广西水牛瘤胃中的细菌多样性

[目的]了解广西水牛瘤胃中细菌的组成及其可能的降解纤维素细菌的主要类群。[方法]提取水牛瘤胃内容物和高效降解滤纸的水牛瘤胃内容物的富集培养物的宏基因组DNA,以宏基因组DNA为模板,扩增16S rRNA基因序列,构建该两种样品的细菌的16S rRNA基因文库。通过对16S rRNA基因序列的分析,了解这两种样品的细菌群体种类及数量。 [结果] 水牛瘤胃内容物与其富集培养物中均主要含有LGCGPB (low G+C Gram-Positive Bacteria)、CFB (Cytophaga-Flexibacter-Bacteroides)两大类菌群和少数的螺旋体菌（Spirochaetes）,且LGCGPB所占的比例都是最高的,LGCGPB在水牛瘤胃内容物细菌中的比例为56.66%,而在富集培养物细菌中的比例升高为73.33%。在水牛瘤胃内容物中,丝状杆菌（Fibrobacteres）占3.33%,但在富集培养物中未被检测到。而在富集培养物中占13.33%的变形杆菌（Proteobacteria）,在水牛瘤胃内容物中未被检测到。本研究还发现了分类地位尚未明确的一菌群（R46）。[结论]细菌类群LGCGPB、Proteobacteria可能在水牛瘤胃中的纤维素降解过程中起重要作用。此外,水牛瘤胃中的细菌组成和牦牛、牛、羊瘤胃中的细菌组成较相似但比例有所不同。     

Cellulolytic activity of luminous bacteria

Summary The luminous bacteriaVibrio harveyi andV. fischeri degrade cellulose. Cellulase activity was high when carboxymethyl cellulose and cellobiose were used as substrates but low with cellulose powder. The role of these microbes in the digestion of food material in fish gut is also discussed.

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Resumen Las bacterias luminosasVibrio harveyi yV. fischeri degradan celulosa. La actividad celulásica fue alta cuando se utilizó Carboxy-metil-celulosa o celobiosa como sustrato y baja cuando se utilizó polvo de celulosa. Se discute el papel de estos microorganismos en la digestión intestinal de los alimentos en peces.

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Résumé Les bactéries lumineusesVibrio harveyi etV. fischeri dégradent la cellulose. L'activité cellulolytique était élevée lorsque la carboxméthylcellulose et la cellobiose servaient de substrats, mais elle était faible avec la poudre de cellulose. On discute également de rôle de ces microorganismes dans la digestion du matériau alimentaire.

     

Cellulolytic bacteria from soils in harsh environments

It is believed that the exposure of organisms to harsh climate conditions may select for differential enzymatic activities, making the surviving organisms a very promising source for bioprospecting. Soil bacteria play an important role in degradation of organic matter, which is mostly due to their ability to decompose cellulose-based materials. This work focuses on the isolation and identification of cellulolytic bacteria from soil found in two environments with stressful climate conditions (Antarctica and the Brazilian semi-arid caatinga). Cellulolytic bacteria were selected using enrichments at high and low temperatures (4 or 60°C) in liquid media (trypic soy broth—TSB and minimum salt medium—MM) supplemented with cellulose (1%). Many of the isolates (119 out of 254—46.9%) displayed the ability to degrade carboxymethyl-cellulose, indicating the presence of endoglucolytic activity, while only a minority of these isolates (23 out of 254—9.1%) showed exoglucolytic activity (degradation of avicel). The obtained isolates revealed a preferential endoglucolytic activity according to the temperature of enrichments. Also, the identification of some isolates by partial sequencing of the 16S rRNA gene indicated that the Bacteroidetes (e.g., Pedobacter, Chryseobacterium and Flavobacterium) were the main phylum of cellulolytic bacteria isolated from soil in Antarctica; the Firmicutes (e.g., Bacillus) were more commonly isolated from samples from the caatinga; and Actinobacteria were found in both types of soil (e.g., Microbacterium and Arthrobacter). In conclusion, this work reports the isolation of bacteria able to degrade cellulose-based material from soil at very low or very high temperatures, a finding that should be further explored in the search for cellulolytic enzymes to be used in the bioenergy industry.     

