Anaerobic Roll Tube Media for Nonselective Enumeration and Isolation of Bacteria in Human Feces

Medium 10 (M10), developed for rumen bacteria and containing small amounts of sugars, starch, volatile fatty acids, hemin, Trypticase, yeast extract, cysteine, and sulfide, plus agar, minerals and CO(2)-HCO(3)-buffer, was used with the Hungate anaerobic method as a basal medium to evaluate the efficacy of various ingredients. Three-day-old colony counts from adults on normal diets (17 samples) were 0.55 x 10(11) to 1.7 x 10(11) per g (mean, 1.15 x 10(11)) for M10. Single deletion of volatile fatty acids, Trypticase, yeast extract, or sulfide did not reduce counts. Deletion of hemin or both Trypticase and yeast extract significantly lowered counts. Addition of fecal extract, rumen fluid, 1% dehydrated Brain Heart Infusion (BHI) or 2 to 6% liver infusion did not increase counts; 1% dehydrated bile or 3.7% BHI markedly depressed them. Decreasing the gas-phase CO(2) concentration from 100 to 5% with N(2) and correspondingly lowering the HCO(3) had little effect. Counts in supplemented Brewer Thioglycollate (Difco), BHI, and Trypticase soy agar were similar or lower than in M10; ease in counting was best in M10. Comparison of features of 88 predominant strains of fecal bacteria randomly isolated indicated that M10 supported growth of as many or more species of bacteria as compared to supplemented BHI. The results suggest that predominant bacteria of human feces, in general, are not as nutritionally fastidious as rumen bacteria and indicate that media for counts or isolation containing large amounts of rich organic materials are neither necessary nor desirable when adequate anaerobic techniques are used.     

Medium Without Rumen Fluid for Nonselective Enumeration and Isolation of Rumen Bacteria

Colony counts which approximated those in a habitat-simulating, rumen fluid-agar medium (RFM) were obtained in medium 10, a medium identical to the RFM except for the replacement of rumen fluid with 1.5 x 10(-6)m hemin, 0.2% Trypticase, 0.05% yeast extract, and a 6.6 x 10(-2)m volatile fatty acid mixture qualitatively and quantitatively similar to that in rumen fluid. Single deletion of Trypticase, yeast extract, or the volatile fatty acid mixture from medium 10 significantly reduced colony counts. Colony counts were also reduced when medium 10 was modified to contain higher concentrations of Trypticase or volatile fatty acids. Significant differences were found between colony counts obtained from diluted rumen contents of animals fed a cracked corn-urea diet, and the colony counts obtained from animals fed either a cracked corn-soyean oil meal or an alfalfa hay-grain diet. Qualitative differences were found between the predominant bacterial strains isolated from rumen contents of animals fed cracked corn diets and strains isolated from animals fed alfalfa hay-grain. Regardless of differences in the predominant flora associated with diet, medium 10 and the RFM supported growth of similar bacterial populations. The results show that medium 10 is suitable for enumeration and isolation of many predominant rumen bacteria.     

Effect of soluble maillard reaction products on rumen microorganisms

After incubation of Maillard reaction polymers (MRPs) with rumen fluid from wethers neither volatile fatty acids nor lactate were produced. Soluble polymeric products of the Maillard reaction were nonmetabolizable by a mixed culture of rumen microorganisms. MRPs added at 0.5 and 2 g/L inhibited the growth of seven ruminal Gram-negative bacteria by 20 and 30%, respectively. In four strains of Gram-positive bacteria, MRPs lowered the cell concentration by 11% (0.5 g/L) and 25% (2 g/L). The rumen fungusOrpinomyces jojonii also did not metabolize soluble MRPs.     

一株瘤胃源乳酸利用菌的分离鉴定及其体外代谢特性

【目的】从饲喂高精料的本地山羊瘤胃内分离到一株利用乳酸并能产生大量丙酸的菌株L9,并进一步研究了该菌在调控瘤胃微生物发酵中的作用。【方法】采用厌氧培养技术,结合形态、生理生化特性和16SrRNA基因序列分析结果。【结果】该菌株被鉴定为反刍兽新月形单胞菌（Selenomonas ruminantium）。该菌株体外代谢特性研究表明,L9可利用乳酸作为唯一碳源,该菌在24h内可对90mmol／L的乳酸完全降解。体外摸拟瘤胃急性酸中毒的发酵试验结果表明,以淀粉为底物时,与对照组相比,添加菌株L9可显著降低瘤胃微生物体外培养体系中乳酸浓度,提高pH值,提高总挥发性脂肪酸和丙酸浓度,并显著降低乙酸与丙酸的浓度比（P〈0．05）。【结论】结果显示,菌株L9是一株可代谢乳酸,促进丙酸生成,提高总挥发性脂肪酸浓度的有益瘤胃细菌。     

