• Asian-Australasian Journal of Animal Sciences
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• v.20 no.5
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• pp.802-810
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• 2007

Fibrobacter succinogenes, a Gram-negative, anaerobic ruminal bacterium is a major fibre digesting species in the rumen. It intensively degrades plant cell walls by an erosion type of mechanism, burrowing its way through the complex matrix of cellulose and hemicellulose with the release of digestible and undigested cell wall fragments. The enzymes involved in this process include a combination of glucanases, xylanases, arabinofuranosidase(s) and esterases. The genome of the bacterium has been sequenced and this has revealed in excess of 100 putative glycosyl hydrolase, pectate lyase and carbohydrate esterase genes, which is greater than the numbers reported present in other major cellulolytic organisms for which genomes have been sequenced. Modelling of the amino acid sequences of two glycanases, CedA and EGB, by reference to crystallized homologs has enabled prediction of the major features of their tertiary structures. Two dimensional gel electrophoresis in conjunction with mass spectroscopy has permitted the documentation of proteins over expressed in F. succinogenes grown on cellulose, and analysis of the cell surfaces of mutant strains unable to bind to cellulose has enabled the identification of candidate proteins with roles in adhesion to the plant cell wall substrate, the precursor to cellulose biodegradation.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.1
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• pp.131-138
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• 2009

Among rumen microbes, bacteria play important roles in the biological degradation of plant fiber due to their large biomass and high activity. To maximize the utilization of fiber components such as cellulose and hemicellulose by ruminant animals, the ecology and functions of rumen bacteria should be understood in detail. Recent genome sequencing analyses of representative fibrolytic bacterial species revealed that the number and variety of enzymes for plant fiber digestion clearly differ between Fibrobacter succinogenes and Ruminococcus flavefaciens. Therefore, the mechanism of plant fiber digestion is also thought to differ between these two species. Ecology of individual fibrolytic bacterial species has been investigated using pure cultures and electron microscopy. Recent advances in molecular biology techniques complement the disadvantages of conventional techniques and allow accurate evaluation of the ecology of specific bacteria in mixed culture, even in situ and in vivo. Molecular monitoring of fibrolytic bacterial species in the rumen indicated the predominance of F. succinogenes. Nutritive interactions between fibrolytic and non-fibrolytic bacteria are important in maintaining and promoting fibrolytic activity, mainly in terms of crossfeeding of metabolites. Recent 16S rDNA-based analyses suggest that presently recognized fibrolytic species such as F. succinogenes and two Ruminococcus species with fibrolytic activity may represent only a small proportion of the total fibrolytic population and that uncultured bacteria may be responsible for fiber digestion in the rumen. Therefore, characterization of these unidentified bacteria is important to fully understand the physiology and ecology of fiber digestion. To achieve this, a combination of conventional and modern techniques could be useful.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.2
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• pp.200-207
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• 2007

In vitro rumen incubation studies were conducted to determine effects of initial pH on bacterial attachment and fiber digestion. Ruminal fluid pH was adjusted to 5.7, 6.2 and 6.7, and three major fibrolytic bacteria attached to rice straw in the mixed culture were quantified with real-time PCR. The numbers of attached and unattached Fibrobacter succinogenes, Ruminococcus flavefaciens and Ruminocococcus albus were lower (p<0.05) at initial pH of 5.7 without significant difference between those at higher initial pH. Lowering incubation media pH to 5.7 also increased bacterial numbers detached from substrate regardless of bacterial species. Dry matter digestibility, gas accumulation and total VFA production were pH-dependent. Unlike bacterial attachment, maintaining an initial pH of 6.7 increased digestion over initial pH of 6.2. After 48 h in vitro rumen fermentation, average increases in DM digestion, gas accumulation, and total VFA production at initial pH of 6.2 and 6.7 were 2.8 and 4.4, 2.0 and 3.0, and 1.2 and 1.6 times those at initial pH of 5.7, respectively. The lag time to reach above 2% DM digestibility at low initial pH was taken more times (8 h) than at high and middle initial pH (4 h). Current data clearly indicate that ruminal pH is one of the important determinants of fiber digestion, which is modulated via the effect on bacterial attachment to fiber substrates.

