Establishment of Bacteroides succinogenes and measurement of the main digestive parameters in the rumen of gnotoxenic lambs

We attempted to determine the degree of diversification of the microflora that allow the establishment of Bacteroides succinogenes S85 in the rumen of gnotoxenic lambs. Four lambs (group I) received an inoculum orally, composed of 182 noncellulolytic bacterial strains (inoculum 1) previously isolated from the rumen of conventional young lambs. Two lambs (group II) were inoculated with 32 strains (inoculum 2) selected among the 182 strains of inoculum 1. Two lambs (group III) received an inoculum (inoculum 3) composed of 106 noncellulolytic bacterial strains previously isolated from the rumen of meroxenic lambs. Two lambs (group IV) were inoculated with 16 strains (inoculum 4) chosen among the 106 strains of inoculum 3. All lambs were inoculated from birth except two lambs of group I, which were inoculated from 1 month of age. Each lamb then received orally a pure culture of B. succinogenes. This strain became established more easily in the rumen of lambs that had received complex inocula (group I). Its population reached a level close to that generally observed in conventional lambs (10(7)-10(8) bacteria.mL-1). In contrast, B. succinogenes became established in only one lamb of group II, but bacterial numbers varied considerably. In group III, repeated inoculations were necessary to obtain its definitive establishment (10(7)-10(8) bacteria.mL-1 after weaning). In spite of several inoculations, this cellulolytic species failed to establish in the rumen of lambs of group IV, which had received the less complex inoculum. The volatile fatty acid levels were very different from one lamb group to another. The more complex the inoculum administered to the animals, the higher the concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antibiotic activity of an isocyanide metabolite of Trichoderma hamatum against rumen bacteria

A metabolite of Trichoderma hamatum, 3-(3-isocyanocyclopent-2-enylidene)propionic acid, was tested for its effects on growth of and carbohydrate metabolism in 11 strains of functionally important rumen bacteria. To standardize the biological activity of this unstable metabolite, a rapid, aerobic disc diffusion assay was developed using Escherichia coli ATCC 11775. In an anaerobic broth dilution assay using a medium lacking rumen fluid and containing a soluble carbohydrate, the minimum inhibitory concentration of the metabolite which completely inhibited growth of the rumen bacteria for 18 h at 39 degrees C was generally less than 10 micrograms X mL-1; however, the minimum inhibitory concentrations for Megasphaera elsdenii B159 and Streptococcus bovis Pe(1)8 were 10-25 and 25-64 micrograms X mL-1, respectively. In general, the Gram-negative strains were more sensitive than the Gram positive. The minimum inhibitory concentration for Bacteroides ruminicola 23 grown with glucose was 1 micrograms X mL-1; for B. ruminicola GA33 (glucose), B. succinogenes S85 (cellobiose), and Succinivibrio dextrinosolvens 24 (maltose), it was 2 microgram X mL-1. When added to a cellulose-containing rumen fluid medium, 1-4 micrograms X mL-1 of the metabolite delayed cellulose hydrolysis by B. succinogenes S85, Ruminococcus albus 7, and R. flavefaciens FD1 for up to 4 days, and 6-7 micrograms X mL-1 prevented hydrolysis for at least 1 month. In the presence of the metabolite, the proportion of acetate produced from soluble carbohydrate by the majority of strains increased, but with some strains net production of acetate decreased relative to production of other acidic fermentation products.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions between Treponema bryantii and cellulolytic bacteria in the in vitro degradation of straw cellulose

To assess the contribution of individual bacterial species to the overall process of cellulose digestion in the rumen, cellulolytic bacteria (Bacteroides succinogenes and Ruminococcus albus) were tested as pure cultures and as cocultures with noncellulolytic Treponema bryantii. In studies of in vitro barley straw digestion, Treponema cocultures surpassed pure cultures of the cellulolytic organisms in dry matter disappearance, volatile fatty acid generation, and in the production of succinic acid, lactic acid, and ethanol. Morphological examination, by electron microscopy, showed that cells of T. bryantii associate with the plant cell wall materials in straw, but that cellulose digestion occurs only when these organisms are present with cellulolytic species such as B. succinogenes. These results show that cellulolytic bacteria interact with noncellulolytic Treponema to promote the digestion of cellulosic materials.     