Function of Growth Factors for Rumen Microorganisms I. Nutritional Characteristics of Selenomonas ruminantium

Nutritional characteristics of Selenomonas ruminantium var. lactilytica isolated from a sheep rumen were studied. The organism required for growth the addition of a clarified rumen fluid to a Trypticase-yeast extract medium with either lactate or glucose as an energy source. The requirement for rumen fluid was found to be satisfied by volatile fatty acids in glucose media and by biotin in lactate media. Straight-chain saturated fatty acids with C(3) to C(10) carbon skeleton had been found to be effective. Among them, n-valerate was most effective at the lowest concentration. An abnormal morphology was observed with n-valerate-deficient glucose media. n-Valerate was essential in glucose media, and it was stimulatory in lactate media. Fermentation products from glucose were lactate, propionate, and acetate, and fermentation products from lactate were propionate and acetate. When cells were grown in a glucose medium containing n-valerate-C(14), the label was present in cell fractions. Almost all of the activity was found in lipid materials.     

Plant-Derived Oils Reduce Pathogens and Gaseous Emissions from Stored Cattle Waste

Carvacrol and thymol in combination at 6.7 mM each completely inhibited the production of short-chain volatile fatty acids and lactate from cattle waste in anoxic flasks over 23 days. Fecal coliforms were reduced from 4.6 × 10⁶ to 2.0 × 10³ cells per ml 2 days after treatment and were nondetectable within 4 days. Total anaerobic bacteria were reduced from 8.4 × 10¹⁰ to 1.5 × 10⁷ cells per ml after 2 days and continued to be suppressed to that level after 14 days. If the concentration of carvacrol or thymol were doubled (13.3 mM), either could be used to obtain the same inhibitory fermentation effect. We conclude that carvacrol or thymol may be useful as an antimicrobial chemical to control pathogens and odor in stored livestock waste.     

Growth Factor Requirements of Ruminococcus flavefaciens Isolated from the Rumen of Cattle Fed Purified Diets

Eight strains of cellulolytic cocci were isolated from a 10^-8 dilution of rumen ingesta and were presumptively identified as Ruminococcus flavefaciens. Four strains were isolated from a steer fed a purified diet which contained isolated soy protein, and four strains were isolated from a steer fed a purified diet which contained urea. Certain growth factor requirements of these bacteria were determined. All strains grew with clarified rumen fluid added to the medium. However, fatty acids could substitute for rumen fluid in four strains. Two strains isolated from each steer either required or their growth was stimulated by isobutyric and/or isovaleric and/or 2-methyl-butyric acid. These results indicate that, even when a diet was fed which contained no branched-chain amino acids, the carbon skeleton precursors of branched-chain fatty acids, the cattle were still able to maintain a large population of cellulolytic bacteria that require fatty acids for growth. Therefore, the fatty acids appear to be provided by other bacteria, by protozoa, or by the host animal.     

Lipid Metabolism of Rumen Ciliates and Bacteria: II. Uptake of Fatty Acids and Lipid Analysis of Isotrichia intestinalis and Rumen Bacteria with Further Information on Entodinium simplex

The total lipid and free fatty acid contents of Isotricha intestinalis, Entodinium simplex, and the rumen bacterial flora of the respective protozoa were determined. Warburg manometric data showed that the sodium salts of tributyrin, oleic, and acetic acids stimulated gas production in I. intestinalis, whereas tributyrin was stimulatory with E. simplex and less active with oleic and acetic acids. Rumen bacteria provided fatty acids produced lower manometric gaseous increases when compared with the protozoa. Volatile fatty acids were produced by I. intestinalis and rumen bacteria with tributyrin, but not with tripalmitin. Sodium oleate gave little volatile fatty acid response with I. intestinalis or rumen bacteria. Washed suspensions of I. intestinalis and rumen bacteria concentrated C¹⁴-labeled oleic, palmitic, stearic, and linoleic acids within the cells during short incubation periods. Autoradiographs demonstrated the conversion of C¹⁴-labeled oleic, palmitic, stearic, linoleic, and acetic acids in the rumen protozoa and bacterial cells.     

