Anaerobic Cocci from the Bovine Alimentary Tract, the Amino Acids of their Cell Wall Peptidoglycans and those of various Species of Anaerobic Streptococcus

Physiological characteristics of 45 strains of anaerobic cocci isolated from the rumen, duodenum, jejunum, ileum, caecum and faeces of pre-ruminant and ruminating calves and cows were examined. Among these strains were unusually anaerobic strains of Streptococcus bovis , Gram negative cocci resembling Megasphaera elsdenii but not fermenting lactic acid and strains resembling Coprococcus catus. The amino acid composition of the cell wall peptidoglycans of these and strains of anaerobic streptococci was determined.     

Use of the Hungate anaerobic technique in the isolation of phloroglucinol-negative mutants of Coprococcus species.

The Hungate anaerobic technique was used with a standard procedure for bacterial mutagenesis employing N-methyl-N-nitro-N'-nitrosoguanidine to obtain mutants of an obligate anaerobe. Three mutant strains were derived from a Coprococcus sp., strain Pe15, a rumen anaerobe capable of growing on phloroglucinol. The mutants did not grow on phloroglucinol but did degrade the compound in anaerobic washed-cell suspensions, producing the same end products in approximately the same proportions as the wild type. It was concluded that the mutants were blocked in a unique step or steps necessary for carbon skeleton or energy synthesis from phloroglucinol and not in formation of an enzyme involved in the pathway of phloroglucinol degradation.     

A method for the selective enumeration and isolation of ruminal Lactobacillus and Streptococcus

Ruminal lactic acid-producing bacteria were selectively isolated and enumerated using a one hour aerobic exposure prior to incubation on a semi-selective Lactobacillus medium, MRS, under anaerobic conditions. The technique allowed growth of pure cultures of ruminal Lactobacillus spp. and Streptococcus bovis without supporting the growth of pure cultures of any of the prominent ruminal bacterial species. In mixed cultures, the one hour aerobic pre-incubation inhibited the growth of the obligate anaerobic ruminal bacteria which can otherwise grow on the MRS medium, and the subsequent anaerobic incubation permitted maximal recovery of the weakly aerotolerant ruminal lactic acid-producing Lactobacillus spp. and Streptococcus spp. The efficacy of this technique in selecting exclusively for the lactic acid-producing bacteria was also demonstrated from populations of rumen bacteria from mixed culture end-point in vitro fermentation, continuous in vitro culture and isolations from fresh ruminal samples.     

Urease assay and urease-producing species of anaerobes in the bovine rumen and human feces.

A growth medium and test were developed for rapid detection of urease in fermentative anaerobic bacteria. Using nonselective rumen fluid roll-tube agar medium and the new test, it was confirmed that Peptostreptococcus productus is often the most numerous urease-forming species in human feces. Also, some fecal strains of Ruminococcus albus, Clostridium innocuum, and Clostridium beijerinckii produced urease. Single strains of Fusobacterium prausnitzii, Coprococcus catus, and Streptococcus mitis that were strongly ureolytic on isolation later lost this ability. Urease activity was also detected in many strains of nonselectively isolated rumen species. They include Succinivibrio dextrinosolvens, Treponema sp., Ruminococcus bromii (not previously known to be present in the rumen), Butyrivibrio sp., Bifidobacterium sp., Bacteroides ruminicola, and P. productus. Most P. productus strains contain urease; however, the uniformity of this feature in the other species noted above is not known. The urease in many of these species was not detected if the growth medium contained 0.2% or more (each) yeast extract and Trypticase.     