Cellulolytic bacteria from pig large intestine

An anaerobic, cellulose-degrading, gram-negative rod and a gram-positive coccus, identified as Bacteroides succinogenes and Ruminococcus flavefaciens, respectively, were isolated from pig fecal samples. These organisms were isolated from cellulolytic most-probable-number dilutions which represented 4 or 6% of the viable bacterial count when pigs were fed a low- or high-fiber diet, respectively.     

Cellulolytic bacteria from pig large intestine.

An anaerobic, cellulose-degrading, gram-negative rod and a gram-positive coccus, identified as Bacteroides succinogenes and Ruminococcus flavefaciens, respectively, were isolated from pig fecal samples. These organisms were isolated from cellulolytic most-probable-number dilutions which represented 4 or 6% of the viable bacterial count when pigs were fed a low- or high-fiber diet, respectively.     

Deoxyribonuclease activity in rumen bacteria

Deoxyribonuclease activity was surveyed in 22 strains belonging to 12 species of rumen bacteria, with lambda bacteriophage DNA as substrate. Activity was readily detected in broken cell preparations from 15 of these strains. Particularly high levels of activity were present in cells and culture supernatant of all 5 strains of Bacteroides succinogenes, and 2 out of 6 strains of Bacteroides ruminicola, examined.     

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.     

Cellulolytic and non-cellulolytic bacteria in rat gastrointestinal tracts.

Lactobacillus and Bifidobacterium species were the predominant organisms isolated from small intestinal (jejunal) contents of rats, and lactic acid was the only organic acid detected. The numbers of cellulolytic bacteria in small intestines were low (approximately 10(3)/g). The fermentation in ceca was different from that in intestines, as, in addition to small amounts of lactic acid, high concentrations of volatile fatty acids were detected. The mixed cecal microflora was able to digest cellulose (pebble-milled Whatman no. 1) and cabbage. High numbers of cellulolytic bacteria were found (0.5 X 10(8) to 12.2 X 10(8)/g; 6% of total viable bacteria). The predominant celluloytic organism isolated was Bacteroides succinogenes. Ruminococcus flavifaciens was isolated from a few animals. The kinds and numbers of the predominant non-cellulolytic organisms isolated from rat ceca were similar to those described by previous workers.     

Intracellular nuclease activities of buffalo rumen bacterial population

Nuclease activities of the predominantly bacterial population obtained from buffalo rumen were investigated. Optimum temperature for hydrolysis of both DNA and RNA was 50°C whereas DNAase activity was observed to be stable up to 50°C, a decrease in RNAase activity was observed even after 40°C. Two pH optima, one at 5.5 and the other at 7.5, were recorded for hydrolysis of DNA. RNAase activity was maximum between pH 6.0 to 7.0. Whereas DNAase activity was stable near its optimum pH, RNAase activity was stable between pH 7.0 to 8.5. Mn²⁺ ions stimulated DNAase activity. It was strongly inhibited by Hg²⁺, Zn²⁺, Pb²⁺ and Ag⁺. RNAase activity was stimulated by Mg²⁺ ions and was strongly inhibited by Hg²⁺, Cu²⁺, Zn²⁺ and Ag⁺. Cysteine hydrochloride and 2-mercaptoethanol stimulated DNAase activity. The activity was strongly inhibited by N-ethylmaleimide, 4-chloromercuribenzoate, 8-quinolinol, iodoacetic acid and 1,10-phenanthroline. RNAase activity was stimulated by cysteine hydrochloride, reduced glutathione and 2-mercaptoethanol and was strongly inhibited by 4-chloromercuribenzoate, 8-quinolinol and 2,2′-bipyridyl. Part of PhD Thesis submitted by the first author to Kurukshetra University.     

Ureolytic bacteria in sheep rumen.

