球毛壳菌降解天然木质纤维素能力差异及酶系基因分析

目的:以不同植物中分离到的4株内生球毛壳菌NK102、NK103、NK104和NK105为对象,研究不同生态来源球毛壳菌降解木质素和纤维素的能力。方法:首先采用羧甲基纤维素和纤维素刚果红平板检测各菌株的纤维素降解能力,并利用Bavendamm平板反应检测各菌株的木质素降解能力;将4株菌分别培养在以微晶纤维素、杨树叶和木屑为惟一碳源的液体培养基中,通过检测培养液中纤维素酶和漆酶的酶活力,比较各菌株分解利用天然木质纤维素材料的能力,连续培养12 d后检测培养液中次级代谢产物的合成情况;利用已测序的球毛壳菌CBS148.51的基因组信息,寻找编码木质纤维素降解酶类的基因,为球毛壳菌分解利用木质纤维素提供分子生物学依据。结果:NK102、NK103、NK104和NK105在羧甲基纤维素培养基和纤维素刚果红培养基上都能够生长并形成水解圈;Bavendamm平板反应显示4株菌降解木质素的能力由强到弱依次是NK103、NK102、NK105和NK104。4株菌都能分解利用微晶纤维素、杨树叶和木屑,分泌纤维素酶和漆酶,其中NK102在以木屑为碳源的培养基上纤维素酶活力最强,达到0.76 U/mL发酵液,NK103在以杨树叶为碳源的培养基上漆酶活力最强。与此同时,4株菌在发酵培养过程中都能够稳定地合成球毛壳甲素(ChA),ChA产量受到碳源影响,在以杨树叶为碳源的培养基上,NK104的ChA产量最高,可达到14.88 mg/L发酵液。利用已测序的球毛壳菌CBS148.51的基因组信息,寻找到119个编码纤维素半纤维素酶的基因、8个编码漆酶的基因和2个编码锰过氧化物酶的基因,球毛壳菌具有完整的降解纤维素半纤维素的酶体系,在木质纤维素降解真菌的开发过程中具有重要的研究价值。结论:本研究为球毛壳菌木质纤维素降解过程的研究及该菌种的开发利用奠定了基础。     

球毛壳菌降解天然木质纤维素能力差异及酶系基因分析简

目的：以不同植物中分离到的4株内生球毛壳菌NK102、NK103、NK104和NK105为对象,研究不同生态来源球毛壳菌降解木质素和纤维素的能力。方法：首先采用羧甲基纤维素和纤维素刚果红平板检测各菌株的纤维素降解能力,并利用Bavendamm平板反应检测各菌株的木质素降解能力;将4株菌分别培养在以微晶纤维素、杨树叶和木屑为惟一碳源的液体培养基中,通过检测培养液中纤维素酶和漆酶的酶活力,比较各菌株分解利用天然木质纤维素材料的能力,连续培养12d后检测培养液中次级代谢产物的合成情况;利用已测序的球毛壳菌CBS148．51的基因组信息,寻找编码木质纤维素降解酶类的基因,为球毛壳菌分解利用木质纤维素提供分子生物学依据。结果：NK102、NK103、NK104和NK105在羧甲基纤维素培养基和纤维素刚果红培养基上都能够生长并形成水解圈;Bavendamm平板反应显示4株菌降解木质素的能力由强到弱依次是NK103、NK102、NK105和NK104。4株菌都能分解利用微晶纤维素、杨树叶和木屑,分泌纤维素酶和漆酶,其中NK102在以木屑为碳源的培养基上纤维素酶活力最强,达到0．76U/mL发酵液,NK103在以杨树叶为碳源的培养基上漆酶活力最强。与此同时,4株菌在发酵培养过程中都能够稳定地合成球毛壳甲素（ChA）,ChA产量受到碳源影响,在以杨树叶为碳源的培养基上,NK104的ChA产量最高,可达到14．88mg/L发酵液。利用已测序的球毛壳菌CBS148．51的基因组信息,寻找到119个编码纤维素半纤维素酶的基因、8个编码漆酶的基因和2个编码锰过氧化物酶的基因,球毛壳菌具有完整的降解纤维素半纤维素的酶体系,在木质纤维素降解真菌的开发过程中具有重要的研究价值。结论：本研究为球毛壳菌木质纤维素降解过程的研究及该菌种的开发利用奠定了基础。     

植物生物质生物降解组合菌株的筛选研究

本研究用平板混合培养和实际降解相结合的方法筛选了较优的降解植物生物质的菌株组合。首先将6株纤维素降解菌和7株木质素降解菌两两平行划线接种到基础平板上,通过观察菌落形态,挑选出了9对能较好共存的菌株组合。随后对这9个组合进行实际降解稻草的研究,培养结束后体系中半纤维素、纤维素和木质素的含量分析结果表明AF93252 M5和XP M5这两个菌株组合较理想,它们混合培养时对稻草的降解效果均优于其单独降解稻草的效果。     

