真菌产纤维素酶培养基中刚果红转移机理研究*

通过对产纤维素酶真菌在纤维素刚果红液体培养基中刚果红染料移动情况研究,表明刚果红染料进入真菌的机制为纤维素分解真菌首先分解纤维素物质为含有葡聚糖等结构的多聚糖类物质,多聚糖与刚果红形成多聚糖-刚果红复合物,复合物不仅被吸附到产纤维素酶活的菌丝外表面,而且能被进一步转运吸收至该部分菌丝内部,使菌丝体和菌落呈现红色。所以,纤维素刚果红培养基可作为分离、筛选纤维素分解真菌的特异性培养基。     

一种纤维素分解菌鉴别培养基

一种新的鉴别性纤维素刚果红培养基,含酸洗纤维素为唯一碳源和染料刚果红,产纤维素酶菌株在其上形成浓郁红色水解圈,明显区别于其它微生物类群,方便纤维素分解菌的筛选和计数。     

稻草秸秆纤维素分解菌的分离筛选

本研究基于获得高效木质纤维素分解菌的目的,以刚果红纤维素琼脂和滤纸条培养基为初筛培养基,从分离获得的124株真菌中筛选出透明圈与菌落直径比值较大、滤纸条分解能力较强的11个菌株.经液体发酵,测定其酶活力,复筛得到羧甲基纤维素酶活和滤纸酶活均较高的4个菌株;并进行了不同碳源和不同pH对筛选菌株产酶能力的影响试验,发现不同菌株对不同纤维素物质的分解能力不一样,同一菌株对不同纤维素碳源的利用能力也不相同.     

三种霉菌产纤维素酶能力分析与培养条件优化

对实验室现有3种真菌产纤维素酶能力的分析及培养条件优化。比较了3种菌在刚果红培养基上的透明圈大小、并分析产纤维素酶酶活;通过单因素与响应面分析的方法优化毛酶产纤维素酶的培养条件。通过试验得出3种真菌均能产纤维素酶,毛霉能产较多的纤维素酶。毛霉产纤维素酶的最佳条件为:pH 5.0,转速220 r/min,发酵时间47 h,发酵温度35℃,纤维素酶活为6.99U/mL。毛霉、青霉、曲霉均产纤维素酶,毛霉能降解玉米芯纤维素。     

纤维素酶产生菌及其发酵条件优化

通过刚果红染色产脱色圈初筛出能分解纤维素的菌株,再利用DNS法分别测定纤维素酶的酶活,得到分解纤维素能力最强的一株真菌Lv-1。该菌株菌丝为黄绿色,孢子为深绿色,有多分支的无隔菌丝,长孢子囊孢子,用DNS法测定其酶活为62.42 U/m L,后对其进行了产酶条件优化。经单因素试验和正交试验得到该菌的最佳产酶条件为:在以10 g/L羧甲基纤维素钠为碳源,4 g/L蛋白质,2 g/L硫酸铵为氮源,1 g/L硫酸二氢钾为无机盐的最适产酶培养基中,初始p H为6.0,培养温度37℃,装液量100 m L/250 m L,发酵36 h。在此发酵条件下,其产酶活力可达96.898U/m L,试验结果显示,该菌株在产纤维素酶能力上具有显著优势,且菌株Lv-1产酶活力较优化前高出34.478 U/m L,提高了55.24%。     

多孔菌Trichaptum abietinum 1302BG自然条件下对合成染料刚果红和酸性品红的高效降解

选用杭州竹林土壤分离并筛选能够降解多种类型染料的真菌。经大量筛选发现一株编号为1302BG的真菌能够在固体培养基上分解所测试的全部9种染料(苯胺蓝、刚果红、橙黄G、甲基红、甲基橙、结晶紫、酸性品红、番红花红、碱性品红、甲基紫)。经形态学和分子生物学方法鉴定, 该菌1302BG为冷杉附毛孔菌(Trichaptum abietinum)。在液体培养基中研究了pH、温度、碳源、氮源、碳氮源组合、碳氮源浓度等参数对该菌脱色效果的影响, 以寻找最适最经济的脱色条件。在液体培养基中研究表明, 冷杉附毛孔菌1302BG既能在酸性又能在碱性条件下有效分解2种测试染料(酸性品红和刚果红)。该真菌能以仅含有0.5 g/L淀粉和0.05 g/L硫酸铵的经济、环境友好的培养基为底物, 能在灭菌和非灭菌(自然)的条件下高效脱色, 在24 h内对2种染料的脱色率均在90%以上。紫外/可见光谱及微核试验分析显示, 该菌脱色主要是以生物降解为主, 2种染料经该菌分解后的毒性也同时大大降低。这些优异特点显示了该菌具有非常广阔的工业染料废水处理应用潜力。     