Fermentation of cellodextrins by cellulolytic and noncellulolytic rumen bacteria.

Water-soluble cellodextrins were prepared from microcrystalline cellulose by using fuming hydrochloric acid and acetone precipitation. This cellodextrin preparation contained only trace amounts of glucose and cellobiose and was primarily composed of cellotetraose and cellopentaose. When various species of cellulolytic and noncellulolytic bacteria were cultured with cellodextrins, their growth rates and maximal optical densities were in most cases similar to those observed with cellobiose. Time course samplings and analyses of cellodextrins by high-pressure liquid chromatography indicated that longer-chain cellodextrins were hydrolyzed extracellularly to cellobiose and cellotriose. Cellodextrin utilization by noncellulolytic rumen bacteria and extracellular hydrolysis of cellodextrins increase the possibility that cross-feeding occurs in the rumen and help to explain the high numbers of noncellulolytic bacteria in ruminants fed fibrous diets.     

Fermentation of cellodextrins by cellulolytic and noncellulolytic rumen bacteria

Water-soluble cellodextrins were prepared from microcrystalline cellulose by using fuming hydrochloric acid and acetone precipitation. This cellodextrin preparation contained only trace amounts of glucose and cellobiose and was primarily composed of cellotetraose and cellopentaose. When various species of cellulolytic and noncellulolytic bacteria were cultured with cellodextrins, their growth rates and maximal optical densities were in most cases similar to those observed with cellobiose. Time course samplings and analyses of cellodextrins by high-pressure liquid chromatography indicated that longer-chain cellodextrins were hydrolyzed extracellularly to cellobiose and cellotriose. Cellodextrin utilization by noncellulolytic rumen bacteria and extracellular hydrolysis of cellodextrins increase the possibility that cross-feeding occurs in the rumen and help to explain the high numbers of noncellulolytic bacteria in ruminants fed fibrous diets.     

Specialized cell surface structures in cellulolytic bacteria.

The cell surface topology of various gram-negative and -positive, anaerobic and aerobic, mesophilic and thermophilic, cellulolytic and noncellulolytic bacteria was investigated by scanning electron microscopic visualization using cationized ferritin. Characteristic protuberant structures were observed on cells of all cellulolytic strains. These structures appeared to be directly related to the previously described exocellular cellulase-containing polycellulosomes of Clostridium thermocellum YS (E. A. Bayer and R. Lamed, J. Bacteriol. 167:828-836, 1986). Immunochemical evidence and lectin-binding studies suggested a further correlation on the molecular level among cellulolytic bacteria. The results indicate that such cell surface cellulase-containing structures may be of general consequence to the bacterial interaction with and degradation of cellulose.     

Microbial bioassay of fungal compounds that stimulate the growth of a consortium of anaerobic cellulolytic bacteria

Summary A bioassay is developed for testing growth factors present in fungal extracts and acting on a consortium of cellulolytic bacteria produced in a continuous anaerobic digester inoculated with rumen liquor and fed with carboxymethylcellulose. Fungal extracts stimulated overall biomass production of the cellulolytic bacterial consortium without changing specific cellulolytic activity.     

