Cellulolytic Bacterium Fibrobacter Succinogenes Research Articles

50 Effects of cottonseed supplementation on the rumen microbiota of parasite-challenged meat goats

Abstract Cotton serves as a vital economic driver within the agricultural sector of Mississippi. The seeds of cotton are rich in fat and protein content, making them a cost-effective alternative to soybean meal and other expensive protein sources. Additionally, cottonseed is the primary source of gossypol, a phenolic compound with significant potential for natural parasite control. The rumen microbiota, a diverse community of microorganisms, has a pivotal role in nutrient digestion and detoxification. However, the impact of cottonseed on rumen microbial ecosystems remains largely unexplored. The aim of this study is to investigate the effects of cottonseed supplementation on the rumen microbiota of meat goats with and without the challenge of parasites. Forty female weaned Boer × Spanish crossed meat goats were assigned to one of the four treatments (n = 10 per treatment) in a 2 × 2 factorial design consisting of control group (CON), parasite challenge group (P), cottonseed supplementation (0.5% body weight) without parasite challenge group (CS-NP), and CS supplementation with parasite challenge group (CS-P). The parasite challenge was performed with a dose of 4,000 infective third-stage larvae of Haemonchus contortus per animal. After 25 d following the first dose, the treatments were applied for 10 wk. The goats were slaughtered at the end of the experiment, and rumen digesta samples were collected for microbiota analysis using high-resolution full-length 16S rRNA gene amplicon sequencing. The results revealed that the parasite challenge significantly reduced the number of bacterial species in the rumen (P = 0.032), regardless of cottonseed supplement. Anaeromassilibacillus senegalensis, which has been isolated from kwashiorkor (severe protein malnutrition) patient, Hungateiclostridium aldrichii, and Mogibacterium diversum were enriched in rumen of P group, while Anaerocella delicata and the rumen lipolytic organism Anaerovibrio lipolyticus were reduced compared with the CON group [linear discriminant analysis (LDA) scores > 3.6]. Cottonseed supplementation did not affect the number of bacterial species in the rumen of parasite-challenged goats, but it did lead to an increase of Anaerovibrio lipolyticus (P = 0.008) and a reduction of Anaeromassilibacillus senegalensis (P = 0.007). Moreover, cottonseed supplementation tended to increase the number of bacterial species in the rumen of meat goats without parasite challenge (P = 0.08) and increased the relative abundance of butyrate-producing bacteria such as Pseudobutyrivibrio xylanivorans and Intestinimonas butyriciproducens, along with the butanol-producing bacteria Clostridium saccharoperbutylacetonicum (LDA scores > 2). Conversely, cottonseed supplementation inhibited cellulolytic bacteria Fibrobacter succinogenes, polysaccharide-degrading bacteria Butyrivibrio proteoclasticus, and several Prevotella species, including P. oris, P. loescheii, P. copri, and P. albensis (LDA score > 2). In conclusion, parasite challenge affects the rumen microbial diversity and certain bacterial species that utilize protein and lipids, and cottonseed supplementation partially restores the altered rumen microbial ecosystem of the parasite-challenged meat goats.

Effects of rearing mode on gastro-intestinal microbiota and development, immunocompetence, sanitary status and growth performance of lambs from birth to two months of age

BackgroundArtificial rearing system, commonly used in prolific sheep breeds, is associated to increased mortality and morbidity rates before weaning, which might be linked to perturbations in digestive tract maturation, including microbiota colonization. This study evaluated the effect of rearing mode (mothered or artificially reared) on the establishment of the rumen and intestinal microbiome of lambs from birth to weaning. We also measured immunological and zootechnical parameters to assess lambs’ growth and health. GIT anatomy as well as rumen and intestinal epithelium gene expression were also analysed on weaned animals to assess possible long-term effects of the rearing practice.ResultsTotal VFA concentrations were higher in mothered lambs at 2 months of age, while artificially-reared lambs had lower average daily gain, a more degraded sanitary status and lower serum IgG concentration in the early growth phase. Metataxonomic analysis revealed higher richness of bacterial and eukaryote populations in mothered vs. artificially-reared lambs in both Rumen and Feces. Beta diversity analysis indicated an evolution of rumen and fecal bacterial communities in mothered lambs with age, not observed in artificially-reared lambs. Important functional microorganisms such as the cellulolytic bacterium Fibrobacter succinogenes and rumen protozoa did not establish correctly before weaning in artificially-reared lambs. Enterobacteriaceae and Escherichia coli were dominant in the fecal microbiota of mothered lambs, but main E. coli virulence genes were not found differential between the two groups, suggesting they are commensal bacteria which could exert a protective effect against pathogens. The fecal microbiota of artificially-reared lambs had a high proportion of lactic acid bacteria taxa. No difference was observed in mucosa gene expression in the two lamb groups after weaning.ConclusionsThe rearing mode influences gastrointestinal microbiota and health-associated parameters in offspring in early life: rumen maturation was impaired in artificially-reared lambs which also presented altered sanitary status and higher risk of gut dysbiosis. The first month of age is thus a critical period where the gastrointestinal tract environment and microbiota are particularly unstable and special care should be taken in the management of artificially fed newborn ruminants.