土壤霉菌菌丝球的筛选、鉴定及固定化菌丝球的应用

从土壤中分离纯化真菌并鉴定为烟曲霉L-3。以菌株L-3作为固定化载体,将地衣芽孢杆菌固定在真菌上组成固定化体系。研究了混菌菌丝球,菌丝饼,发酵混合液,粗漆酶液对刚果红染料废水的降解情况并对染料废水进行毒性试验。结果表明,菌丝球对染料废水的降解效果最显著,降解率高达99.96%,菌丝饼仅用20 s降解率为91%,发酵混合液与粗漆酶液的处理效果并不显著。该体系对染料废水的去毒率较明显,尤其是菌丝球的去毒率可达到78%。可见,固定化体系对染料废水不但有较高的降解能力,也有较高的去毒率。     

果胶分解菌的简便筛选方法

果胶醇是分解果胶的一类酶的总称,被广泛应用于食品工业中,也可用于麻类脱胶及木材防腐等。能产生果胶酶的微生物种类很多,果胶分解菌的筛选方法已有一些报道,如透明带法、十六烷基三甲基澳化铁（多糖沉淀剂）的透明圈法、钉红染色的透明圈法。这些方法或者效果不明显,或者果胶用量较大,或者成本较高,在实际工作中受到限制。刚果红染料可与多糖水解物形成有浓郁色泽的复合物［7］,因此Hen－driCkS等人及叶姜瑜分别利用刚果红对纤维素分解菌进行筛选和计数阳。我们经过试验,发现刚果红也可用于果胶分解菌的筛选和计数。1果胶琼脂…     

一株纤维素降解真菌的筛选、鉴定及酶学性质分析

通过对富含枯枝败叶的土壤样品进行富集培养,利用刚果红纤维素培养基初筛和酶活测定复筛得到产纤维素酶的一株真菌,将其命名为GC2-2,并对该菌株进行鉴定及酶学性质研究。结果表明该菌株是一株耐高温、碱性纤维素酶的真菌GC2-2。通过18S rDNA分子克隆测定,该菌为球孢枝孢菌,其滤纸酶的活力优于CMC酶的活力。该菌所产酶的最适反应条件为温度35°C,最适pH值7.5。     

纤维素降解的菌株筛选及其运用

目的:筛选降解稻草纤维素菌株,为纤维素的高效降解提供理论依据.方法:采用羧甲基纤维素钠刚果红培养基与滤纸条培养基从采集的腐木、腐土和腐叶等样品中筛选出纤维素降解菌.然后经液态发酵后测定其羧甲基纤维素酶活力与降解稻草的天然纤维素酶活力,综合考虑这两种酶活力,对其进行单独与混合发酵培养.筛选分解稻草能力较强的菌株组合.结果:初筛到5株真菌和5株细菌纤维素降解力较优的菌株.经酶活力测定后,得到分解纤维素能力较强的两株真菌F3和F5与两株细菌B1和B5,其中F3和B1的羧甲基纤维素酶活分别为705.6U、214.6U;F5和B5天然纤维素酶活分别为466.5U、204.8 U.混合培养在一定程度上能提高纤维素酶活,F3/F5具有稳定而较高的酶活力,某时间段酶活高达646.8U,且后续酶活力也保持在较高水平.而F3/B5在某时间段的酶活高达788.6U.结论:菌株的混合培养可以提高纤维素酶活.     

