Molecular biological detection and characterization of Clostridium populations in municipal landfill sites

Primer sets specific for 16S rRNA genes were designed for four phylogenetic groups of clostridia known to contain mesophilic cellulolytic species. Specific amplification of these groups from landfill leachate DNA extracts demonstrated the widespread occurrence of clostridia from the Clostridium thermocellum and C. leptum groups. In contrast, the C. botulinum group was never detected, and the C. coccoides-C. lentocellum group was only occasionally detected. Amplification products were analyzed by temporal thermal gel electrophoresis to generate profiles of the clostridial groups and to identify dominant bands. Sequence analysis of 17 landfill clones confirmed that the primers were specific for the clostridial subgroups and that the cloned sequences had a close relationship with known cellulose-degrading clostridia. The primers have therefore been authenticated for use in the rapid identification of clostridia in anaerobic environments.     

Biology of gut anaerobic fungi

The obligately anaerobic nature of the gut indigenous fungi distinguishes them from other fungi. They are distributed widely in large herbivores, both in the foregut of ruminant-like animals and in the hindgut of hindgut fermenters. Comparative studies indicate that a capacious organ of fermentative digestion is required for their development. These fungi have been assigned to the Neocallimasticaceae, within the chytridiomycete order Spizellomycetales. The anaerobic fungi of domestic ruminants have been studied most extensively. Plant material entering the rumen is rapidly colonized by zoospores that attach and develop into thalli. The anaerobic rumen fungi have been shown to produce active cellulases and xylanases and specifically colonise and grow on plant vascular tissues. Large populations of anaerobic fungi colonise plant fragment in the rumens of cattle and sheep on high-fibre diets. The fungi actively ferment cellulose which results in formation of a mixture of products including acetate, lactate, ethanol, formate, succinate, CO2 and H2. The properties of the anaerobic fungi together with the extent of their populations on plant fragments in animals on high-fibre diets indicates a significant role for the fungi in fibre digestion.     

Molecular Biological Detection of Anaerobic Gut Fungi (Neocallimastigales) from Landfill Sites

Oligonucleotide primers were designed for the 18S rRNA genes of members of the Neocallimastigales and used in a nested PCR protocol to amplify 787-bp fragments of DNA from landfill site samples. The specificities of the primers were confirmed by phylogenetic analysis of the environmental clone sequences, and this method can therefore now be used to investigate the ecology of the obligately anaerobic fungi. To our knowledge, this is the first demonstration of the occurrence of members of the Neocallimastigales outside the mammalian gut, and their distribution across the landfill samples examined here suggests that they are actively involved in cellulose degradation.     

Hydrolytic activities of anaerobic fungi from wild blue bull (Boselaphus tragocamelus)

The anaerobic fungi play an active role in the plant fibre degradation by producing a wide array of potential hydrolytic enzymes in the rumen. In present study, 12 anaerobic fungal strains were isolated from the faecal samples of wild blue bull, and identified as species of Piromyces, Anaeromyces, Orpinomyces and Neocallimastix based on their morphological characteristics. Isolate WNG-12 (Piromyces sp.), showed maximum filter paper cellulase (23 mIU ml(-1)) and xylanase (127 mIU ml(-1)) activity, while WNG-5 (Piromyces sp.) showed maximum carboxymethyl cellulase activity (231 mIU ml(-1)). Based on the results obtained, it can be stated that Piromyces sp. WNG-12 may be a promising isolate in utilizing fibre rich diets in the rumen as evidenced by the production of hydrolytic enzymes in vitro.     

一种褐飞虱病原真菌的分子生物学鉴定

从田间褐飞虱Nilaparvata lugens( St(a)l)罹病虫体分离得到一株绿僵菌,以该菌为供试菌体,采用氯化苄法、CTAB法以及裂解液法分别提取了该菌的基因组DNA;以ITS1和ITS4为绿僵菌通用引物,对供试菌株的rDNA ITS序列进行PCR扩增、琼脂糖凝胶电泳检测和序列分析,并在核酸序列数据库中进行同...     

Identification of spores in the polycentric anaerobic gut fungi which enhance their ability to survive

Two new isolates of the gut fungi were obtained from the rumen digesta and faeces of a cow. These isolates, designated Anaeromyces following rDNA typing, displayed a polycentric growth habit but differed from all other gut fungi in that they were able to survive in the laboratory for considerable periods without the need for sub-culture. Light microscopy of preparations from old liquid-grown cultures revealed the presence of DNA-containing spores with two or four chambers. A comparative evaluation of the growth produced when fresh media were inoculated with a sample originating from young or old cultures revealed that active growth was delayed with the inoculum from the older culture. We propose that the chambered spores observed in these cultures provide an alternative path in the life cycle of these fungi and may function as a resting stage within the anaerobic environment of the herbivore gut.     