Selection of plant oil as a supplemental energy source by monitoring rumen profiles and its dietary application in Thai crossbred beef cattle.

ObjectiveThe present study was conducted to select a plant oil without inhibitory effects on rumen fermentation and microbes, and to determine the optimal supplementation level of the selected oil in a series of in vitro studies for dietary application. Then, the selected oil was evaluated in a feeding study using Thai crossbred beef cattle by monitoring growth, carcass, blood and rumen characteristics.MethodsRumen fluid was incubated with substrates containing one of three different types of plant oil (coconut oil, palm oil, and soybean oil) widely available in Thailand. The effects of each oil on rumen fermentation and microbes were monitored and the oil without a negative influence on rumen parameters was selected. Then, the dose-response of rumen parameters to various levels of the selected palm oil was monitored to determine a suitable supplementation level. Finally, an 8-month feeding experiment with the diet supplemented with palm oil was carried out using 12 Thai crossbred beef cattle to monitor growth, carcass, rumen and blood profiles.ResultsBatch culture studies revealed that coconut and soybean oils inhibited the most potent rumen cellulolytic bacterium Fibrobacter succinogenes, while palm oil had no such negative effect on this and on rumen fermentation products at 5% or higher supplementation level. Cattle fed the diet supplemented with 2.5% palm oil showed improved feed conversion ratio (FCR) without any adverse effects on rumen fermentation. Palm oil-supplemented diet increased blood cholesterol levels, suggesting a higher energy status of the experimental cattle.ConclusionPalm oil had no negative effects on rumen fermentation and microbes when supplemented at levels up to 5% in vitro. Thai crossbred cattle fed the palm oil-supplemented diet showed improved FCR without apparent changes of rumen and carcass characteristics, but with elevated blood cholesterol levels. Therefore, palm oil can be used as a beneficial energy source.

Digestibility of Juncus acutus and its effects on ruminal cellulolytic bacteria

The objectives of this study were to estimate the digestibility of Juncus acutus and to investigate its effects on rumen cellulolytic bacteria to consider J. acutus as an alternative roughage source in ruminant nutrition. Juncus acutus samples were collected from three different stations in Kizilirmak Delta and their proximate analysis was carried out. Organic matter digestibility (OMD) and metabolisable energy (ME) values of J. acutus were estimated from gas measured by the in vitro gas production method. The effects of J. acutus on the abundance of rumen cellulolytic bacteria Fibrobacter succinogenes, Ruminococcus flavefaciens and Ruminococcus albus were determined by real-time PCR method. Crude nutrient values of J. acutus collected from three stations were analysed. Mean OMD %, MEOMD and MEGP values of J. acutus from three stations were ranged from 42.64 to 42.48%, 6.78 to 6.82 MJ/kg DM and 5.05 to 5.26 MJ/kg DM respectively. Ruminal F. succinogenes, R. flavefaciens and R. albus abundance calculated from threshold (CT) values in rumen fluids obtained from 0 to 96 h incubations showed an increases in following order F. succinogenes> R. flavefaciens> R. albus. The CP % of J. acutus was found higher from cereal straw and close to low-quality alfalfa hay and dry meadow grass. Juncus acutus increased the amount of rumen cellulolytic bacteria. In the light of obtained results, it can be concluded that J. acutus may be considered as a new alternative roughage source in ruminant nutrition.