Protozoa involved in butyric rather than lactic fermentative pattern during latent acidosis in sheep

We used six ruminally cannulated Texel wethers to study the relative role of protozoa and lactate-metabolizing bacteria in ruminal fermentative patterns during an induced latent acidosis. The sheep were fed an alfalfa hay diet (H) and latent acidosis was induced, following a short transition period of one week, with a grain-rich acidotic diet (W, 60% wheat + 40% alfalfa hay). Ruminal pH, ruminal volatile fatty acids (VFA), lactate and NH3 concentrations, protozoa and lactate-utilizing bacterial counts, the relative proportions of three main bacteria implicated in lactate metabolism (a lactate-producing species, Streptococcus bovis, and two lactate-utilizing species, Selenomonas ruminantium, and Megasphaera elsdenii) using specific 16S-rRNA-targeting oligonucleotide probes, and lactate dehydrogenase (LDH) activity were determined for both diets. The pH parameters (mean, minimum, maximum, time and area under pH 6.0 and 5.5) measured with the W diet were indicative of a latent (i.e., subacute and maintained) acidosis. However, a butyric rather than lactic latent acidosis was observed in this study. Total ruminal lactate concentration remained at low levels with the acidotic diet (< 4 mmol x L(-1)), but changes were observed in VFA composition, which was oriented towards butyrate at the expense of acetate (P < 0.05), while propionate remained constant. In agreement with the low ruminal lactate concentration, no changes in the proportion of S. bovis 16S-rRNA were observed. The lactate-metabolizing bacterial population also remained fairly constant in number, proportion and activity. The increase in butyrate concentration was accompanied by a proliferation of entodiniomorphs (P < 0.01). These results suggest that the protozoa limited lactate accumulation and possibly also the decrease in pH during latent acidosis. Experiments with defaunated and faunated sheep could provide further evidence of the role of protozoa in the development of rumen latent acidosis.     

Changes in Lactate-Producing and Lactate-Utilizing Bacteria in Relation to pH in the Rumen of Sheep During Stepwise Adaptation to a High-Concentrate Diet

Changes in the numbers and types of lactate-producing and lactate-utilizing bacteria in the rumen of sheep were followed during stepwise adaptation from a low- to a high-concentrate diet. The mean numbers of bacteria increased after each change in diet when increasing amounts of maize grain were substituted for maize stover. A surge in number of amylolytic bacteria always preceded an increase in lactate-utilizing bacteria, and with the final diet containing 71% grain and molasses the two groups tended to balance each other, which resulted in low lactic acid accumulation. The lactate utilizers thus played a key role in controlling the fermentation. Orderly shifts occurred among the predominating amylolytic and lactate-utilizing bacteria in response to the gradual decrease in ruminal pH as the amount of maize meal in the diet increased. Among the lactate utilizers, the succession began with acid-sensitive Veillonella and Selenomonas, which were superseded by more acid-tolerant Anaerovibrio and Propionibacterium. Among the amylolytic bacteria, Bacteroides was superseded by more acid-tolerant Lactobacillus and Eubacterium. The ecological succession of predominating genera was shown to be correlated significantly with ruminal pH and, more specifically, with the length of time as well as the extent to which the pH remained below a certain critical undefined value in the rumen, arbitrarily set at pH 6.00.     

Continuous cultivation of rumen microorganisms,a system with possible application to the anaerobic degradation of lignocellulosic waste materials

Summary An in vitro continuous fermentation device is described which allows the maintenance of a mixed rumen microbial population under conditions similar to those in the rumen. The differences in flow rates of solids and liquids found in the rumen were established in vitro by means of a simple filter construction. A grass-grain mixture was used as a solid growth substrate. During a test period of 65 days the artificial rumen fermenter showed stable operation with respect to ciliate numbers, fibre degradation and volatile fatty acids production. Values obtained were comparable to those found in vivo. Optimal fibre degradation and volatile fatty acids production were maintained when hydraulic retention times (HRT) ranged from 11 to 14 h. At these HRT-values ciliate numbers were maintained at about 8.5×10⁴ cells per ml. Ciliate numbers declined drastically at HRT-values above 14h. A fermenter inoculated with a small volume of rumen fluid (1:100, v/v) reached normal protozoal numbers, fibre degradation and volatile fatty acids productions after a start up period of only 8 to 10 days. The possible application of rumen microorganisms for an efficient degradation of lignocellulosic waste material in an artificial rumen digester is discussed.     