Capacities and limits of three different technologies for the biological treatment of leachate from solid waste landfill sites

Leachate from a municipal waste landfill site was treated using an activated sludge bioreactor, a fluidized bed biofilm reactor and a packed-bed column reactor (trickling filter). The leachate contained high organic matter (2.0–2.6 g/l of COD), high ammonium (300–700 mg/l) and sulphide (200–800 mg/l) concentrations, as well as low metal concentrations. The continuously operating reactors were employed to study the effects of TOC loading on the removal of TOC as well as on the nitrification and denitrification processes. Among the three biological treatment technologies investigated, the fluidized bed biofilm reactor was best with respect to removing ammonia and TOC. More than 90% of TOC and 99% of ammonia were removed when TOC loading was less than 0.5 kg/m³ × d. At a TOC loading of 4 kg/m³ × d, the removal of TOC and ammonia was 80% and 99%, respectively. In contrast, the treatment of leachate with the packed-bed reactor was successful in TOC removing only at TOC loading less than 0.3 kg/m³ × d (TOC elimination decreased from 86% at 0.06 kg/m³ × d to 60% at 0.3 kg/m³ × d). However, the reactor was active in nitrification even at a higher TOC loading (more than a 98% ammonia elimination at a TOC loading of 0.5 kg/m³ × d). Leachate was processed in the activated sludge reactor when TOC loading was less than 0.5 kg/m³ × d (with a removal of TOC and ammonia up to 83% and 99%, respectively). The activated sludge reactor was also effective in TOC removal at a higher TOC loading (e.g. a 74% TOC removal at a TOC loading of 1 kg/m³ × d), but for ammonia elimination, the activity continuously decreased (less than 60% ammonia removal at a TOC loading of 1 kg/m³ × d). Overloading in the activated sludge system was indicated by a high concentration of ammonia and nitrite in the effluent. In the packed bed reactor, overloading was characterized by a progressively incomplete TOC removal. No significant overloading was found in the fluidized bed reactor up to a TOC loading of 4 kg/m³ × d.     

土壤中甲烷厌氧氧化菌多样性的分子检测

甲烷厌氧氧化作用是减少海洋底泥甲烷释放的重要生物地球化学过程,然而在陆地生态系统中甲烷厌氧氧化作用及其功能菌群的生态功能仍然不确定。对甲烷厌氧氧化菌多样性的研究可为减少甲烷排放提供重要科学依据。与传统的分离培养方法比较,分子检测方法是一种更为快速和高效的研究手段,可直接和全面的反映参与甲烷厌氧氧化作用的功能微生物。以DNA分子标记物为研究对象,重点探讨三类主要的分子标记基因,即16S rRNA,mcr A和pmo A,所采用的相关探针和引物信息,同时从定性和定量两个角度综述土壤甲烷厌氧氧化菌的多样性研究的主要进展,最后提出厌氧甲烷氧化菌多样性研究中存在的一些问题和相应的解决思路。     

Trichomycete gut fungi from tropical regions of the world

Fifty-nine species of gut fungi in the orders Harpellales and Asellariales, Class Trichomycetes, have been collected in eight tropical regions of the world, some species occurring in more than one geographic region. Regarding the Harpellales, the rather low number of taxa, compared to reports in more temperate localities, is due primarily to relatively few collections in the tropics, as well as the usually warmer waters found in tropical regions that often have a lower species richness of potential immature insect hosts. Asellariales in freshwater, marine, and terrestrial habitats, likewise, have been seldom inventoried in the tropics. Nonetheless, it is clear that the tropics are fertile grounds for discovering new genera and species, and future investigations will undoubtedly reveal many new taxa that will lead to a better understanding of the evolution and biogeography of Trichomycetes.     

The design of oligonucleotide primers for the universal amplification of the N-acetylglucosaminidase gene (nag1) in Chytridiomycetes with emphasis on the anaerobic Neocallimastigales

The common feature of all chytridiomycetous fungi, aerobic as well as anaerobic, is an abundance of chitin in their cell wall. The genes coding for chitinases have therefore been widely used as phylogenetic markers in ascomycetes. As their utility for Chytridiomycetes has not been determined we chose the gene encoding an enzyme involved in chitin degradation and energy metabolism, the beta-(1,4)-N-acetylglucosaminidase (nag1). Primer pair Nag-forward and Nag-reverse was used to create PCR product from 5 strains of anaerobic and 7 strains of aerobic chytrids. However, Blast search of sequenced amplicons showed that these primers are specific only for fungus Emericella nidulans. Amino acid alignment of Nag1 proteins of fungal, protozoal and bacterial origin available in GenBank database was therefore performed. Five amino acid regions were found to be conserved enough to serve as a suitable domain for the design of a set of primers for the universal amplification of the nag1 gene in the Neocallimastigales fungi.     