Estimates were made of the numbers of viable bacteria in the rumens of sheep receiving different rations. Representative colonies were isolated and tested for urease production. Some urease-positive isolates were characterized and identified. The ureolytic activities of the urease-producing isolates were determined and compared with the activity of rumen fluid. The rations fed to the sheep did not exert a significant influence on the relative numbers of the urease-producting organisms in the rumen. No obligately anaerobic ureolytic bacteria were found. All urease-positive isolates were facultatively anaerobic, Gram-positive, catalase-positive cocci. Out of ten isolates, nine were identified as Staphylococcus saprophyticus and one as Micrococcus varians. The total urease activity of the different isolates based on the lowest numbers in which they were present in the rumen, compared favourably with the urease activity of rumen fluid. The facultatively anaerobic Gram-positive cocci were probably responsible for a large proportion of the urease activity of the rumen fluid. Conditions prevailing in the rumen were found to be conducive to urease production by the isolates tested.     

Some rumen bacteria degrading fructan

Degradation of fructan obtained from timothy ( Phleum pratense L.) by the following six species of bacteria isolated from sheep rumen was studied: Streptococcus bovis, Bacteroides ruminicola, Selenomonas ruminantium, Butyrivibrio fibrisolvens, Treponema bryantii and Treponema saccharophilum. The enzymatic activity of the bacteria was analysed by TLC. The highest activity was found in whole cells and in the strains B. fibrisolvens No. 3 and T. saccharophilum S.     

Urease activity of adherent bacteria and rumen fluid bacteria

In experiments on six sheep fed on a low nitrogen diet (3.7 g N/day), urease (EC 3.5.1.5) activity (nkat X mg-1 bacterial dry weight) 3 h after feeding was found to be highest in the bacteria adhering to the rumen wall (13.25 +/- 2.10), lower in the rumen fluid bacteria (8.96 +/- 1.35) and lowest in the bacteria adhering to feed particles in the rumen (5.69 +/- 2.13). The urease activity of bacteria adhering to the rumen wall and of the rumen fluid bacteria of six sheep fed on a high nitrogen diet (21 g N/day) was significantly lower than in sheep with a low N intake and in both cases was roughly the same (3.81 +/- 1.37 and 3.76 +/- 1.02 respectively); it was lowest in bacteria adhering to feed particles in the rumen (1.92 +/- 0.90). It is concluded from the results that the urease activity of rumen fluid bacteria and of bacteria adhering to the rumen wall and to feed particles in the rumen is different and that it falls significantly in the presence of a high nitrogen intake. From the relatively high ureolytic activity of bacteria adhering to the rumen wall in the presence of a low nitrogen intake it is assumed that this is one of the partial mechanisms of the hydrolysis of blood urea entering the rumen across the rumen wall and of its reutilization in the rumen-liver nitrogen cycle in ruminants.     

Alkaline phosphatase activity of rumen bacteria

Of the 54 strains of rumen bacteria examined for alkaline phosphatase (APase) production, 9 of 33 gram-negative strains and none of 21 gram-positive strains produced the enzyme. The APase of the cells of the three strains of Bacteroides ruminicola that produced significant amounts of the enzyme was located in the periplasmic area of the cell envelope, whereas the enzyme was located in the strains of Selenomonas ruminantium and Succinivibrio dextrinosolvens was associated with the outer membrane. The localization of APase production in the cells of natural populations of rumen bacteria from hay-fed sheep was accomplished by reaction product deposition, and both the proportion of APase-producing bacteria and the location of the enzyme in the cell envelope of the producing cells could be determined. We suggest that this procedure is useful in detecting shifts in the bacterial population and the release of cell-bound APase that accompany feedlot bloat and other sequelae of dietary manipulation in ruminants.     

PCR detection of uncultured rumen bacteria

16S rRNA sequences of ruminal uncultured bacterial clones from public databases were phylogenetically examined. The sequences were found to form two unique clusters not affiliated with any known bacterial species: cluster of unidentified sequences of free floating rumen fluid uncultured bacteria (FUB) and cluster of unidentified sequences of bacteria associated with rumen epithelium (AUB). A set of PCR primers targeting 16S rRNA of ruminal free uncultured bacteria and rumen epithelium adhering uncultured bacteria was designed based on these sequences. FUB primers were used for relative quantification of uncultured bacteria in ovine rumen samples. The effort to increase the population size of FUB group has been successful in sulfate reducing broth and culture media supplied with cellulose.