Assessment of microbial diversity in effluent treatment plants by culture dependent and culture independent approaches

Microbial community structure of two distinct effluent treatment plants (ETPs) of pesticide and pharmaceutical industries was assessed and defined by (i) culture dependent and culture independent approaches on the basis of 16S rRNA gene sequencing, (ii) diversity index analysis - operational taxonomic units (OTUs). A total of 38 and 44 bacterial OTUs having 85-99% similarity with the closest match in the database were detected among pharmaceutical and pesticide sludge samples, respectively. Fifty percent of the OTUs were related to uncultured bacteria. These OTUs had a Shannon diversity index value of 2.09-2.33 for culturables and in the range of 3.25-3.38 for unculturables. The high species evenness values of 0.86 and 0.95 indicated the vastness of microbial diversity retrieved by these approaches. The dominant cultured bacteria indicative of microbial diversity in functional ETPs were Alcaligenes, Bacillus and Pseudomonas. Brevundimonas, Citrobacter, Pandoraea and Stenotrophomonas were specific to pesticide ETP and Agrobacterium, Brevibacterium, Micrococcus, Microbacterium, Paracoccus and Rhodococcus were specific to pharmaceutical ETP. These microbes can thus be maintained and exploited for efficient functioning and maintenance of ETPs.     

Effect of fumarate reducing bacteria on in vitro rumen fermentation,methane mitigation and microbial diversity

The metabolic pathways involved in hydrogen (H₂) production, utilization and the activity of methanogens are the important factors that should be considered in controlling methane (CH₄) emissions by ruminants. H₂ as one of the major substrate for CH₄ production is therefore should be controlled. One of the strategies on reducing CH₄ is through the use of hydrogenotrophic microorganisms such as fumarate reducing bacteria. This study determined the effect of fumarate reducing bacteria, Mitsuokella jalaludinii, supplementation on in vitro rumen fermentation, CH4 production, diversity and quantity. M. jalaludinii significantly reduced CH₄ at 48 and 72 h of incubation and significantly increased succinate at 24 h. Although not significantly different, propionate was found to be highest in treatment containing M. jalaludinii at 12 and 48 h of incubation. These results suggest that supplementation of fumarate reducing bacteria to ruminal fermentation reduces CH₄ production and quantity, increases succinate and changes the rumen microbial diversity.     

Bifid bacteria in bovine rumen

Summary About 500 bifid isolates from 150 samples of bovine rumen liquor were examined for their morphology, physiology and biochemistry. Diagnosis as bifid bacteria was based upon the peculiar pathway of glucose anaerobic metabolism i.e. the fructose-6-phosphate shunt. Four phenetic types were recognized. These types can be differentiated from those found in human habitats because their cell-free extracts are aldolase and HMP dehydrogenases positive: they are potential heterofermenters; furthermore the rumen types are nutritionally different. The distinction of the rumen bifids from the Bifidobacterium species of the intestine of Apis mellifica and Apis indica is still more consistent for a lot of characters. The characters of two rumen types warranted the creation of two new species of the genus Bifidobacterium. One of these, B. globosum n. sp., has a proper morphology, is serologically distinct and has a deoxyribonucleic acid base composition, in % GC, of 64.5. The other, B. ruminale n. sp., found so far only in rumen, is characteristically lactose non fermenter, at variance with all the bifids from human habitats and has peculiar morphological traits. A third type is probably a rough variant of B. ruminale and a fourth is serologically distinct and mannitol fermenter; their taxonomic definition is still, however, premature.This investigation was supported by a grant received from Consiglio Nazionale delle Ricerche (C.N.R.), Roma.     

Diversity of cold-active protease-producing bacteria from arctic terrestrial and marine environments revealed by enrichment culture

A new approach for enrichment culture was applied to obtain cold-active protease-producing bacteria for marine and terrestrial samples from Svalbard, Norway. The method was developed for the enrichment of bacteria by long-term incubation at low temperatures in semi-solid agar medium containing meat pieces as the main source of carbon and energy. ZoBell and 0.1× nutrient broth were added for marine and terrestrial microorganisms, respectively, to supply basal elements for growth. One to three types of colonies were observed from each enrichment culture, indicating that specific bacterial species were enriched during the experimental conditions. Among 89 bacterial isolates, protease activity was observed from 48 isolates in the screening media containing skim milk. Good growth was observed at 4°C and 10°C while none of the isolates could grow at 37°C. At low temperatures, enzyme activity was equal to or higher than activity at higher temperatures. Bacterial isolates were included in the genera Pseudoalteromonas (33 isolates), Arthrobacter (24 isolates), Pseudomonas (16 isolates), Psychrobacter (6 isolates), Sphingobacterium (6 isolates), Flavobacterium (2 isolates), Sporosarcina (1 isolate), and Stenotrophomonas (1 isolate). Protease activity was observed from Pseudoalteromonas (33 isolates), Pseudomonas (10 isolates), Arthrobacter (4 isolates), and Flavobacterium (1 isolate).     

