• 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.

• Korean Journal of Soil Science and Fertilizer
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• v.45 no.6
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• pp.1049-1057
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• 2012

The study investigated the biochemical methane potential (BMP) assay of pig slurry supplemented with mixed methanogens and cellulolytic bacteria to improve anaerobic digestion for methane production. For the BMP assay, 7 different microbial supplementation groups consisted of the cultures of mixed methanogens (M), Fibrobacter succinogenes (FS), Ruminococcus flavefaciensn (RF), R. albus (RA), RA+FS, M+RA+FS, and 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 60 days at $38^{\circ}C$ using anaerobic digestate obtained from an anaerobic digester with pig slurry as inoculum. In results, 5% RF and RA+FS increased total biogas up to 8.1 and 8.4%, respectively, compared with that of control (p<0.05). All 5% microbial culture supplements significantly increased methane production up to 12.1~17.9% compared with that of control (p<0.05). Total solid (TS) and volatile solid (VS) digestion efficiencies showed no relationship to the increased supplementation levels of microbial cultures. After incubation, pH values in all treatment groups ranged between 7.527 and 7.657 indicating that methanogensis was not inhibited during the incubation. In conclusion, the results indicated that both hydrolysis and methanogenesis stages for methane production in anaerobic batch reactors were influenced by the supplemented microorganisms due to the chemical characteristics of pig slurry, but only the 5% supplementation level of all microbial culture supplements used in the experiment affected methane production.

• Asian-Australasian Journal of Animal Sciences
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• v.25 no.1
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• pp.66-74
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• 2012

The effects of exogenous fibrolytic enzymes (EFE; a mixture of two preparations from Trichoderma spp., with predominant xylanase and ${\beta}$-glucanase activities, respectively) on colonization and digestion of ground barley straw and alfalfa hay by Fibrobacter succinogenes S85 and Ruminococcus flavefaciens FD1 were studied in vitro. The two levels (28 and 280 ${\mu}g$/ml) of EFE tested and both bacteria were effective at digesting NDF of hay and straw. With both substrates, more NDF hydrolysis (p<0.01) was achieved with EFE alone at 280 than at 28 ${\mu}g$/ml. A synergistic effect (p<0.01) of F. succinogenes S85 and EFE on straw digestion was observed at 28 but not 280 ${\mu}g$/ml of EFE. Strain R. flavefaciens FD1 digested more (p<0.01) hay and straw with higher EFE than with lower or no EFE, but the effect was additive rather than synergistic. Included in the incubation medium, EFE showed potential to improve fibre digestion by cellulolytic ruminal bacteria. In a second batch culture experiment using mixed rumen microbes, DM disappearance (DMD), gas production and incorporation of $^{15}N$ into particle-associated microbial N ($^{15}N$-PAMN) were higher (p<0.001) with ammoniated (5% w/w; AS) than with native (S) ground barley straw. Application of EFE to the straws increased (p<0.001) DMD and gas production at 4 and 12 h, but not at 48 h of the incubation. EFE applied onto S increased (p<0.01) $^{15}N$-PAMN at 4 h only, but EFE on AS increased (p<0.001) $^{15}N$-PAMN at all time points. Prehydrolysis increased (p<0.01) DMD from both S and AS at 4 and 12 h, but reduced (p<0.01) $^{15}N$-PAMN in the early stage (4 h) of the incubation, as compared to non-prehydrolyzed samples. Application of EFE to barley straw increased rumen bacterial colonization of the substrate, but excessive hydrolytic action of EFE prior to incubation decreased it.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.11
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• pp.1705-1712
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• 2007