Protoplast formation and regeneration of dehydrodivanillin-degrading strains of Fusobacterium varium and Enterococcus faecium

Two strains of rumen anaerobes isolated from dehydrodivanillin-degrading cultures were identified as Fusobacterium varium and Enterococcus faecium. These organisms degraded dehydrodivanillin synergistically to 5-carboxymethylvanillin and vanillic acid. Specific conditions for protoplast formation and cell wall regeneration for both bacteria were determined, under strictly anaerobic conditions, to be as follows. The cell wall of each bacterium in yeast extract medium was loosened by adding penicillin G during early log-phase growth. The cell wall of F. varium was lysed by lysozyme (1 mg/ml) in glycerol (0.2 M)-phosphate buffer (0.05 M; pH 7.0). The addition of NaCl (0.08 M) with lysozyme was necessary for lysis of E. faecium in this solution. Almost all cells were converted to protoplasts after 2 h of incubation at 37 degrees C. Regeneration of both protoplasts was 20 to 30% on an agar-containing yeast extract medium.     

Protoplast formation and regeneration of dehydrodivanillin-degrading strains of Fusobacterium varium and Enterococcus faecium.

Two strains of rumen anaerobes isolated from dehydrodivanillin-degrading cultures were identified as Fusobacterium varium and Enterococcus faecium. These organisms degraded dehydrodivanillin synergistically to 5-carboxymethylvanillin and vanillic acid. Specific conditions for protoplast formation and cell wall regeneration for both bacteria were determined, under strictly anaerobic conditions, to be as follows. The cell wall of each bacterium in yeast extract medium was loosened by adding penicillin G during early log-phase growth. The cell wall of F. varium was lysed by lysozyme (1 mg/ml) in glycerol (0.2 M)-phosphate buffer (0.05 M; pH 7.0). The addition of NaCl (0.08 M) with lysozyme was necessary for lysis of E. faecium in this solution. Almost all cells were converted to protoplasts after 2 h of incubation at 37 degrees C. Regeneration of both protoplasts was 20 to 30% on an agar-containing yeast extract medium.     

Anaerobic degradation of biphenyl by the facultative anaerobic strain Citrobacter freundi BS2211

Using a synthetic medium supplemented with biphenyl (a polycyclic aromatic hydrocarbon), a new bacterial strain of Citrobacter freundii was isolated from enrichment cultures containing soil and industrial wastewater samples of the Serpukhov Condenser Factory. This strain was found to be capable of degrading biphenyl under anaerobic conditions in the course of nitrate reduction. When the initial concentration of biphenyl in culture medium equaled 150 mg/l, the culture with a titer of 10(9) cells/ml degraded up to 26-28% of biphenyl in 3 days (28 degrees C). At 250 mg/l, the culture with a titer of 10(7) cells/ml degraded 15% of biphenyl in 21 days. Approximately 10% of the substrate consumed was utilized completely, whereas the remainder underwent transformation.     

降解有毒的含羞草素和二羟基吡啶化合物的瘤胃细菌

从采食银合欢不引起中毒症状的我国广西北海市涠洲岛的黄牛瘤胃中,分离出4株厌氧细菌.经高效液相色谱分析证实,菌株BR-1,BR-2,BR-5和BR-7能降解有毒的含羞草素(mimosine)、3-羟基-4(1氢)吡啶酮(3,4 DHP)和2,3-二羟基吡啶(2,3DHP).在宿主体外进行纯菌和混菌液体培养,3天分别降解含羞草素44—59%,3,4DHP 30—47%和2,3DHP58—60%.经分类鉴定,确定为三个种:BR-1和BR-2很相似,为乳杆菌属(Lactobacillus),可能是一个新种.BR-5为牛链球菌(Streptococcus bovis)和BR-7为生孢梭菌(Clostridiumsporogenes).上述这些兼性和严格厌氧的革兰氏阳性细菌对含羞草素和二羟基吡啶类毒素具有降解活性,这是以前没有报道过的.由于这些脱毒细菌栖居于瘤胃的微生态系统中,从而保护宿主动物免受银合欢的毒害.     