粗毛栓菌降解麦草木质纤维素的试验研究

正交试验结果表明 ,培养基质中葡萄糖的存在 ,抑制粗毛栓菌对麦草木质纤维素的降解作用 ;适量添加酒石酸 ,可提高该菌对木素的降解程度。粗毛栓菌有较强的降解麦草木质纤维素的能力 ,培养 6 0d后 ,原麦草中6 6 .2 1%纤维素、71.96 %半纤维素和 70 .14%木质素将分别消失     

贝叶多孔菌营养生理生化特性的研究

本文对贝叶多孔菌(Polyporus frondosus)在柞树木屑——麦麸基物上生长期间基物的降解特性和培养物中胞外纤维素酶、半纤维素酶和淀粉酶在培养过程中的活性变化规律及其它一些生物化学性质进行了研究。认为贝叶多孔菌是白腐型木腐真菌。在培养初期主要利用基物中可溶性糖类为碳源,在子实体发育期,碳源基本由降解基物中木质纤维素所提供。     

粗糙脉孢菌木质纤维素降解利用研究进展简

粗糙脉孢菌作为木质纤维素降解真菌,不仅具有完整的木质纤维素降解酶系,而且还拥有全基因组基因敲除突变体库,是研究丝状真菌纤维素酶表达分泌和木质纤维素降解机制的优秀体系。近年来,国内外利用粗糙脉孢菌系统,在木质纤维素降解机制方面取得了显著进展,包括纤维素酶信号传导、调控以及生物质降解后糖的转运利用等。笔者就相关方面的进展进行综述,并对利用粗糙脉孢菌研究木质纤维素降解利用进行展望,总结和分析木质纤维素降解机制研究的国际前沿动态,有助于加深本领域研究人员对真菌体系纤维素降解机制的理解。     

粗糙脉孢菌木质纤维素降解利用研究进展

粗糙脉孢菌作为木质纤维素降解真菌,不仅具有完整的木质纤维素降解酶系,而且还拥有全基因组基因敲除突变体库,是研究丝状真菌纤维素酶表达分泌和木质纤维素降解机制的优秀体系。近年来,国内外利用粗糙脉孢菌系统,在木质纤维素降解机制方面取得了显著进展,包括纤维素酶信号传导、调控以及生物质降解后糖的转运利用等。笔者就相关方面的进展进行综述,并对利用粗糙脉孢菌研究木质纤维素降解利用进行展望,总结和分析木质纤维素降解机制研究的国际前沿动态,有助于加深本领域研究人员对真菌体系纤维素降解机制的理解。     

解糖热解纤维素菌F32降解未预处理秸秆及产纤维素酶特性分析

【目的】明确极端嗜热厌氧木质纤维素降解菌解糖热解纤维素菌F32代谢特征,并分析其产酶特性。【方法】使用细胞计数法绘制菌株的生长曲线,使用离子色谱及气相色谱进行产物和残糖量分析,以DNS法及对硝基苯酚法检测菌株胞外蛋白的酶活性。【结果】解糖热解纤维素菌F32在以葡萄糖、微晶纤维素和未经预处理小麦秸秆为碳源时生长状况优于解糖热解纤维素菌DSM 8903。在以葡萄糖为碳源进行培养时,与菌株DSM 8903相比,菌株F32具有产乳酸较多,而产氢气较少的特点。在以微晶纤维素和未经预处理小麦秸秆为碳源进行培养时,与菌株DSM 8903相比,菌株F32胞外蛋白具有较高的内切纤维素酶活性和木聚糖酶活性。【结论】解糖热解纤维素菌F32表现出较强的木质纤维素降解能力,其与DSM 8903的产物组成及胞外蛋白的酶活性具有明显差异。     

白腐菌混合发酵产酶及对秸秆木质纤维素的降解研究

采用平板显色法从五株白腐菌中筛选出一组相容性较好且产漆酶的组合,进行了混合发酵产酶及秸秆降解实验,结果发现混合发酵相对于纯种培养具有较明显优势,不仅体系中漆酶的活性大为增强,而且拥有更全的胞外酶组成;同比情况下,混合发酵对农作物秸秆木质纤维素的降解程度也最大.     