• Asian-Australasian Journal of Animal Sciences
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• v.7 no.3
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• pp.389-396
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• 1994

Effects of chemical treatments on in sacco and in vitro digestibility of barley straw by rumen fluid and pure cultures of cellulolytic bacteria were studied to evaluate the pretreatment and to improve the poor quality feed. Chemicals were applied by dissolving them in water equivalent to 40% of the weight of the straw (dry matter basis). Pretreatment with 5% NaOH yielded the largest increase in sacco digestion followed by pretreatment with 2% $(NH_4)_2SO_3$, 2.6% $NH_4OH$, 1.6% $NaHSO_3$ and untreated straw (control). In sacco dry matter digestibility of straw treated with NaOH and $(NH_4)_2SO_3$ continued to increase as the concentration of chemical increased (1 to 7.5%), as it was the in vitro dry matter loss by leaching. Treatment of barley straw with 5% NaOH enhanced significantly (p < 0.01) in vitro digestibility by rumen fluid, Fibrobacter suceinogenes and Ruminococcus albus though the fermentation products by cellulolytic bacteria were low, whereas the treatment with 5% $(NH_4)_2SO_3$ inhibited in vitro digestibility by F. succinogenes and R. albus together with lower fermentation products. Dry matter loss by leaching and bacterial digestion from barley straw treated with NaOH and $(NH_4)_2SO_3$ suggested the effect of pretreatment with these chemicals were based on leaching, and the cellulolytic bacteria had little to do with digestion.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.6
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• pp.818-823
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• 2008

The interaction of fibre degrading microbes and methanogens was studied using two forages, lucerne (Medicago sativa) hay and maize (Zea mays) hay, as substrate and 2-bromoethanesulphonic acid (BES) as an additive in an in vitro gas production test. Gas and methane production (ml/g dry matter) were significantly higher (p<0.05) on lucerne as compared to maize hay. Inclusion of BES in the incubation medium significantly suppressed methane emission irrespective of substrate. The population density of total bacteria, fungi, Ruminococcus flavefaciens and Fibrobacter succinogenes was higher, whereas that of methanogens was lower with maize hay as compared to lucerne as substrate. BES suppressed methanogen population by 7 fold on lucerene and by 8.5 fold on maize at 24 h incubation as estimated by real time-PCR. This suppression was accompanied by almost complete (>98% of control) inhibition of methanogenesis. The proportion of acetate decreased, whereas that of propionate increased significantly by inclusion of BES, resulting in narrowing of acetate to propionate ratio. In vitro true digestibility (IVTD) of lucerne was significantly higher as compared to maize but BES inclusion did not affect IVTD.

• Asian-Australasian Journal of Animal Sciences
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• v.31 no.1
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• pp.54-62
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• 2018

Objective: Due to the threat of global warming, the livestock industry is increasingly interested in exploring how feed additives may reduce anthropogenic greenhouse gas emissions, especially from ruminants. This study investigated the effect of Rhodophyta supplemented bovine diets on in vitro rumen fermentation and rumen microbial diversity. Methods: Cannulated Holstein cows were used as rumen fluid donors. Rumen fluid:buffer (1:2; 15 mL) solution was incubated for up to 72 h in six treatments: a control (timothy hay only), along with substrates containing 5% extracts from five Rhodophyta species (Grateloupia lanceolata [Okamura] Kawaguchi, Hypnea japonica Tanaka, Pterocladia capillacea [Gmelin] Bornet, Chondria crassicaulis Harvey, or Gelidium amansii [Lam.] Lamouroux). Results: Compared with control, Rhodophyta extracts increased cumulative gas production after 24 and 72 h (p = 0.0297 and p = 0.0047). The extracts reduced methane emission at 12 and 24 h (p<0.05). In particular, real-time polymerase chain reaction analysis indicated that at 24 h, ciliate-associated methanogens, Ruminococcus albus and Ruminococcus flavefaciens decreased at 24 h (p = 0.0002, p<0.0001, and p<0.0001), while Fibrobacter succinogenes (F. succinogenes) increased (p = 0.0004). Additionally, Rhodophyta extracts improved acetate concentration at 12 and 24 h (p = 0.0766 and p = 0.0132), as well as acetate/propionate (A/P) ratio at 6 and 12 h (p = 0.0106 and p = 0.0278). Conclusion: Rhodophyta extracts are a viable additive that can improve ruminant growth performance (higher total gas production, lower A/P ratio) and methane abatement (less ciliateassociated methanogens, Ruminococcus albus and Ruminococcus flavefaciens and more F. succinogenes.

• Korean Journal of Soil Science and Fertilizer
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• v.45 no.4
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• pp.515-523
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• 2012