Metabolism of tannin-protein complex by facultatively anaerobic bacteria isolated from koala feces

The metabolic pathways involved in degradation of tannin-protein complex (T-PC) were investigated in various facultatively anaerobic bacteria, with specific reference to fecal isolates from the koala including T-PC-degrading enterobacteria (T-PCDE),Streptococcus bovis, Klebsiella pneumoniae, andK. oxytoca. It was demonstrated that T-PCDE andS. bovis biotype I were capable of degrading protein complexed with gallotannin (a hydrolyzable tannin), but not that complexed with quebracho (a condensed tannin). Subsequent studies showed that these strains metabolized gallic acid to pyrogallol. Strains ofKlebsiella pneumoniae andK. oxytoca, which did not degrade T-PC, also metabolized gallic acid into pyrogallol. Pyrogallol was not degraded by any strains studied, but it was not detected in fresh feces of the koalas. The majority of strains isolated from feces could degrade phloroglucinol. Based on these findings, we propose that members of the gut microflora of the koala cooperate in the degradation of T-PC.     

Glucose-1-phosphate as a selective substrate for enumeration of Bacteroides species in the rumen.

When glucose-1-phosphate was used as the only added energy source in a selective roll tube medium, colony counts for rumen contents ranged from 17.8 to 84.8% of the total culturable count. Percentages were highest in rumen contents from sheep fed high-concentrate rations. From a total of 73 cultures isolated from glucose-1-phosphate roll rubes, only 15.1% were presumptively identified as Bacteroides species. Strains presumptively identified as Butyrivibrio, Selenomonas, Treponema, Streptococcus bovis, and Lachnospira also fermented glucose-1-phosphate. Thus, glucose-1-phosphate would not be useful as a selective substrate for isolation or enumeration of Bacteroides species from the rumen.     

Identification and enumeration of oleic acid and linoleic acid hydrating bacteria in the rumen of sheep and cows

The diversity and population densities of facultative anaerobic bacteria with the capacity to hydrate oleic acid and linoleic acid in the rumen of sheep and dairy cows were determined. The screening of representative colonies, from rumen fluid plated aerobically on a range of agar media, revealed that sheep rumen fluid contained hydration-positive strains of Streptococcus, Staphylococcus, Enterococcus, Lactobacillus and Pediococcus, whereas cow rumen fluid contained hydration-positive strains of Streptococcus, Lactobacillus and Staphylococcus. Mean counts of facultative anaerobic bacteria in sheep and cattle rumen were log10 7.29 and log10 6.40, respectively, and were independent of diet. Approximately 56% of facultative anaerobic bacteria were able to hydrate oleic and/or linoleic acid in anaerobic broth culture. For both sheep and cows, the most numerous hydration-positive isolates were strains of Strep. bovis. The results, which are the first to show that pediococci have the capacity to hydrate unsaturated fatty acids, suggest that lactic acid bacteria are the major unsaturated fatty acid hydrating bacteria in the rumen.     

Anaerobic Degradation of Biphenyl by the Facultative Anaerobic Strain Citrobacter freundii BS2211

Using a synthetic medium supplemented with biphenyl (a polycyclic aromatic hydrocarbon), a new bacterial strain of Citrobacter freundiiwas isolated from enrichment cultures containing soil and industrial wastewater samples of the Serpukhov Condenser Factory. This strain was found to be capable of degrading biphenyl under anaerobic conditions in the course of nitrate reduction. When the initial concentration of biphenyl in the culture medium equaled 150 mg/ml, the culture with a titer of 10⁹ cells/ml degraded up to 26–28% of biphenyl in 3 days (28°C). At 250 mg/ml, the culture with a titer of 10⁷ cells/ml degraded 15% of biphenyl in 21 days. Approximately 10% of the substrate consumed was utilized completely, whereas the remainder underwent transformation.     

