different levels (250-600 mg of N/L of broth) of organic and inorganic nitrogen sources. e.g., cattle urine, urea, cornsteep liquor, ammonium sulfate, ammonium nitrate, ammonium iron sulfate, ammonium chloride, and sodium nitrate. Among different levels of alkali-treated bagasse substrate concentrations (0.5-4.0% w/v) tested, 1.0% substrate yielded the highest crude protein content, protein recovery, and cellulolytic activity. The biomass recovery with 1.0% substrate ranged from 290-380 mg/500 mg bagasse substrate in a 50-mL broth with a nitrogen level of 250-600 mg of N/L in all the sources except ammonium iron sulfate, which yielded 402-439 mg/500 mg bagasse substrate. However, crude protein content of biomass obtained with an ammonium iron sulfate nitrogen source was the lowest. Cornsteep liquor nitrogen source at the rate of 600 mg of N/L yielded the maximum crude protein of 32.9%, protein recovery of 22.2 g/100 g of bagasse, and carboxymethyl cellulase and filter paper enzyme activities of 1.1 and 0.09 units/mL, among the organic and inorganic nitrogen sources studied. In general, the organic nitrogen sources and inorganic nonammonium nitrogen sources were utilized preferentially for protein production over the inorganic ammonium nitrogen sources. The fermentation time required under optimum cultural and nutritional conditions for A. terreus GN1 was also evaluated. The crude protein content of the biomass increased gradually up to the seventh day of fermentation, but the protein recovery rate was high up to two or three days. It was observed that the cellulose utilization rate increased after an initial lag of one day up to the third day and gradually increased further, which corresponded positively with protein content, biomass protein recovery, and cellulase enzyme activity. On the seventh day of fermentation, the crude protein content, biomass protein recovery, water-soluble carbohydrate, bagasse cellulose utilization, CMCase, and FPase activities were 32.8%, 20.1 g/100 g of bagasse, 6.2%, 82.7%, 1.0. and 0.08 U/mL, respectively. The final biomass recovered contained 32.8% crude protein content and had an in vitro rumen digestibility (IVRD) coefficient of 68.8%. The biomass contained almost all the essential and nonessential amino acids and was comparable with FAO reference protein. It is concluded that a fermentation time of 72 h gave a faster rate of protein production of 16.9 g/100 g of bagasse with 69.8% bagasse cellulose utilization with 76.0% IVRD. and contained almost all the essential and nonessential amino acids.     

Bioconversion of cellulose into ethanol by Clostridium thermocellum--product inhibition

Direct anaerobic bioconversion of cellulosic substances into ethanol by Clostridium thermocellum ATCC 27405 has been carried out at 60 degrees C and pH 7.0 (initial for 100 L) under continuous sparging of oxygen free nitrogen in a culture vessel. Raw bagasse, mild alkali-treated bagasse, and solka floc were used as substrates. The extent of conversion of raw bagasse (cellulose, 50%; hemicellulose, 25%; lignin, 19%) was observed as 52% (w/w) and 79% (w/w) in the case of mild alkali and steam-treated bagasse (cellulose, 72%; hemicellulose, 11%; lignin, 12%), respectively. Use of bagasse concentration above 10 g/L showed a decreased rate in ethanol production. An inoculum age between 28-30 h and cell mass content of 0.027-0.036 g/L (dry basis) were used. The results obtained with raw and pretreated bagasse have been compared with those of highly pure Solka Floc (hemicellulose, 10%). Studies on the product inhibition indicated a linear fall of the percent of survivors with time. An Arrhenius type correlation between the cell decay rate constant and the product concentration was predicted. Even at low levels, the inhibitory effects of products on cell viability, the specific growth rate, and extracellular cellulase enzyme were observed.     