Shifts in xylanases and the microbial community associated with xylan biodegradation during treatment with rumen fluid

Treatment with rumen fluid improves methane production from non-degradable lignocellulosic biomass during subsequent methane fermentation; however, the kinetics of xylanases during treatment with rumen fluid remain unclear. This study aimed to identify key xylanases contributing to xylan degradation and their individual activities during xylan treatment with bovine rumen microorganisms. Xylan was treated with bovine rumen fluid at 37°C for 48 h under anaerobic conditions. Total solids were degraded into volatile fatty acids and gases during the first 24 h. Zymography showed that xylanases of 24, 34, 85, 180, and 200 kDa were highly active during the first 24 h. Therefore, these xylanases are considered to be crucial for xylan degradation during treatment with rumen fluid. Metagenomic analysis revealed that the rumen microbial community’s structure and metabolic function temporally shifted during xylan biodegradation. Although statistical analyses did not reveal significantly positive correlations between xylanase activities and known xylanolytic bacterial genera, they positively correlated with protozoal (e.g., Entodinium, Diploplastron, and Eudiplodinium) and fungal (e.g., Neocallimastix, Orpinomyces, and Olpidium) genera and unclassified bacteria. Our findings suggest that rumen protozoa, fungi, and unclassified bacteria are associated with key xylanase activities, accelerating xylan biodegradation into volatile fatty acids and gases, during treatment of lignocellulosic biomass with rumen fluid.     

Isolation and identification of fecal bacteria from adult swine.

An examination of the fecal microflora of adult swine was made with regard to the efficiency of several roll tube media in enumeration and recovery of anaerobes, the effects of medium constituents on recovery, and the isolation and identification of the predominant kinds of bacteria. Total number of organisms by microscopic bacterial counts varied among fecal samples from 4.48 X 10(10) to 7.40 X 10(10) bacteria/g (wet weight). Comparison of different nonselective roll tube media indicated that about 30% of the fecal bacteria could be recovered with a rumen fluid (40%, vol/vol) medium (M98-5). Recoveries of 21 and 15%, respectively, were obtained with M10 and rumen fluid-glucose-cellobiose agar (RGCA) media. Rumen fluid, Trypticase, sugars, and CO2 gas phase were important components required for maximum recovery with this medium. Similar high recoveries of anaerobes were also obtained with M98-5 containing swine cecal extract of place in rumen fluid or M10 plus swine cecal extract. Significantly lower recoveries were observed with RCGA, media supplemented with swine fecal extracts, reinforced clostridial medium, brain heart infusion agar, and prereduced blood agar. Ninety percent of the bacteria isolated from roll tube media were gram positive and consisted of facultatively anaerobic streptococci, Eubacterium sp., Clostridium sp., and Propionibacterium acnes. The remainder of the flora (8%) included several other species of anaerobes and Escherichia coli. Rumen fluid (or volatile fatty acids), Trypticase, and yeast extract additions to basal media stimulated the growth of anaerobic strains. Variation in the relative proportions of the predominant fecal microflora was observed. This work indicates that satisfactory enumeration, isolation and cultivation of the predominant microflora in swine feces can be obtained when strict anaerobic culture methods and a rumen fluid medium are used.     

Proximate Analyses of Rumen Fluid Preparations Used in Microbiological Media

Proximate analyses of clarified rumen fluid No. 1 (CRF1) and No. 2 (CRF2). and of an aqueous extract of Escherichia coli cells are reported. Differences in the procedures for preparing CRF2 and CRF1 from the same batch of whole rumen fluid were reflected by increased levels of carbohydrate, protein and total nitrogen in CRF2. Variations in the composition of CRF2 with time after feeding are also reported. Results showed CRF2 to be an inconsistent and non-reproducible medium supplement. The extract of E. coli cells contained very low levels of volatile fatty acids and ammonia and thus by itself could not replace CRF2 in medium for many species of rumen bacteria. It could be used as an alternative to CRF2 in media where more consistent and reproducible composition was required if the medium were supplemented with defined mixtures of nutrients known to be required by rumen bacteria.     

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.     

Transfer of plasmids between strains of Escherichia coli under rumen conditions

K. P. SCOTT AND H.J. FLINT. 1995. Strains of Escherichia coli originally isolated from the rumen of sheep were shown to be capable of exchanging a 60kb plasmid, conferring resistance to tetracycline and ampicillin, at low frequencies (below 10^-6 per recipient) under anaerobic conditions in the presence of (a) autoclaved and clarified rumen fluid, (b) raw clarified rumen fluid, or (c) whole rumen fluid. Under anaerobic conditions the two rumen strains showed no inhibition of growth rate when 50 mmol 1^-1 volatile fatty acids were added to LB medium at pH 7, although significant inhibition resulted with 100 mmol 1^-1 VFA. The two rumen strains, and four strains from the pig gut, showed less inhibition of anaerobic growth by volatile fatty acids than did three laboratory strains examined for comparison. These findings indicate that plasmid transfer between certain E. coli strains can occur under conditions that closely simulate an anaerobic gut environment.     