Detection and identification of rumen bacteria constituting a fibrolytic consortium dominated by Fibrobacter succinogenes

A fibrolytic consortium, dominated by the rumen cellulolytic bacterium Fibrobacter succinogenes, was artificially constructed on hay stems to detect and identify rumen bacteria that can potentially interact with F. succinogenes. Consortium-bacterial members were determined by DGGE and sequencing analysis targeted bacterial 16S rDNA. An artificial consortium was formed in a 2-step incubation of hay stems; the first step with group 1, 2 or 3 F. succinogenes strains, the second step with rumen fluid. After consortium formation, morphologically different bacteria were observed in association with F. succinogenes. DGGE exhibited more than 30 bands, the pattern of which depended on the F. succinogenes group. Sequencing suggested that Butyrivibrio fibrisolvens, Pseudobutyrivibrio ruminis, Clostridium sp., F. succinogenes group 2, Prevotella ruminicola and unclassified Bacteroides were prominent in the group 1 consortium and that Treponema bryantii, B. fibrisolvens, Acinetobacter sp, and Wolinella succinogenes were prominent in the group 2 consortium. However, in the group 3 consortium, F. succinogenes-like bacteria were microscopically undetectable, whereas cellulolytic Ruminococcus albus and F. succinogenes group 1 were prominent, suggesting that the group 3 cannot be a core member of this consortium. This study is the first attempt to identify bacterial members of a fibrolytic consortium dominated by a specific bacterium.

Isolation and identification of cellulose-binding proteins from sheep rumen contents.

To extend our understanding of the mechanisms of plant cell wall degradation in the rumen, cellulose-binding proteins (CBPs) from the contents of a sheep rumen were directly isolated and identified using a metaproteomics approach. The rumen CBPs were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and some CBPs revealed endoglucanase activities toward carboxymethyl cellulose. Using mass spectrometry analyses, four CBPs were identified and annotated as known proteins from the predominant rumen cellulolytic bacterium Fibrobacter succinogenes: tetratricopeptide repeat domain protein, OmpA family protein, fibro-slime domain protein, and cellulose-binding endoglucanase F (EGF). Another CBP was identified as the cellulosomal glycosyl hydrolase family 6 exoglucanase, Cel6A, of Piromyces equi. F. succinogenes cells expressing EGF were found to be major members of the bacterial community on the surface or at the inner surface of hay stems by immunohistochemical analyses using anti-EGF antibody. The finding that four of the five CBPs isolated and identified from sheep rumen contents were from F. succinogenes indicates that F. succinogenes is significantly involved in cellulose degradation in the rumen.

Ruminal distribution of the cellulolytic bacterium Fibrobacter succinogenes in relation to its phylogenetic grouping

ABSTRACTTo investigate the ecological importance of the cellulolytic bacterium Fibrobacter succinogenes in fiber digestion, ruminal distribution of F. succinogenes was determined in relation to its phylogenetic grouping. Rumen digesta from wethers and steers fed orchardgrass hay, rice straw or fresh orchardgrass were employed as the materials for the analyses. Orchardgrass hay stem incubated in the rumen was also used. By using total DNA extracted from these materials, population sizes of total F. succinogenes and of four different phylogenetic groups of this species were quantitated through competitive polymerase chain reaction (PCR), and restriction fragment length polymorphism analysis of PCR products targeted the bacterial 16S rDNA. Rumen digesta and ruminally incubated hay stems had a reasonably high population size of F. succinogenes (×107−8/g) that was composed of strains belonging to the phylogenetic groups 1 and 3. The relative abundance of each group was different among the samples; group 1 dominated on the ruminally incubated hay stem and in the rumen of wethers fed fresh orchardgrass, while group 3 was major in the rumen of wethers and steers on hay diet. These results suggest that there could be phenotypic differences among the phylogenetic groups of F. succinogenes, and group 1 dominating on hay stem might contribute to rumen fiber digestion more than the other groups.

Mechanical resistance of wheat straw after incubation in cultures of ruminal cellulolytic microorganisms

The mechanical properties of wheat straw fragments were measured using tensile and shear tests before and after incubation in pure cultures of the rumen fungus Neocallimastix frontalis MCH3, in pure cultures of the rumen cellulolytic bacteria Fibrobacter succinogenes S85 and Ruminococcus flavefaciens 007 and in co-cultures associating the fungus with each of the two bacterial species. N. frontalis considerably reduced the tensile strength of the straw; on the contrary R. flavefaciens increased it and F. succinogenes did not affect it. In co-cultures, F. succinogenes and N. frontalis interacted synergistically to weaken straw resistance, whereas R. flavefaciens inhibited the fungal action. The shear test also revealed that physical and mechanical properties were strongly affected by the action of the three microorganisms. After six days of incubation the straws resistance to shearing was lower after incubation with N. frontalis or R. flavefaciens than in the presence of F. succinogenes. No synergism or antagonism between microorganisms was observed in the shear test. These results thus clearly demonstrate a specific action of the three rumen microorganisms in physical degradation of plant material.