Bacteriophages that infect the cellulolytic ruminal bacterium Ruminococcus albus AR67

AIM: To isolate bacterial viruses that infect the ruminal cellulolytic bacterium Ruminococcus albus. METHODS: Four phages infecting R. albus AR67 were isolated under anaerobic conditions using the soft-agar overlay technique. The phages were characterized on morphology, solvent stability, nucleic acid type and digestion characteristics. Two phages, phiRa02 and phiRa04 comprised icosahedral virions with linear double-stranded DNA and appeared to belong to the family Podoviridae [corrected] The other two phages are most likely filamentous phages with circular single-stranded DNA of the family Inoviridae. SIGNIFICANCE OF THE STUDY: Viruses of the family Inoviridae [corrected] have not previously been isolated from rumen bacteria. The phages isolated in this study are the first phages shown to infect the cellulolytic bacteria of the rumen. This suggests that the cellulolytic populations of the rumen are subject to lytic events that may impact on the ability of these bacteria to degrade plant fibre and on the nutrition of the animal.     

Transformation of mercuric chloride and methylmercury by the rumen microflora

The microflora in strained rumen fluid did not methylate or volatilize 203Hg2+ at detectable rates. However, there was an exponential decay in the concentration of added CH3Hg+, which was attributed to demethylation. The major product of demethylation was metallic mercury (Hg0), and it was released as a volatile product from the reaction mixture. Demethylation occurred under both anaerobic and aerobic conditions. The rate of demethylation was proportional to the concentration of added CH3Hg+-Hg from 0.02 to 100 microgram of Hg per ml. The presence of HgCl2 had almost no inhibitory effect on the rate of cleavage of the carbon-mercury bond of CH2HgCl, but it completely inhibited volatilization of the Hg formed, when the concentration of HgCl2-Hg reached 100 micrograms/ml. Three of 11 species of anaerobic rumen bacteria catalyzed demethylation. These were Desulfovibrio desulfuricans, Selenomonas ruminantium, and Megasphaera elsdenii. None of the 11 species caused detectable methylation, and only two caused limited volatilization of Hg2+. Three species of bacteria out of 90 fresh aerobic isolates from rumen contents were demethylators: two were identified as Pseudomonas sp., and the third was a Micrococcus sp. Demethylation by the rumen microflora appeared to be carried out by both aerobic and anaerobic bacteria and, on the basis of Hg2+ sensitivity, probably resulted from the activity of two enzymes, a CH3-Hg+ hydrolase and a Hg2+ reductase.     

Transformation of mercuric chloride and methylmercury by the rumen microflora.

The microflora in strained rumen fluid did not methylate or volatilize 203Hg2+ at detectable rates. However, there was an exponential decay in the concentration of added CH3Hg+, which was attributed to demethylation. The major product of demethylation was metallic mercury (Hg0), and it was released as a volatile product from the reaction mixture. Demethylation occurred under both anaerobic and aerobic conditions. The rate of demethylation was proportional to the concentration of added CH3Hg+-Hg from 0.02 to 100 microgram of Hg per ml. The presence of HgCl2 had almost no inhibitory effect on the rate of cleavage of the carbon-mercury bond of CH2HgCl, but it completely inhibited volatilization of the Hg formed, when the concentration of HgCl2-Hg reached 100 micrograms/ml. Three of 11 species of anaerobic rumen bacteria catalyzed demethylation. These were Desulfovibrio desulfuricans, Selenomonas ruminantium, and Megasphaera elsdenii. None of the 11 species caused detectable methylation, and only two caused limited volatilization of Hg2+. Three species of bacteria out of 90 fresh aerobic isolates from rumen contents were demethylators: two were identified as Pseudomonas sp., and the third was a Micrococcus sp. Demethylation by the rumen microflora appeared to be carried out by both aerobic and anaerobic bacteria and, on the basis of Hg2+ sensitivity, probably resulted from the activity of two enzymes, a CH3-Hg+ hydrolase and a Hg2+ reductase.     