Ruminal Fermentation and Sugar Concentrations

The maximal amounts of growth of Selenomonas ruminantium were examined in the media containing various amounts of glucose. The yields of cells per unit weight of glucose are linear functions to glucose concentrations in the ranges between zero to 0.005% and 0.005 to 0.7%, Cell yields per glucose are greater in the former range, indicating greater a-mounts of energy are available per glucose at lower concentrations. Growth responses in lactate media containing various amounts of glucose showed that the preincubation with larger amounts of glucose is inhibitory for the following growth and metabolism of lactate. The organism produces predominantly lactate in the glucose medium. However, volatile fatty acid productions increase when the initial concentrations of glucose become low. Isotopic studies showed that the lactate utilization yielding volatile fatty acids is inhibited by the preceding metabolism of high concentrations of glucose. These results were discussed with regard to normal and abnormal fermentations in the rumen.     

Volatile Fatty Acids and the Inhibition of Escherichia coli Growth by Rumen Fluid

Concentrations of volatile fatty acids (VFA) normally found in bovine rumen fluid inhibited growth of Escherichia coli in Antibiotic Medium 3. Acetic, propionic, and butyric acids each produced growth inhibition which was markedly pH-dependent. Little inhibition was observed at pH 7.0, and inhibition increased with decreasing pH. A combination of 60 mumoles of acetate, 20 mumoles of propionate, and 15 mumoles of butyrate per ml gave 96, 69, and 2% inhibition at pH 6.0, 6.5, and 7.0, respectively. Rumen fluid (50%) gave 89 and 48% inhibition at pH 6.0 and 6.5, respectively, and growth stimulation (22%) at pH 7.0. Rumen fluid inhibitory activity was heat-stable, was not precipitated by 63% ethyl alcohol, and was lost by dialysis and by treatment with anion-exchange resins but not with cation-exchange resins. These results are consistent with the idea that VFA are the inhibitory substances in rumen fluid. Previous results which indicated that rumen fluid VFA did not inhibit E. coli growth were due to lack of careful control of the final pH of the growth medium. The E. coli strain used does not grow in rumen fluid alone at pH 7.0.     

Mureinolytic ability of the rumen ciliate <Emphasis Type="Italic">Diploplastron affine</Emphasis>

Rumen ciliate protozoa intensively engulf bacteria. However, their ability to utilize murein which is the main polysaccharide of bacterial cell wall has hardly been recognized. The present study concerns the ability of the rumen protozoa Diploplastron affine to digest and ferment murein. The ciliates were isolated from the rumen fluid and grown in vitro or inoculated into the rumen of defaunated sheep. The results of long-term cultivation of protozoa showed a positive correlation between their number and murein content in the culture medium. It was also found that bacteria-free D. affine ciliates incubated with or without murein produced volatile fatty acids at the rate of 12.3 and 8.7 pmol/h per protozoan, respectively, acetic, butyric and propionic acids being the three main acids released to the medium. Enzyme studies performed with the use of protozoan cell extract prepared from bacteria-free ciliates degraded murein at a rate of 25 U/mg protein per h; two mureinolytic enzymes were identified by zymographic technique in the examined preparation.     

Characterization of the rumen lipidome and microbiome of steers fed a diet supplemented with flax and echium oil

Developing novel strategies for improving the fatty acid composition of ruminant products relies upon increasing our understanding of rumen bacterial lipid metabolism. This study investigated whether flax or echium oil supplementation of steer diets could alter the rumen fatty acids and change the microbiome. Six Hereford × Friesian steers were offered grass silage/sugar beet pulp only (GS), or GS supplemented either with flax oil (GSF) or echium oil (GSE) at 3% kg⁻¹ silage dry matter in a 3 × 3 replicated Latin square design with 21-day periods with rumen samples taken on day 21 for the analyses of the fatty acids and microbiome. Flax oil supplementation of steer diets increased the intake of polyunsaturated fatty acids, but a substantial degree of rumen biohydrogenation was seen. Likewise, echium oil supplementation of steer diets resulted in increased intake of 18:4n-3, but this was substantially biohydrogenated within the rumen. Microbiome pyrosequences showed that 50% of the bacterial genera were core to all diets (found at least once under each dietary intervention), with 19.10%, 5.460% and 12.02% being unique to the rumen microbiota of steers fed GS, GSF and GSE respectively. Higher 16S rDNA sequence abundance of the genera Butyrivibrio, Howardella, Oribacterium, Pseudobutyrivibrio and Roseburia was seen post flax feeding. Higher 16S rDNA abundance of the genus Succinovibrio and Roseburia was seen post echium feeding. The role of these bacteria in biohydrogenation now requires further study.