The effect of growth and starvation on the lysis of the ruminal cellulolytic bacterium Fibrobacter succinogenes.

Growing cultures of Fibrobacter succinogenes assimilated more ammonia than could be accounted for by cellular protein, RNA, or DNA and released large amounts of nonammonia nitrogen. The difference between net and true growth was most dramatic at low dilution rates, but mathematical derivations indicated that the lysis rate was a growth rate-independent function. The lysis rate was sevenfold greater than the true maintenance rate (0.07 h-1 versus 0.01 h-1). Because slowly growing cells had as much proton motive force and ATP as fast-growing cells, lysis was not a starvation response per se. Stationary-phase cells had a lysis rate that was 10-fold less than that of growing cells. Rapidly growing cells were not susceptible to phenylmethylsulfonyl fluoride, but phenylmethylsulfonyl fluoride increased the lysis rate of the cultures when they reached the stationary phase. This latter result indicated that autolysins of stationary-phase cells were being inactivated by a serine proteinase. When growing cells were treated with the glycolytic inhibitor iodoacetate, the proteinase-dependent transition to the stationary phase was circumvented, and the rate of lysis could be increased by as much as 50-fold.

Effects of dilution rate and pH on the ruminal cellulolytic bacterium Fibrobacter succinogenes S85 in cellulose-fed continuous culture.

The ruminal cellulolytic bacterium Fibrobacter succinogenes S85 was grown in cellulose-fed continuous culture at 22 different combinations of dilution rate (D, 0.014-0.076 h-1) and extracellular pH (6.11-6.84). Effects of pH and D on the fermentation were determined by subjecting data on cellulose consumption, cell yield, product yield (succinate, acetate, formate), and soluble sugar concentration to response surface analysis. The extent of cellulose conversion decreased with increasing D. First-order rate constants at rapid growth rates were estimated as 0.07-0.11 h-1, and decreased with decreasing pH. Apparent decreases in the rate constant with increasing D was not due to inadequate mixing or preferential utilization of the more amorphous regions of the cellulose. Significant quantities of soluble sugars (0.04-0.18 g/l, primarily glucose) were detected in all cultures, suggesting that glucose uptake was rather inefficient. Cell yields (0.11-0.24 g cells/g cellulose consumed) increased with increasing D. Pirt plots of the predicted yield data were used to determine that maintenance coefficient (0.04-0.06 g cellulose/g cells.h) and true growth yield (0.23-0.25 g cells/g cellulose consumed) varied slightly with pH. Yields of succinate, the major fermentation endproduct, were as high as 1.15 mol/mol anhydroglucose fermented, and were slightly affected by dilution rate but were not affected by pH. Comparison of the fermentation data with that of other ruminal cellulolytic bacteria indicates that F. succinogenes S85 is capable of rapid hydrolysis of crystalline cellulose and efficient growth, despite a lower mu max on microcrystalline cellulose.

Futile cycling of glycogen in Fibrobacter succinogenes as shown by in situ 1H-NMR and 13C-NMR investigation.

Glycogen was synthesized during all the growth phases in the rumen anaerobic cellulolytic bacterium Fibrobacter succinogenes. Glycogen synthesis and degradation were monitored using in situ 13C and 1H-NMR spectroscopy in resting cells of F. succinogenes. The cells were incubated at 37 degrees C under anaerobic conditions with [1-13C]glucose and [2-13C]glucose. 1H-NMR spectra were used to quantify enrichment by 13C of metabolism products. Glucose was utilized for energy requirements of the bacterium, essentially via the Embden-Meyerhof pathway, leading to the synthesis of succinate and acetate, while glycogen was stored. From [1-13C]glucose, labeling occurred on C2 of succinate and acetate, and on both C1 and C6 of glycogen, the labeling on C1 being predominant. The C6-labeling of glycogen may be explained by scrambling and reversal of the glycolytic pathway at the triose-phosphate and fructose 1,6-bisphosphate level. When the bacteria were incubated first with [1-13C]glucose, then washed and incubated with [2-13C]glucose, the pattern of 13C labeling in the products of the metabolism, as shown by 13C and 1H-NMR spectra, indicated that glycogen was degraded at the same time as it was being stored, suggesting futile cycling of glycogen. The hydrolysis of previously stored glycogen can provide, in the presence of glucose, up to 30% of the carbon source for the bacteria.