Interactions among cellulolytic bacteria from an anaerobic digester

High cellulolytic activity of particular strains did not cause dominance of one, or a few, species of fiber-digesting bacteria in a cattlewaste anaerobic digester. The population contained a large number of species and varieties with different cellulolytic and fiber-digesting activities. Although mixed cultures of some of these bacteria showed no intereffects, with others, cellulolysis was less or in some cases greater than that shown by individual components of the cultures. The interactions were probably related to effects on growth of the bacteria rather than on activities of components of the cellulase enzyme complex, and culture filtrates of two of the more numerous cellulolytic species ofClostridium affected growth of other cellulolytic bacteria. The inhibitory factor(s) appeared to be of bacteriocin type, but the stimulatory factor(s) was unknown. It was suggested that these interactions are localized or short-lived in the digester, and so the population remains in a dynamic steady state.Some inhibitions of growth of rumen cellulolytic bacteria were caused by the digester bacteria, but it was suggested that factors other than these inhibitions are responsible for the absence of rumen bacteria from anaerobic digesters.     

利用定量杂交法检测绵羊瘤胃纤维细菌的研究

实验利用通用细菌探针和3株纤维分解菌的特异性探针,初步建立起对瘤胃细菌进行检测的16SrRNA定量杂交的方法。试验将提取的总RNA按浓度系列稀释后与通用细菌探针进行杂交,检测结果所做的回归分析表明,杂交信号与尼龙膜上的所点RNA的量具有明显线性关系。同时对几份瘤胃样品进行3种纤维分解菌的初步定量检测,结果显示3种纤维分解菌的相对丰度与前人报道相似,表明该方法能够对瘤胃细菌进行定量分析,可在后续相关研究中使用。     

An investigation of the relationship between fecal and rumen bacterial concentrations in sheep

With no acceptable method for collecting fresh rumen fluid from zoo ruminants, it was proposed that fecal bacterial concentrations may be correlated with rumen bacteria. If so, fecal bacterial concentrations could be used to study both the effects of diet on rumen bacteria as well as rumen abnormalities. Total and cellulolytic bacterial concentrations were determined in whole rumen contents and feces of sheep using a most‐probable‐number (MPN) assay. In a Latin square design, four crossbred ewes were fed diets of 100% long or chopped orchardgrass hay (OH) and 60% ground or whole shelled corn plus 40% chopped OH. In a second trial, the sheep were fed a pelleted complete feed at varying levels of intake i.e., control at 2.0% of body weight and at 1.8, 1.6, and 1.2% of body weight. Higher total rumen bacterial concentrations (P<0.01) were found on the high concentrate diets as compared with the high forage diets. Grinding the corn also increased total bacterial concentrations (P<0.05). Fecal concentrations of total bacteria were higher (P<0.01) with the high concentrate diets. Chopping the forage decreased the concentration of fecal cellulolytic bacteria (P<0.05) but had no effect on their concentration in the rumen. An inverse linear relationship (P<0.01) was observed between total bacterial concentrations in the feces and diet intake. Although relationships were observed between the rumen and feces for total and cellulolytic bacterial concentrations, they were dependent on diet, particle size, and level of intake. Thus, fecal bacterial concentrations cannot be used to reliably predict rumen bacterial concentrations. Zoo Biol 27:100–108, 2008. © 2008 Wiley‐Liss, Inc.     

Interactions between anaerobic cellulolytic bacteria and fungi in the presence of Methanobrevibacter smithii

A mixed inoculum of cellulolytic rumen bacteria depressed straw degradation by a mixed culture of cellulolytic fungi grown in the presence of Methanobrevibacter smithii. The inhibitory effect appeared to be caused by Ruminococcus albus strain JI and R. flavefaciens strain 007. Ruminococcus albus strain J1 also depressed straw degradation by the fungi, but R. albus strain SY3 and three strains of Bacteroides (Fibrobacter) succinogenes tested showed little or no inhibitory activity. It seems that some ruminococci show competitive or antagonistic activity towards certain rumen fungi.     