Pectinolytic enzymes of anaerobic fungi

Pectinolytic enzymes of four rumen fungi have been described. Three fungal species were monocentric Neocallimastix spp. H15, JL3 and OC2, and one isolate was a polycentric strain of Orpinomyces joyonii , A4. They differed in degree of pectin degradation and utilization. Only the strain Neocallimastix sp. H15 and partially Orpinomyces joyonii A4 were able to utilize pectin to a higher extent. The most important pectinolytic activity in all these isolates represented pectin lyase (EC 4.2.2.10) and polygalacturonase (EC 3.2.1.15). Their specific activities were in the range of 100–900 and 10–450 μg galacturonic acid h^-1 mg protein^-1 for pectin lyase and polygalacturonase, respectively. Polygalacturonase, located mainly in the endocellular fraction, was inhibited by calcium ions and had the main pH optimum at pH 6.0. All strains produced pectate lyase (EC 4.2.2.2). None of the strains tested produced pectinesterase (EC 3.1.1.11).     

Molecular identification of mycorrhizal fungi in Neuwiedia veratrifolia (Orchidaceae)

We here apply a previously described method for identification of single peloton orchid mycorrhiza to a key orchid group and extend the usefulness in the heterobasidiomycetes of an existing fungal database for identification of mycorrhizal fungi. We amplified and sequenced mitochondrial ribosomal large subunit DNA from fungi in roots of Neuwiedia veratrifolia (Orchidaceae), a member of the small subfamily Apostasioideae that is sister to the remainder of Orchidaceae, and used the extended database to identify the mycorrhizal fungi. Sequences from fungi cultured from Neuwiedia roots and from direct peloton amplifications were analyzed cladistically with sequences determined from reference fungal collections and published sequences. The fungi from Neuwiedia are referred to the heterobasidiomycetous orders Tulasnellales and Ceratobasidiales, indicating that apostasioids utilize the same fungi as other photosynthetic orchids. The majority of Neuwiedia mycobionts came together in a clade with Tulasnella species, but some were most closely related to Thanatephorus. In some cases members of these two clades were isolated from the same orchid plant, providing another example of multiple mycobionts occurring in a single plant.     

日本弓背蚁消化道厌氧细菌的分离培养

【目的】采用自行设置的厌氧装置,分离培养日本弓背蚁Camponotus japonicus消化道厌氧细菌。【方法】改进了一种简易的厌氧装置——平皿夹层法,利用4种培养基对日本弓背蚁消化道内厌氧或兼性厌氧菌进行分离培养。【结果】从日本弓背蚁消化道共分离到22个不同的菌株,隶属于厚壁菌门、放线菌门和变形菌门3大类群的17个属;4种培养基分离到的细菌种类存在明显差异,选择性较强,其中从LB和牛肉膏蛋白胨培养基上分离到的细菌种类较多,分别为10种和8种;从MRS和LBS培养基上分离到的细菌较少,分别为3种和1种;大工蚁和小工蚁的消化道细菌组成也存在差异,可能与其在巢群中担负的功能和职责有关,还有待于进一步研究分析。【结论】利用平皿夹层法可以成功分离到日本弓背蚁肠道的厌氧细菌,该种方法对于其他昆虫消化道厌氧菌的分离培养具有一定的参考价值。     

Molecular detection and diversity of polycyclic aromatic hydrocarbon-degrading bacteria isolated from geographically diverse sites

Nineteen polycyclic aromatic hydrocarbon (PAH)-degrading bacteria were isolated from environmental samples in Kuwait, Indonesia, Thailand, and Japan by enrichment with either naphthalene or phenanthrene as a sole carbon source. Sequence analyses of the 16-S rRNA gene indicated that at least seven genera (Ralstonia, Sphingomonas, Burkholderia, Pseudomonas, Comamonas, Flavobacterium, and Bacillus) were present in this collection. Determination of the ability of the isolates to use PAH and its presumed catabolic intermediates suggests that the isolates showed multiple phenotypes in terms of utilization and degradation pathways. The large subunit of the terminal oxygenase gene (phnAc) from Burkholderia sp. strain RP007 hybridized to 32% (6/19) of the isolates, whilst gene probing using the large subunit of terminal oxygenase gene (pahAc) from Pseudomonas putida strain OUS82 revealed no pahAc-like genes amongst the isolates. Using three degenerated primer sets (pPAH-F/NR700, AJ025/26, and RieskeF/R), targeting a conserved region with the genes encoding the large subunit of terminal oxygenase successfully amplified material from 6 additional PAH-degrading isolates. Sequence analyses showed that the large subunit of terminal oxygenase in 4 isolates was highly homologous to the large subunit of naphthalene dioxygenase gene from Ralstonia sp. strain U2. However, we could not obtain any information on the oxygenase system involved in the naphthalene and/or phenathrene degradation by 7 other strains. These results suggest that PAH-degrading bacteria are diverse, and that there are still many unidentified PAH-degrading bacteria.     