Isolation and identification of adherent epimural bacteria during succession in young lambs

Successive changes in aerobic and anaerobic bacterial counts and changes in the generic composition of the epimural community in lambs from 1 to 10 weeks were determined. Bacterial culture counts revealed a predominantly anaerobic community, with the mean anaerobic count being 1.4 X 10(7) CFU/cm2 of tissue surface. The aerobic count was highest at 1 week of age and declined significantly thereafter to a mean of 1.8 X 10(4) CFU/cm2, thus representing only 0.13% of the mean anaerobic count after week 1. Of the 345 strains isolated anaerobically at 1, 2, 4, 6, 8, and 10 weeks of age, 47, 32, 12, 32, 2, and 5% were capable of growth in a partially reduced medium, indicating a reduction in the number of facultative anaerobes with time. The majority of isolated strains were identified as belonging to genera commonly isolated from rumen contents. In some instances, however, strains did not correspond to previously described species, and some genera were present in proportions different from those expected in rumen fluid. At three of the sampling times, one genus was dominant, constituting 45 to 55% of the isolates. These dominant isolates were Streptococcus bovis, Bacteroides sp., and an anaerobic Streptococcus sp. for weeks 1, 2, and 10, respectively. During the transition period (weeks 4 to 8), two or more groups were codominant.     

Isolation and identification of adherent epimural bacteria during succession in young lambs.

Successive changes in aerobic and anaerobic bacterial counts and changes in the generic composition of the epimural community in lambs from 1 to 10 weeks were determined. Bacterial culture counts revealed a predominantly anaerobic community, with the mean anaerobic count being 1.4 X 10(7) CFU/cm2 of tissue surface. The aerobic count was highest at 1 week of age and declined significantly thereafter to a mean of 1.8 X 10(4) CFU/cm2, thus representing only 0.13% of the mean anaerobic count after week 1. Of the 345 strains isolated anaerobically at 1, 2, 4, 6, 8, and 10 weeks of age, 47, 32, 12, 32, 2, and 5% were capable of growth in a partially reduced medium, indicating a reduction in the number of facultative anaerobes with time. The majority of isolated strains were identified as belonging to genera commonly isolated from rumen contents. In some instances, however, strains did not correspond to previously described species, and some genera were present in proportions different from those expected in rumen fluid. At three of the sampling times, one genus was dominant, constituting 45 to 55% of the isolates. These dominant isolates were Streptococcus bovis, Bacteroides sp., and an anaerobic Streptococcus sp. for weeks 1, 2, and 10, respectively. During the transition period (weeks 4 to 8), two or more groups were codominant.     

Deoxyribonuclease activity in Streptococcus bovis

Deoxyribonuclease activity was tested with lambda bacteriophage DNA as a substrate in three Streptococcus bovis strains isolated from the rumen of sheep and cow. Non-specific nuclease activity was detected in the cell extract of Strep, bovis BM 114. Specific endonuclease activity was detected in the cell extract of the strain Strep. bovis II/I, isolated from the rumen of a sheep. A rapid technique is proposed for the detection of endonuclease activity of rumen bacteria.     

Anaerobic degradation of veratrylglycerol-beta-guaiacyl ether and guaiacoxyacetic acid by mixed rumen bacteria.

Veratrylglycerol-beta-guaiacyl ether (0.2 g/liter), a lignin model compound, was found to be degraded by mixed rumen bacteria in a yeast extract medium under strictly anaerobic conditions to the extent of 19% within 24 h. Guaiacoxyacetic acid, 2-(o-methoxyphenoxy)ethanol, vanillic acid, and vanillin were detected as degradation products of veratrylglycerol-beta-guaiacyl ether by thin-layer chromatography, gas chromatography, and gas chromatography-mass spectrometry. Guaiacoxyacetic acid (0.25 g/liter), when added into the medium as a substrate, was entirely degraded within 36 h, resulting in the formation of phenoxyacetic acid, guaiacol, and phenol. These results suggest that the beta-arylether bond, an important intermonomer linkage in lignin, can be cleaved completely by these rumen anaerobes.     