Cellulolytic bacteria in buffalo rumen

The influence of three different feeds, wheat straw, sorghum and berseem, on total and cellulolytic bacterial counts in the buffalo rumen at different time intervals from 0 to 8 h after feeding was studied. Berseem feeding supported maximum growth of rumen bacteria in general and cellulolytic bacteria in particular. Wheat straw supported the poorest growth.
The types of cellulolytic bacteria recovered from the rumen of adult buffaloes were Ruminococcus albus, R. flavefaciens, Bacteroides succinogenes, Butyrivibrio fibrisolvens, Clostridium lochheadii, Cl. longisporum and other Clostridium spp. Cellulolytic cocci were present in smaller numbers than rod forms in the rumen of wheat-straw-fed buffaloes, whereas the cocci outnumbered rod forms in sorghum-and berseem-fed buffaloes.     

Removal of anaerobic fungi from the rumen of sheep by chemical treatment and the effect on feed consumption and in vivo fibre digestion

The population of anaerobic fungi in the rumen of sheep was reduced by the addition of tetronasin (an ionophore antibiotic) to a herbage diet. Fungi were reduced to undetectable levels (< 1 fungal zoospore per ml rumen fluid) by the combined addition of tetronasin and cycloheximide (a protein synthesis inhibitor) and the absence of fungi was maintained with low levels of tetronasin. Sheep with fungi present in the rumen ate 40% more of a straw-based diet (with a fibre digestibility in vivo of 51%) than they ate when without fungi (47% fibre digestibility). Counts of total viable bacteria, cellulolytic bacteria and ciliate protozoa in the rumen were not significantly different when anaerobic fungi were either present or absent.     

利用基本培养基初步筛选牛瘤胃中纤维素降解菌

目的初步筛选牛瘤胃中纤维素降解菌。方法分别采用基本培养基（牛肉膏蛋白胨培养基、马丁培养基）,利用好氧、兼性和厌氧3种不同的培养方法进行初选,初步分离牛瘤胃中的细菌与真菌,再通过复选培养基（加入微晶纤维素）,筛选降解纤维素的菌种。结果筛选分离出降解纤维素的1株厌氧细菌和1株厌氧真菌。结论此实验方法简单易行,能够有效地从牛瘤胃中筛选出生长良好的纤维素降解菌。     

Effects of glycerol on the growth,adhesion, and cellulolytic activity of rumen cellulolytic bacteria and anaerobic fungi

The effect of glycerol on the growth, adhesion, and cellulolytic activity of two rumen cellulolytic bacterial species,Ruminococcus flavefaciens andFibrobacter succinogenes subsp.succinogenes, and of an anaerobic fungal species,Neocallimastix frontalis, was studied. At low concentrations (0.1–1%), glycerol had no effect on the growth, adhesion, and cellulolytic activity of the two bacterial species. However, at a concentration of 5%, it greatly inhibited their growth and cellulolytic activity. Glycerol did not affect the adhesion of bacteria to cellulose. The growth and cellulolytic activity ofN. frontalis were inhibited by glycerol, increasingly so at higher concentrations. At a concentration of 5%, glycerol totally inhibited the cellulolytic activity of the fungus. Thus, glycerol can be added to animal feed at low concentrations.     

Isolation of Pseudobutyrivibrio ruminis and Pseudobutyrivibrio xylanivorans from rumen of Creole goats fed native forage diet

We isolated and identified functional groups of bacteria in the rumen of Creole goats involved in ruminal fermentation of native forage shrubs. The functional bacterial groups were evaluated by comparing the total viable, total anaerobic, cellulolytic, hemicellulolytic, and amylolytic bacterial counts in the samples taken from fistulated goats fed native forage diet (Atriplex lampa and Prosopis flexuosa). Alfalfa hay and corn were used as control diet. The roll tubes method increased the possibility of isolating and 16S rDNA gene sequencing allowed definitive identification of bacterial species involved in the ruminal fermentation. The starch and fiber contents of the diets influenced the number of total anaerobic bacteria and fibrolytic and amylolytic functional groups. Pseudobutyrivibrio ruminis and Pseudobutyrivibrio xylanivorans were the main species isolated and identified. The identification of bacterial strains involved in the rumen fermentation helps to explain the ability of these animals to digest fiber plant cell wall contained in native forage species.