A Rapid and Easy Method for the Detection of Microbial Cellulases on Agar Plates Using Gram’s Iodine

Screening for cellulase-producing microorganisms is routinely done on carboxymethylcellulose (CMC) plates. The culture plates are flooded either with 1% hexadecyltrimethyl ammonium bromide or with 0.1% Congo red followed by 1 M NaCl. In both cases, it takes a minimum of 30 to 40 minutes to obtain the zone of hydrolysis after flooding, and the hydrolyzed area is not sharply discernible. An improved method is reported herein for the detection of extracellular cellulase production by microorganisms by way of plate assay. In this method, CMC plates were flooded with Gram's iodine instead of the reagents just mentioned. Gram's iodine formed a bluish-black complex with cellulose but not with hydrolyzed cellulose, giving a sharp and distinct zone around the cellulase-producing microbial colonies within 3 to 5 minutes. The new method is rapid and efficient; therefore, it can be easily performed for screening large numbers of microbial cultures of both bacteria and fungi. This is the first report on the use of Gram's iodine for the detection of cellulase production by microorganisms using plate assay.     

南方红豆杉内生真菌产油及降解纤维素的研究

从油脂植物南方红豆杉Taxusmairei茎中分离到26株内生真菌,用形态学方法鉴定表明,交链孢霉属Alternaria、无孢菌群MyceliaSterilia和硬内囊霉属Sclerocystis为优势类群;用苏丹黑染液对这26株内生真菌菌丝染色后,在光学显微镜下观察,发现其中14株内生真菌菌丝中有明显的油滴存在,选出其中油滴较大较多的7株在PDA液体培养基中培养6d后提取油脂,结果它们的油脂含量为细胞干重的13.2%~29.5%;这26株内生真菌在以微晶纤维为唯一碳源的液体培养基中培养8d后,测定发酵液的滤纸酶活,发现酶活为6.5~17.8μg葡萄糖/mL·min。研究结果表明南方红豆杉内生真菌中存在大量产油菌株,且它们有以纤维素为碳源生长的潜力。本研究为筛选能以秸秆中纤维素为碳源积累油脂的菌株打下基础。     

Ultrastructural and cytochemical aspects of the interaction between the ectomycorrhizal fungus Laccaria laccata and the saprotrophic fungi, Trichoderma harzianum and T. virens

Different interactions between soil fungi competing in the rhizosphere with each other are necessary to understand their influence on plant growth and health. The interactions between the ectomycorrhizal (ECM) fungus Laccaria laccata and soil saprotrophic fungi (T. harzianum, T. virens) were studied by transmission electron microscopy, and by gold cytochemistry to assess the potential role of cell wall lytic enzymes in mycoparasitism. Anti-β-1,3-glucan antibody, WGA/ovomucoid-gold complex and PATAg test were used to localize β-1,3-glucan, chitin and polysaccharides. Cytoplasm disorganisation of the saprotrophic fungi occurred concurrently with dissolution of β-1,3-glucan in walls of hyphae and conidia of the saprotrophic fungi. Then digestion of polysaccharides and chitin of colonised fungal structures occurred. The studies suggest sequential contribution of cell wall lytic enzymes and importance of disturbing the host's cell integrity during mycoparasitism. We conclude that the ECM fungus can parasitise on the saprotrophic fungi not only in dual culture on artificial medium but also in the rhizosphere of Scots pine.     

Metschnikowia strains isolated from botrytized grapes antagonize fungal and bacterial growth by iron depletion

Noble-rotted grapes are colonized by complex microbial populations. I isolated pigment-producing Metschnikowia strains from noble-rotted grapes that had antagonistic activity against filamentous fungi, yeasts, and bacteria. A red-maroon pigment was formed from a diffusible colorless precursor released by the cells into the medium. The conversion of the precursor required iron and could occur both in the cells (red colonies) and in the medium (red halos around colonies). The intensity of pigmentation was correlated with the intensity of the antimicrobial activity. Mutants that did not form pigment also lacked antifungal activity. Within the pigmented halos, conidia of the sensitive fungi did not germinate, and their hyphae did not grow and frequently lysed at the tips. Supplementation of the medium with iron reduced the size of the halos and the inhibition zones, while it increased the pigment accumulation by the colonies. The iron-binding agent tropolone had a similar effect, so I hypothesize that pigmented Metschnikowia isolates inhibit the growth of the sensitive microorganisms by pigment formation, which depletes the free iron in the medium. As the pigment is a large nondiffusible complex produced in the presence of both low and high concentrations of ferric ions, the proposed mechanism is different from the mechanisms operating in microbes that release siderophores into the environment for iron acquisition.     