Molecular detection,community structure and phylogeny of ericoid mycorrhizal fungi

Through traditional culturing and molecular characterization, we have determined that five putative species and 2 polyphyletic assemblages of fungi produce ericoid mycorrhizae in Gaultheria shallon, other Ericaceae and Epacridaceae. Using phylogenetic analysis of ITS2 sequences in GenBank, we have confirmed that most of these fungi occur in North America, Europe, and Australia. The low recovery rate of culturable ericoid mycorrhizal fungi from Gaultheria shallon may partly be explained by the fact that most mycorrhizal root segments contain an unculturable basidiomycete, revealed by direct amplification, cloning, and sequencing of LSU fungal DNA from root. Molecular characterization and phylogenetic analysis are powerful tools in revealing the geographic distribution and identity of ericoid mycorrhizal fungi.     

垃圾填埋场恶臭污染与控制研究进展

垃圾填埋场是城市公共设施恶臭的重要来源.随着城市化进程的加快和城镇居民对生活环境质量要求的提高,垃圾填埋过程产生的恶臭污染问题日益突出,垃圾填埋场恶臭污染控制已成为目前的研究热点.本文概述了垃圾填埋场恶臭气体的主要成分和浓度范围,重点阐述了垃圾填埋场恶臭原位控制的研究进展,并对今后垃圾填埋场恶臭气体控制的研究方向进行了展望.     

The microbial ecology of anaerobic cellulose degradation in municipal waste landfill sites: evidence of a role for fibrobacters

Cellulose is reputedly the most abundant organic polymer in the biosphere, yet despite the fundamental role of cellulolytic microorganisms in global carbon cycling and as potential sources of novel enzymes for biotechnology, their identity and ecology is not well established. Cellulose is a major component of landfill waste and its degradation is therefore a key feature of the anaerobic microbial decomposition process. Here, we targeted a number of taxa containing known cellulolytic anaerobes (members of the bacterial genus Fibrobacter, lineages of Clostridium clusters I, III, IV and XIV, and anaerobic fungi of the Neocallimastigales) in landfill leachate and colonized cellulose 'baits' via PCR and quantitative PCR (qPCR). Fibrobacter spp. and Clostridium clusters III, IV and XIV were detected in almost all leachate samples and cluster III and XIV clostridia were the most abundant (1-6% and 1-17% of total bacterial 16S rRNA gene copies respectively). Two landfill leachate microcosms were constructed to specifically assess those microbial communities that colonize and degrade cellulose substrates in situ. Scanning electron microscopy (SEM) of colonized cotton revealed extensive cellulose degradation in one microcosm, and Fibrobacter spp. and Clostridium cluster III represented 29% and 17%, respectively, of total bacterial 16S rRNA gene copies in the biofilm. Visible cellulose degradation was not observed in the second microcosm, and this correlated with negligible relative abundances of Clostridium cluster III and Fibrobacter spp. (≤ 0.1%), providing the first evidence that the novel fibrobacters recently detected in landfill sites and other non-gut environments colonize and degrade cellulose substrates in situ.     

Vitamin K composition of anaerobic gut bacteria

The menaquinone (vitamin K₂) composition of a wide range of obligately anaerobic bacteria was investigated using chromatographic and physicochemical techniques. Members of the genera Bifidobacterium, Butyrivibrio, Clostridium, Fusobacterium, Lachnospira, Megasphaera, Propionispira, Ruminococcus, Selenomonas, Succinovibrio and Succinomonas were found to lack menaquinones, In contrast, all species of the genera Actinomyces, Arachnia, Bacteroides, Propionibacterium, Veillonella and Wolinella examined possessed menaquinones. Considerable variation in length and degree of saturation of the C-3 isoprenyl side-chains of the menaquinones isolated from these taxa were evident. In addition to menaquinone, strains of Wolinella and Eubacterium lentum contained methylated menaquinones. The possible contribution of bacterial menaquinones to the total pool of vitamin K in the human gut is discussed.