Submerged cultivation of scytalidium thermophilum on complex lignocellulosic biomass for endoglucanase production

Scytalidium thermophilum endoglucanase production was analyzed on lignocellulosic biomass in submerged cultures at 45 degrees C and 155 rpm for 8 days. Endoglucanase, adsorbability of endoglucanase onto avicel, as well as exoglucanase, and filter paper activities were determined and compared with those on microcrystalline cellulose (avicel) as the main source of carbon. Lentil bran and sunflower seed bagasse yielded c. 1.5 fold more endoglucanase and avicel-adsorbable endoglucanase activity than avicel, and activities on grass clippings were similar. Grass clippings yielded the highest percentage of avicel-adsorbable endoglucanase among all lignocellulosic substrates tested. By the time when endoglucanase activities reached maximal levels, exoglucanase activities on lentil bran, sunflower seed bagasse and grass clippings were c. 1.5-3 fold lower than those on avicel, although a significant difference in filter paper activities was not observed. On lignocellulosic biomass, maximum levels of endoglucanase activity were reached within 3-4 days, and within 6-7 days on avicel.     

Enzymic saccharification of sugarcane bagasse pretreated by autohydrolysis-steam explosion

Pretreatment of bagasse by autohydrolysis at 200 degrees C for 4 min and explosive defibration resulted in the solubilization of 90% of the hemicellulose (a heteroxylan) and in the production of a pulp that was highly susceptible to hydrolysis by cellulases from Trichoderma reesei C-30 and QM 9414, and by a comercial preparation, Meicelase. Saccharification yields of 50% resulted after 24 h at 50 degrees C (pH 5.0) in enzymic digests containing 10% (w/v) bagasse pulps and 20 filter paper cellulase units (FPU). Saccharifications could be increased to more than 80% at 24 h by the addition of exogenous beta-glucosidase from Aspergillus niger. The crystallinity of cellulose in bagasse remained unchanged following autohydrolysis-explosion and did not appear to hinder the rate or extent of hydrolysis of cellulose. Autohydrolysis-exploded pulps extracted with alkali or ethanol to remove lignin resulted in lowere conversions of cellulose (28-36% after 25 h) than unextracted pulps. Alkali extracted pulps arising from autohydrolysis times of more than 10 min at 200 degrees C were less susceptible to enzymic hydrolysis than unextracted pulps and alkali-extracted pulps arising from short autohydrolysis times (e.g., 2 min at 200 degrees C). Autohydrolysis-explosion was as effective a pretreatment method as 0.25M NaOH (70 degrees C/2 h) both yielded pulps that resulted in high cellulose conversions with T. reesei cellulase preparations and Meicelase. Supplementation of T. reesei C-30 cellulose preparations with A. niger beta-glucosidases was effective in promoting the conversion of cellulose into glucose. A ration of FPU to beta-glucosidase of 1:1.25 was the minimum requirement to achieve more than 80% conversion of cellulose into glucose within 24 h. Other factors which influenced the extent of saccharification of autohydrolysis-exploded bagasse pulps were the enzyme-substrate ratio, the substrate concentration, and the saccharification mode.     

Screening and Characterization of the High-Cellulase-Producing Strain Aspergillus glaucus XC9

Cellulose is a kind of renewable resource that is abundant in nature.It can be degraded by microorganisms such as mildew.A mildew strain with high cellulase activity was isolated from mildewy maize cob and classified as Aspergillus glaucus XC9 by morphological and 18S rRNA gene sequence analyses.We studied the effects of nitrogen source,initial pH,temperature,incubation time,medium composition,and surfactants on cellulase production.Maximal activities of carboxymethylcellulase (6,812 U/g dry koji) and filter paperase (172 U/g dry koji) were obtained in conditions as follows:initial pH,5.5-6.0;temperature,30℃;cultivation period,3-4 days;inoculum ratio,6% (vol/vol);sugarcane bagasse/wheat bran ratio,4:6.When bagasse was used as substrate and mixed with wet koji at a 1:1 (wt/wt) ratio,the yield of reducing sugars was 36.4%.The corresponding conversion rate of cellulose to reducing sugars went as high as 81.9%.The results suggest that A.glaucus XC9 is a preferred candidate for cellulase production.