The study investigated the biochemical methane potential (BMP) assay of cellulose supplementing with mixed methanogens and cellulolytic bacteria to improve anaerobic digestion for methane production. For the BMP assay, 7 different microbial supplementation groups were consisted of the cultures of mixed methanogens (M), Fibrobacter succinogenes (FS), Ruminococcus flavefaciensn (RF), R. albus (RA), RA+FS and M+RA+FS including control. The cultures were added in the batch reactors with the increasing dose levels of 1% (0.5 mL), 3% (1.5 mL) and 5% (2.5 mL). Incubation for the BMP assay was carried out for 40 days at $38^{\circ}C$ and anaerobic digestate obtained from an anaerobic digester with pig slurry as inoculum was used. In results, 5% FS increased total biogas and methane production up to 10.4~22.7% and 17.4~27.5%, respectively, compared to other groups (p<0.05). Total solid (TS) digestion efficiency showed a similar trend to the total biogas and methane productions. Generally the TS digestion efficiency of the FS group was higher than that of other groups showing at the highest value of 64.2% in the 5% FS group. Volatile solid (VS) digestion efficiencies of 68.4 and 71.0% in the 5% FS and the 5% RF were higher than other groups. After incubation, pH values in all treatment groups were over 6.4 indicating that methanogensis was not inhibited during the incubation. In conclusion, the results indicated that the hydrolysis stage for methane production in anaerobic batch reactors was the late-limiting stage compared with the methanogenesis stage, and especially, as the supplementation levels of F. succinogenes supplementation increased, the methane production was increased in the BMP assay compared with other microbial culture addition.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.6
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• pp.797-802
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• 2001

Two strains of rumen fungi (Piromyces rhizinflata B157, Orpinomyces joyonii SG4) and three strains of rumen cellulolytic bacteria (Ruminococcus albus B199, Ruminococcus flavefaciens FD1 and Fibrobacter succinogenes S85) were used as mono-cultures or combinationally arranged as co- and sequential-cultures to assess the relative contributions and interactions between rumen fungi and cellulolytic bacteria on rice straw degradation. The rates of dry matter degradation of co-cultures were similar to those of corresponding bacterial mono-cultures. Compared to corresponding sequential-cultures, the degradation of rice straw was reduced in all co-cultures (P<0.01). Regardless of the microbial species, the cellulolytic bacteria seemed to inhibit the degradation of rice straw by rumen fungi. The high efficiency of fungal cellulolysis seems to affect bacterial degradation rates.

• Journal of Animal Science and Technology
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• v.47 no.4
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• pp.615-624
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• 2005

The cPCR technique was used to monitor rumen fermentation and attachment of Fibrobacter succinogenes to cellulose at different pH in the in vitro culture medium. The target fragments of 16S rDNA(445 bp) were amplified from genomic DNA of F. succinogenes with specific primers and internal controls(205 bp) were constructed. Cell counts were estimated from the amounts of genomic DNA, which was calculated from cPCR results. F. succinogenes in pH 6.8 and 6.2 showed apparently higher attachment than in pH 5.8 during all incubation time. There were some difference between pH 6.8 and 6.2 in the degree of attachment, but the different was not significant (P>0.05). Cellulose degradation increased in process of incubation time and the increasing rate was higher when initial pH was higher. The pH in culture medium decreased regardless of initial pH in course of incubation time. After 24 h of incubation, medium pH was dropped by 0.24, 0.58 and 0.16 units from original medium pH 6.8, 6.2 and 5.8, respectively. More gas was produced at higher initial pH in the same manner as in cellulose degradation. In summery, Initial pH of rumen culture in vitro significantly influenced cellulose digestion, gas production, pH change and bacterial attachment. Especially, low pH(5.8) resulted in much lower bacterial attachment and fiber digestion compared to higher medium pH.

• Asian-Australasian Journal of Animal Sciences
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• v.27 no.8
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• pp.1088-1097
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• 2014

The study aimed to investigate the effects of gynosaponin on in vitro methanogenesis under different forage-concentrate ratios (F:C ratios). Experiment was conducted with two kinds of F:C ratios (F:C = 7:3 and F:C = 3:7) and gynosaponin addition (0 mg and 16 mg) in a $2{\times}2$ double factorial design. In the presence of gynosaponin, methane production and acetate concentration were significantly decreased, whereas concentration of propionate tended to be increased resulting in a significant reduction (p<0.05) of acetate:propionate ratio (A:P ratio), in high-forage substrate. Gynosaponin treatment increased (p<0.05) the butyrate concentration in both F:C ratios. Denaturing gradient gel electrophoresis (DGGE) analysis showed there was no apparent shift in the composition of total bacteria, protozoa and methanogens after treated by gynosaponin under both F:C ratios. The real-time polymerase chain reaction (PCR) analysis indicated that variable F:C ratios significantly affected the abundances of Fibrobacter succinogenes, Rumninococcus flavefaciens, total fungi and counts of protozoa (p<0.05), but did not affect the mcrA gene copies of methanogens and abundance of total bacteria. Counts of protozoa and abundance of F.succinogenes were decreased significantly (p<0.05), whereas mcrA gene copies of methanogens were decreased slightly (p<0.10) in high-forage substrate after treated by gynosaponin. However, gynosaponin treatment under high-concentrate level did not affect the methanogenesis, fermentation characteristics and tested microbes. Accordingly, overall results suggested that gynosaponin supplementation reduced the in vitro methanogenesis and improved rumen fermentation under highforage condition by changing the abundances of related rumen microbes.