Diversity and versatility of lipid-protein interactions revealed by molecular genetic approaches

The diversity in structures and physical properties of lipids provides a wide variety of possible interactions with proteins that affect their assembly, organization, and function either at the surface of or within membranes. Because lipids have no catalytic activity, it has been challenging to define many of their precise functions in vivo in molecular terms. Those processes responsive to lipids are attuned to the native lipid environment for optimal function, but evidence that lipids with similar properties or even detergents can sometimes partially replace the natural lipid environment has led to uncertainty as to the requirement for specific lipids. The development of strains of microorganisms in which membrane lipid composition can be genetically manipulated in viable cells has provided a set of reagents to probe lipid functions. These mutants have uncovered previously unrecognized roles for lipids and provided in vivo verification for putative functions described in vitro. In this review, we summarize how these reagent strains have provided new insight into the function of lipids. The role of specific lipids in membrane protein folding and topological organization is reviewed. The evidence is summarized for the involvement of anionic lipid-enriched domains in the organization of amphitropic proteins on the membrane surface into molecular machines involved in DNA replication and cell division.     

Assessment of microbial diversity in biofilms recovered from endotracheal tubes using culture dependent and independent approaches

Ventilator-associated pneumonia (VAP) is a common nosocomial infection in mechanically ventilated patients. Biofilm formation is one of the mechanisms through which the endotracheal tube (ET) facilitates bacterial contamination of the lower airways. In the present study, we analyzed the composition of the ET biofilm flora by means of culture dependent and culture independent (16 S rRNA gene clone libraries and pyrosequencing) approaches. Overall, the microbial diversity was high and members of different phylogenetic lineages were detected (Actinobacteria, beta-Proteobacteria, Candida spp., Clostridia, epsilon-Proteobacteria, Firmicutes, Fusobacteria and gamma-Proteobacteria). Culture dependent analysis, based on the use of selective growth media and conventional microbiological tests, resulted in the identification of typical aerobic nosocomial pathogens which are known to play a role in the development of VAP, e.g. Staphylococcus aureus and Pseudomonas aeruginosa. Other opportunistic pathogens were also identified, including Staphylococcus epidermidis and Kocuria varians. In general, there was little correlation between the results obtained by sequencing 16 S rRNA gene clone libraries and by cultivation. Pyrosequencing of PCR amplified 16 S rRNA genes of four selected samples resulted in the identification of a much wider variety of bacteria. The results from the pyrosequencing analysis suggest that these four samples were dominated by members of the normal oral flora such as Prevotella spp., Peptostreptococcus spp. and lactic acid bacteria. A combination of methods is recommended to obtain a complete picture of the microbial diversity of the ET biofilm.     

骆驼瘤胃内降解含氮杂环化合物细菌的多样性

【目的】作为典型的荒漠动物,骆驼能够采食其他动物不能够食用的具有强烈气味的或有毒的植物,而不影响其正常生理代谢。研究发现骆驼采食的植物毒素与吡啶、喹啉、吲哚等杂环化合物具有相似的化学结构,所以研究骆驼体内是否存在潜在的杂环化合物降解菌具有重要意义。【方法】本研究采集3头骆驼瘤胃内容物,分别以吡啶、喹啉和吲哚3种含氮杂环化合物为唯一碳源和氮源进行5代富集培养。通过高通量测序技术对瘤胃内容物和5代富集培养细菌进行了测序分析。【结果】骆驼肠道中降解杂环化合物(吡啶、喹啉、吲哚)细菌群体样品中变形菌门、放线菌门、拟杆菌门、浮霉菌门和厚壁菌门等5个门类丰富度最高。骆驼瘤胃内降解吡啶的优势菌可能属于鞘氨醇杆菌属和不动杆菌属,降解吲哚的优势菌主要属于芽孢杆菌属,而降解喹啉的优势菌可能以赖氨酸芽孢杆菌属和鞘氨醇杆菌属为主。【结论】骆驼瘤胃原始样品经过吡啶、喹啉、吲哚富集5代后,与原始样品比较优势菌群发生了较大的改变,这说明骆驼瘤胃内蕴含降解吡啶、吲哚和喹啉的细菌,但对吡啶、吲哚和喹啉的降解过程中发挥降解作用的细菌群落存在差异。     