瘤胃中木质纤维素降解菌及降解酶基因的研究进展

摘要：反刍动物瘤胃是公认的木质纤维素高效降解的天然反应器,对瘤胃微生物的研究成为开发生物能源的热点领域之一。其研究手段已经从传统的依赖分离培养从瘤胃中获得木质纤维素降解菌,并对降解菌中的木质纤维素降解酶逐一分析,发展到通过基因组/元基因组技术,直接从瘤胃中发现获得大量新的木质纤维素降解酶基因/基因簇,进而探讨其降解的分子机理。已有的研究结果表明,瘤胃微生物降解木质纤维素的过程非常复杂,其中涉及到大量不同种类的微生物、酶及基因/基因簇,随着新分析技术的建立和完善,对这些微生物、酶和基因的研究已取得了诸多进展。本论文综述报道了近期有关该方向的研究进展。     

Detection of siderophore production from several fungi and bacteria by a modification of chrome azurol S (CAS) agar plate assay.

A well-known and widely used method for detection of siderophore production by microorganisms in solid medium is the universal chrome azurol S (CAS)-agar plate assay. However, the high toxicity of CAS-blue agar medium caused by the presence of a detergent impedes its utilization with many varieties of fungi and Gram-positive bacteria. To solve this problem, a modification of the CAS-agar plate assay was made by incorporating the CAS-blue dye in a medium with no contact with the microorganisms tested. Half of each plate used in our experiments was filled with the most appropriate culture medium for each type of microorganism and the other half with CAS-blue agar. This modification allowed us to study several strains of fungi (basidiomycetes, deuteromycetes, ascomycetes and zygomycetes) and bacteria (Gram positive and negative), some of them appearing for the first time in the literature. All the microorganisms grew properly and reacted in different manners to the CAS assay. Some strains of wood-decaying basidiomycetes (mainly white-rot fungi) and Aspergillus species produced the fastest color-change reactions in the CAS-blue agar. This modified method could facilitate optimization of culture conditions, since both CAS-blue agar and growth medium were prepared and added in the Petri plate separately.     

Detection of Legionella pneumophila in bioaerosols by polymerase chain reaction

Most studies focusing on detecting microorganisms in air by polymerase chain reaction (PCR) have used a liquid impinger to sample bioaerosols, mainly because a liquid sample is easy to be processed by PCR analysis. Nevertheless, the use of multiple-hole impactors for the analysis of bioaerosols by PCR has not been reported despite its great utility in culture analysis. In this study we have modified the impaction onto an agar surface sampling method to impaction onto a liquid medium using the MAS-100 air sampler (Merck) (single-stage multiple-hole impactor). To evaluate the recovery of airborne microorganisms of both sampling methods, a suspension containing Escherichia coli was artificially aerosolized and bioaerosols were collected onto Tergitol-7 agar and phosphate-buffered saline (PBS) with the MAS-100. A linear regression analysis of the results showed a strong positive correlation between both sampling methods (r = 0.99, slope 0.99, and y intercept 0.07). Afterwards, the method of impingement into a liquid medium was used to study airborne Legionella pneumophila by PCR. A total of 64 samples were taken at a wastewater treatment plant, a chemical plant, and an office building and analyzed by culture and PCR. Results showed that three samples were positive both by PCR and plate culture, and that nine samples negative by plate culture were positive by PCR, proving that L. pneumophila was present in bioaerosols from these three different environments. The results demonstrate the utility of this single-stage multiple-hole impactor for sampling bioaerosols, both by culture and by PCR.     

不同培养基对放线菌TRM10325抑制群感效应活性的影响

检测不同培养基条件下,放线菌TRM10325发酵液抑制群感效应的活性,初步了解其活性稳定性,并为筛选最优发酵培养基提供实验依据。选取17种合成培养基、26种天然培养基发酵放线菌TRM10325,采用微孔板半定量法检测其对紫色素杆菌群感效应以及表皮葡萄球菌生物膜形成的抑制作用。不同配方的培养基对放线菌10325抑制群感效应活性的影响也不相同,其中Am6培养基抑制群感效应效果最佳。成分不同的培养基,明显影响微生物不同次级代谢产物的产生。同一微生物在不同培养基中发酵,其次生代谢产物的种类和含量变化很大。最终选定Am6培养基为最适发酵培养基。     

Anaerobic Simulated Mixed Culture System

A compartmented, autoclavable culture vessel has been developed for the purpose of studying interactive associations of microorganisms which are essential to the anaerobic decomposition of sewage sludge. The unit employs sterile filter membranes to subdivide the interior culture space into individual compartments. Bacteria cultured in one compartment are denied access to adjacent compartments, even though rapid interchange of nutrients and metabolic waste products occurs throughout the unit. The obligate methane-forming anaerobe, Methanobacillus omelianskii has been successfully grown and concentrated in this system by use of a synthetic medium reduced with sodium sulfide. The feasibility of using this system to study microbial interactions was, in part, demonstrated by growing M. omelianskii in a thoroughly aerated medium which had been biologically reduced by Escherichia coli prior to inoculation with the anaerobe. Under this condition of simulated mixed culture growth, the cell yield of both microorganisms, as well as specific metabolic activities ascribed to each organism, was readily monitored.     