Anaerobic degradation of veratrylglycerol-beta-guaiacyl ether and guaiacoxyacetic acid by mixed rumen bacteria

Veratrylglycerol-beta-guaiacyl ether (0.2 g/liter), a lignin model compound, was found to be degraded by mixed rumen bacteria in a yeast extract medium under strictly anaerobic conditions to the extent of 19% within 24 h. Guaiacoxyacetic acid, 2-(o-methoxyphenoxy)ethanol, vanillic acid, and vanillin were detected as degradation products of veratrylglycerol-beta-guaiacyl ether by thin-layer chromatography, gas chromatography, and gas chromatography-mass spectrometry. Guaiacoxyacetic acid (0.25 g/liter), when added into the medium as a substrate, was entirely degraded within 36 h, resulting in the formation of phenoxyacetic acid, guaiacol, and phenol. These results suggest that the beta-arylether bond, an important intermonomer linkage in lignin, can be cleaved completely by these rumen anaerobes.     

Isolation and some characteristics of anaerobic oxalate-degrading bacteria from the rumen.

Obligately anaerobic oxalate-degrading bacteria were isolated from an enriched population of rumen bacteria in an oxalate-containing medium that had been depleted of other readily metabolized substrates. These organisms, which are the first reported anaerobic oxalate degraders isolated from the rumen, were gram negative, nonmotile rods. They grew in a medium containing sodium oxalate, yeast extract, cysteine, and minerals. The only substrate that supported growth was oxalate. Growth was directly related to the concentration of oxalate in the medium (1 to 111 mM), and cell yields were approximately 1.1 g (dry weight)/mol of oxalate degraded. Oxalate was stoichiometrically degraded to CO2 and formate. These anaerobes occupy a unique ecological niche and are distinct from any previously described oxalate-degrading bacteria.     

The effect of carbon and nitrogen sources on bovicin HC5 production by Streptococcus bovis HC5

Aims:  To investigate the effect of media composition and agroindustrial residues on bovicin HC5 production by Streptococcus bovis HC5.
Methods and Results:  Batch cultures of S. bovis HC5 were grown in basal medium containing different carbon and nitrogen sources. The activity of cell-free and cell-associated bovicin HC5 was determined in culture supernatants and acidic extracts obtained from cell pellets, respectively. Streptococcus bovis HC5 produced bovicin using a variety of carbon and nitrogen sources. The highest specific activity was obtained in media containing 16 g l⁻¹ of glucose, after 16 h of incubation. The peak in cell-free and cell-associated bovicin HC5 activity was detected when S. bovis HC5 cultures reached stationary phase. The bovicin HC5 specific activity and bacterial cell mass increased approximately 3-fold when yeast extract and trypticase (0·5 and 1·0 g l⁻¹, respectively) were added together to the basal medium. Streptococcus bovis HC5 cultures produced bovicin HC5 in cheese whey and sugar cane juice and maximal volumetric productivity was obtained after 12 h of incubation.
Conclusions:  Streptococcus bovis HC5 is a versatile lactic acid bacterium that can utilize several carbon and nitrogen sources for bovicin HC5 production. This bacterium could be a useful model to study bacteriocin production in the rumen ecosystem.
Significance and Impact of the Study:  The use of agroindustrial residues as carbon sources could have an economical impact on bovicin HC5 production. To our knowledge, this is the first report to show the use of sugar cane juice for bacteriocin production by lactic acid bacteria.     

Anaerobic degradation of coniferyl alcohol by methanogenic consortia

Coniferyl alcohol was shown to be completely biodegradable to carbon dioxide and methane under strictly anaerobic culture conditions. The mineralization of 300 mg of the substrate per liter was observed in acclimated ferulic acid-degrading methanogenic consortia, as well as in anaerobic enrichments on coniferyl alcohol seeded with sewage sludge. Ferulic and phenylpropionic acids were detected in the cultures degrading coniferyl alcohol as the sole carbon and energy source, suggesting that this compound is oxidized to ferulic acid, which is then degraded as previously described.