Metschnikowia Strains Isolated from Botrytized Grapes Antagonize Fungal and Bacterial Growth by Iron Depletion

Noble-rotted grapes are colonized by complex microbial populations. I isolated pigment-producing Metschnikowia strains from noble-rotted grapes that had antagonistic activity against filamentous fungi, yeasts, and bacteria. A red-maroon pigment was formed from a diffusible colorless precursor released by the cells into the medium. The conversion of the precursor required iron and could occur both in the cells (red colonies) and in the medium (red halos around colonies). The intensity of pigmentation was correlated with the intensity of the antimicrobial activity. Mutants that did not form pigment also lacked antifungal activity. Within the pigmented halos, conidia of the sensitive fungi did not germinate, and their hyphae did not grow and frequently lysed at the tips. Supplementation of the medium with iron reduced the size of the halos and the inhibition zones, while it increased the pigment accumulation by the colonies. The iron-binding agent tropolone had a similar effect, so I hypothesize that pigmented Metschnikowia isolates inhibit the growth of the sensitive microorganisms by pigment formation, which depletes the free iron in the medium. As the pigment is a large nondiffusible complex produced in the presence of both low and high concentrations of ferric ions, the proposed mechanism is different from the mechanisms operating in microbes that release siderophores into the environment for iron acquisition.     

Possible role of soil microorganisms in aggregation in soils

In many soils, roots and fungal hyphae, especially those of vesicular arbuscular mycorrhizal (VAM) fungi, stabilize macroaggregates (>250 μm diameter); organic residues, bacteria, polysaccharides and inorganic materials stabilize microaggregates (<250 μm). This review discusses the factors (including other organisms) which affect VAM hyphae and their extracellular polysaccharides in soil, and the subsequent effect on stability of aggregates. The review also discusses the possible role of other organisms, including ectomycorrhizal fungi, in the stability of soil, and suggests future research.     

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。     

链霉菌A048产几丁质酶最佳发酵工艺研究

将链霉菌A048在完全培养基中培养至对数生长末期,离心洗涤收集菌丝体,然后接种入发酵产酶培养基中,进行二步发酵工艺牛产几丁质酶,几丁质酶活力比一步发酵工艺提高1．1倍,发酵周期共54h,比一步发酵工艺缩短66h;把菌丝体与几丁质粉共固定化,接入发酵产酶培养基中培养36h,几丁质酶活力比一步发酵工艺提高1．8倍,发酵周期缩短54h;在二步发酵工岂中另添加0．4％纤维素,几丁质酶活力可提高4倍,比一步发酵工艺提高10倍,酶活力达18．52U／mL。采用几丁质和纤维索双因子诱导二步发酵工艺可能是链霉菌A048生产几丁质酶的最佳工艺。     

A Novel screening method of cellulase-producing bacteria based on <Emphasis Type="Italic">Phytophthora parasitica</Emphasis> var. <Emphasis Type="Italic">nicotianae</Emphasis>

Cellulase is the key to utilize the renewable and abundant cellulose resource, cellulase-producing microorganism is an important source of cellulase. The traditional screening method of cellulase-producing microorganism is low efficacy and not macroscopic. The screening method in this study was based on the interactive culture character between cellulase-producing bacteria and Phytophthora parasitica var. nicotianae on plates, the results indicated that the inhibition zone and cellulase activity of bacterial strains are conformity on the whole, so the screening method is very quickly and apparent     

A novel screening method of cellulase-producing bacteria

Cellulase is the key to utilize the renewable and abundant cellulose resource, cellulase-producing microorganism is an important source of cellulase. The traditional screening method of cellulase-producing microorganism is low efficacy and not macroscopic. The screening method in this study was based on the interactive culture character between cellulase-producing bacteria and Phytophthora parasitica var. nicotianae on plates, the results indicated that the inhibition zone and cellulase activity of bacterial strains are conformity on the whole, so the screening method is very quickly and apparent.