Use of CAS-agar plate modified to study the effect of different variables on the siderophore production by Aspergillus

AIMS: To evaluate the suitability of chrome azurol S (CAS) agar plate assay as a quantitative methodology for siderophore production. METHODS AND RESULTS: Aspergillus species (A. flavus, A. niger, A. tamarii) were inoculated in the CAS-agar plates and the siderophores production was determined and expressed as CAS-reaction rate (mm per day). All the species showed positive CAS reaction with different rates depending on culture conditions and A. flavus showed the highest CAS-reaction rate. The siderophore production in solid medium expressed as CAS-reaction rate was correlated with siderophore production in liquid medium. CONCLUSIONS: The use of CAS-agar plate assay was modified and the evaluation of CAS reaction in mm per day made it possible to study and quantify the effect of several variables on the siderophore production by Aspergillus fungi. SIGNIFICANCE AND IMPACT OF THE STUDY: We describe the CAS-agar plate assay as a quantitative methodology, which make it possible to select and evaluate the siderophore production by several microorganisms (fungi and bacteria) according to different culture conditions.     

Description and evaluation of reciprocating plate bioreactors

The environment in which live microorganisms has a major impact on their productivity. One important factor is the mechanical mixing that is used to promote good heat and mass transfer in bioreactors. In this paper, the performance of reciprocating plate bioreactors is first evaluated for their ability to produce high oxygen transfer coefficients. Pure water and a glycerol water (5050 wt%) solution are used for this evaluation. Then, the performance of reciprocating plate bioreactors for the production of an exocellular polysaccharide (pullulan) by yeast Aureobasidium pullulans is analyzed in terms of quantity and quality of the polysaccharide. Results clearly show that a more efficient substrate utilisation is achieved with reciprocating plate bioreactors.     

Effect of a modification of the medium on the structure of bacterial cells based on electron microscopy data

Electron microscopy was used to study the interactions between microorganisms and the culture medium in the presence of modifying agents. Temperature-dependent changes in the intensity of water-glycerin exchange between the cells and the medium are demonstrated, which is of interest for the optimization of the conditions of using protectors, as well as for the study of the permeability of cell membranes.     

CAS平板覆盖法检测氢氧化细菌铁载体

【目的】用CAS平板覆盖法检测氢氧化细菌铁载体,解决通用CAS琼脂平板法中十六烷基三甲基溴化铵对真菌和某些细菌的生长抑制问题。【方法】将改良的CAS检测培养基覆盖在长满菌落的无铁培养基上,生长抑制问题因微生物未与十六烷基三甲基溴化铵直接接触而解决。【结果】3株氢氧化细菌SDW-5、SDW-9和AaP-13均能产生单菌落,加入CAS检测培养基1 h后,菌落周围产生明显的铁载体晕圈。【结论】本方法成功解决了生长抑制问题,可以作为检测微生物铁载体的通用方法。     

Culturable and metagenomic approaches of wheat bran and wheat straw phyllosphere’s highlight new lignocellulolytic microorganisms

The phyllosphere, defined as the aerial parts of plants, is one of the most prevalent microbial habitats on earth. The microorganisms present on the phyllosphere can have several interactions with the plant. The phyllosphere represents then a unique niche where microorganisms have evolved through time in that stressful environment and may have acquired the ability to degrade lignocellulosic plant cell walls in order to survive to oligotrophic conditions. The dynamic lignocellulolytic potential of two phyllospheric microbial consortia (wheat straw and wheat bran) has been studied. The microbial diversity rapidly changed between the native phyllospheres and the final degrading microbial consortia after 48 h of culture. Indeed, the initial microbial consortia was dominated by the Ralstonia (35·8%) and Micrococcus (75·2%) genera for the wheat bran and wheat straw whereas they were dominated by Candidatus phytoplasma (59%) and Acinetobacter (31·8%) in the final degrading microbial consortia respectively. Culturable experiments leading to the isolation of several new lignocellulolytic isolates (belonging to Moraxella and Atlantibacter genera) and metagenomic reconstruction of the microbial consortia highlighted the existence of an unpredicted microbial diversity involved in lignocellulose fractionation but also the existence of new pathways in known genera (presence of CE2 for Acinetobacter, several AAs for Pseudomonas and several GHs for Bacillus in different metagenomes-assembled genomes). The phyllosphere from agricultural co-products represents then a new niche as a lignocellulolytic degrading ecosystem.