Diversity of DMSP transport in marine bacteria, revealed by genetic analyses

The enzyme product of the dddD gene, found in several different marine bacteria, acts on dimethylsulfoniopropionate (DMSP), liberating dimethyl sulfide (DMS) and generating 3-OH-propionate as the initially detected C3 product. In many bacteria, dddD is near genes whose sequence suggests that they encode a DMSP transporter. These are of two very different types, in the BCCT (betaine-carnitine-choline transporter) family or resembling members of the ABC super-family that import betaines. Even within these two families, the amino acid sequences of these putative transporters are not particularly similar to each other. Genes for the predicted DMSP transporters of Halomonas and Marinomonas (both BCCT type) and of Burkholderia ambifaria AMMD (ABC-type) were each cloned and introduced into an Escherichia coli mutant (MKH13) that is defective in betaine uptake, and so fails to catabolise DMSP even when a cloned dddD gene was present, due to the failure of the substrate to be imported. DMSP-dependent DMS production (Ddd⁺ phenotype) was restored by introducing any of these cloned transporters into MKH13 containing dddD. Other marine bacteria use a range of enzymes, called DddL, DddP, DddQ, DddW and DddY, to cleave DMSP, but the various ddd genes that encode them are usually unlinked to any that are predicted to encode betaine transporters. We identified one gene in Sulfitobacter sp. EE-36 and two in Roseovarius nubinhibens ISM, which, when cloned and introduced into E. coli MKH13, overcame its osmotic sensitivity when it was grown with DMSP or other exogenous betaines. These genes all encoded BCCT transporters, but were unlinked to any known genes involved in DMSP catabolism in these two strains of α-proteobacteria.     

松材线虫伴生细菌多样性的宏基组分析

摘要：【目的】松材线虫是松材线虫病的病原,且与其伴生细菌之间存在互作关系,它们构成一个微生态系统。本研究旨在揭示松材线虫-伴生细菌群落细菌多样性。【方法】采用16S rRNA基因文库和454测序对伴生细菌群落的宏基因组进行初步分析。【结果】依据97％序列相似性划分OTU（Operational Taxonomic Unit）,构建的16S rRNA文库包含25个OTU,分别属于Alphaproteobacteria、Betaproteobacteria、Gammaproteobacteria和Bacter     

Degradation of isolated grass mesophyll, epidermis and fibre cell walls in the rumen and by cellulolytic rumen bacteria in axenic culture

The degradation of cell walls of mesophyll, epidermis and fibre cells isolated from leaves of perennial and Italian ryegrass within the sheep rumen or by selected strains of rumen bacteria in vitro , was followed by estimation of dry matter loss, or loss of neutral sugar residues. Primary cell walls (mesophyll and epidermis) were fully degraded within 12 h in the rumen, while the more heavily lignified fibre cell walls showed only a 40% loss of dry matter over the same period. Neutral sugar residues were lost at a common rate from walls of all three cell types. Incubation of cell walls with cellulolytic bacteria showed that the extent to which cell walls were attacked was constantly ordered (epidermis > mesophyll > fibre). The rate of degradation of cell walls was less in axenic culture than within the rumen. Greatest weight losses were produced by Ruminococcus albus , followed by Bacteroides succinogenes , with Ruminococcus flavefaciens effecting the least change, regardless of the nature of the cell wall provided as a substrate. Xylose was more readily lost from primary cell walls than glucose during the early stages of attack, but both were lost at a common rate from fibre cell walls. Dry matter losses produced by the hemicellulolytic strain, Bacteroides ruminocola , were limited even after extended incubation. Electron microscopy indicated that R. albus was less commonly attached to cell walls than were the other cellulolytic strains, although evidence of capsular material was present. Bacteroides succinogenes was seen with an extensive capsule which enveloped clusters of cells, forming micro-colonies in association with the plant cell wall. Vesicle-like structures, commonly associated with the cellulolytic bacteria R. albus and B. succinogenes , were found on comparatively few occasions in this study.     

Production of alkaline phosphatase by epithelial cells and adherent bacteria of the bovine rumen and abomasum

Three distinct bacterial populations have been defined in the bovine rumen: the rumen fluid population; the population associated with food particles; and the population adherent to the rumen epithelium. Alkaline phosphatase activity has been reported in cells of the first two populations and here we report that assays of rumen epithelial samples containing both tissue and bacteria also contain the enzyme. The reaction product technique has localized the enzyme both in adherent bacteria and in cell of the stratified squamous rumen epithelium. The epithelium of the abomasum shows much lower levels of alkaline phosphatase activity.     

Characterization of several bovine rumen bacteria isolated with a xylan medium

Dehority, B. A. (Ohio Agricultural Research and Development Center, Wooster). Characterization of several bovine rumen bacteria isolated with a xylan medium. J. Bacteriol. 91:1724-1729. 1966.-Studies were conducted to characterize eight strains of bacteria isolated from bovine rumen contents, by use of a medium containing xylan as the only added carbohydrate source. Based on morphology, biochemical reactions, nutritional requirements, and fermentation products, five of the eight strains were identified as Butyrivibrio fibrisolvens. Many properties of the remaining three strains resembled Bacteroides ruminicola; however, propionic acid was consistently found as a fermentation product. When the type strains for B. ruminicola subsp. ruminicola and B. ruminicola subsp. brevis were compared with the present isolates, it was found that propionic acid was a normal fermentation product for the type strain B. ruminicola subsp. ruminicola when grown in a 40% rumen fluid-0.5% glucose broth. Production of propionic acid was markedly reduced for all strains when grown in a 20% rumen fluid-1% glucose broth. The three remaining strains were thus placed in the species B. ruminicola, and further classified into the subspecies ruminicola (one strain) and brevis (two strains) on the basis of their requirement for hemin. Although the type strain of B. ruminicola subsp. brevis did not produce propionic acid, both of the present isolates classified as this subspecies produced substantial amounts. One strain of B. ruminicola subsp. brevis had an absolute requirement for volatile fatty acids. Either isobutyric or dl-2-methylbutyric acid would satisfy this requirement, whereas isovaleric acid was ineffective. It is of interest that xylan-fermenting bacteria isolated from 10(-7) and 10(-8) dilutions of rumen contents by use of a xylan medium are similar to the xylan fermenters isolated at the same dilutions with a nonselective medium.     

Diversity of metallothioneins in the American oyster, Crassostrea virginica, revealed by transcriptomic and proteomic approaches.

Metallothioneins are typically low relative molecular mass (6000-7000), sulfhydryl-rich metal-binding proteins with characteristic repeating cysteine motifs (Cys-X-Cys or Cys-X(n)-Cys) and a prolate ellipsoid shape containing single alpha- and beta-domains. While functionally diverse, they play important roles in the homeostasis, detoxification and stress response of metals. The originally reported metallothionein of the American oyster, Crassostrea virginica showed the canonical molluscan alphabeta-domain structure. Oyster metallothioneins have been characterized as cDNA and as expressed proteins, and here it is shown that the previously reported metallothionein is a prototypical member of a subfamily (designated as CvMT-I) of alphabeta-domain metallothioneins. A second extensive subfamily of oyster metallothioneins (designated as CvMT-II) has apparently arisen from (a) a stop mutation that truncates the protein after the alpha-domain, and (b) a subsequent series of duplication and recombination events that have led to the development of metallothionein isoforms containing one to four alpha-domains and that lack a beta-domain. Analysis of metallothioneins revealed that certain CvMT-I isoforms showed preferential association either with cadmium or with copper and zinc, even after exposure to cadmium. These data extend our knowledge of the evolutionary diversification of metallothioneins, and indicate differences in metal-binding preferences between isoforms within the same family.     

Fermentation of barley straw by anaerobic rumen bacteria and fungi in axenic culture and in co-culture with methanogens

When incubated in axenic culture, strains of anaerobic rumen fungi were more active than cellulolytic bacteria in solubilizing barley straw stem fragments 5 to 10 mm in length. Pretreatment with ammonia had little effect on microbial attack. Of three species of methanogens tested, Methanobrevibacter smithii strain PS formed the most stable and reproducible co-cultures with the fungi and with Ruminococcus albus , and the presence of this organism enhanced the extent of degradation of straw, although this effect was less marked than that previously observed when pure cellulose was used as substrate.     

Effect of pH on the efficiency of growth by pure cultures of rumen bacteria in continuous culture.

A total of 10 strains of rumen bacteria, Selenomonas ruminantium HD4, Megasphaera elsdenii B159, Butyrivibrio fibrisolvens A38, Streptococcus bovis JB1, Lactobacillus vitulinus GA1, Bacteroides ruminicola B14, B. ruminicola GA33, Ruminococcus albus 7, Ruminococcus flavefaciens C94, and Bacteroides succinogenes S85, were grown in energy-limiteH of the medium reservoir was lowered approximately 0.3 pH units, and the energy source concentration remaining in the culture vessel, optical density, cell mass, and pH were determined. A low pH appeared to have a detrimental effect on cell yields. Large variations were seen among strains in both the magnitude of yield depressions at lower pH values and in the pH at which the culture washed out. Lactate analysis indicated ta are discussed in relation to the effect of pH on the efficiency of protein synthesis in the rumen and rumen microbial ecology.