The effect of different tropical feed sources on rumen ecology, cellulolytic bacteria, feed intake and digestibility of beef cattle was investigated. Four fistulated, castrated male crossbred cattle were randomly allocated to a $4{\times}4$ Latin square design. The treatments were: T1) urea-treated (5%) rice straw (UTS); T2) cassava hay (CH); T3) fresh cassava foliage (FCF); T4) UTS:FCF (1:1 dry matter basis). Animals were fed concentrates at 0.3% of body weight on a DM basis and their respective diets on an ad libitum basis. The experimental period was 21 days. The results revealed that the use of UTS, CH, FCF and UTS:FCF as roughage sources could provide effective fiber and maintain an optimal range of ruminal pH and $NH_3-N$. Total viable and cellulolytic bacterial populations were enhanced (p<0.05) with UTS as the roughage source. Animals fed FCF had a higher rumen propionate production (p<0.05) with a lower cellulolytic bacteria count. Moreover, three predominant cellulolytic bacteria species, namely Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens, were found in all treatment groups. Roughage intake and total DM intake were highest with UTS (2.2 and 2.5% BW, respectively) as the roughage source (p<0.05). Nutrient intake in terms of organic matter intake (OMI) was similar in UTS, CH and UTS:FCF treatments (8.0, 6.8 and 8.7 kg/d, respectively), while crude protein intake (CPI) was enhanced in CH, FCF and UTS:FCF as compared to the UTS treatment (p<0.05). Digestion coefficients of DM and organic matter (OM) were similar among treatments, while the CP digestion coefficients were similar in CH, FCF and UTS:FCF treatments, but were higher (p<0.05) in CH than in UTS. CP and ADF digestible intakes (kg/d) were highest (p<0.05) on the CH and UTS treatments, respectively. It was also observed that feeding FCF as a full-feed resulted in ataxia as well as frequent urination; therefore, FCF should only be fed fresh as part of the feed or be fed wilted. Hence, combined use of FCF and UTS as well as CH and FCF were recommended.

• Asian-Australasian Journal of Animal Sciences
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• v.16 no.1
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• pp.104-115
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• 2003

A series of experiments was carried out to determine the possibility for the non-ionic surfactant (NIS) as a feed additive for ruminant animals. The effect of the NIS on (1) the enzyme distribution in the rumen fluids of Hereford bulls, (2) the growth of pure culture of rumen bacteria and (3) rumen anaerobic fungi, (4) the ruminal fermentation characteristics of Korean native cattle (Hanwoo), and (5) the performances of Holstein dairy cows were investigated. When NIS was added to rumen fluid at the level of 0.05 and 0.1% (v/v), the total and specific activities of cell-free enzymes were significantly (p<0.01) increased, but those of cell-bound enzymes were slightly decreased, but not statistically significant. The growth rates of ruminal noncellulolytic species (Ruminobacter amylophilus, Megasphaera elsdenii, Prevotella ruminicola and Selenomonas ruminantium) were significantly (p<0.01) increased by the addition of NIS at both concentrations tested. However, the growth rate of ruminal cellulolytic bacteria (Fibrobacter succinogenes, Ruminococcus albus, Ruminococcus flavefaciens and Butyrivibrio fibrisolvens) were slightly increased or not affected by the NIS. In general, NIS appears to effect Gram-negative bacteria more than Gram-positive bacteria; and non-cellulolytic bacteria more than cellulolytic bacteria. The growth rates of ruminal monocentric fungi (Neocallimastix patriciarum and Piromyces communis) and polycentric fungi (Orpinomyces joyonii and Anaeromyces mucronatus) were also significantly (p<0.01) increased by the addition of NIS at all concentrations tested. When NIS was administrated to the rumen of Hanwoo, Total VFA and ammonia-N concentrations, the microbial cell growth rate, CMCase and xylanase activities in the rumen increased with statistical difference (p<0.01), but NIS administration did not affect at the time of 0 and 9 h post-feeding. Addition of NIS to TMR resulted in increased TMR intake and increased milk production by Holstein cows and decreased body condition scores. The NEFA and corticoid concentrations in the blood were lowered by the addition of NIS. These results indicated that the addition of NIS may greatly stimulate the release of some kinds of enzymes from microbial cells, and stimulate the growth rates of a range of anaerobic ruminal microorganisms, and also stimulate the rumen fermentation characteristics and animal performances. Our data indicates potential uses of the NIS as a feed additive for ruminant animals.

• Asian-Australasian Journal of Animal Sciences
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• v.25 no.7
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• pp.971-979
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• 2012

Four, lactating dairy crossbreds ($50%{\times}50%$ Holstein Friesian${\times}$Native Zebu cattle) were randomly assigned according to a $2{\times}2$ factorial arrangement (two protein levels and two levels of mangosteen peel pellets (Mago-pel)) in a $4{\times}4$ Latin square design to receive four dietary treatments. All cows received concentrate at a proportion of 1 kg concentrate per 2 kg of milk yield, and urea-treated 5% rice straw (UTRS) was given ad libitum. It was found that total dry matter intakes, nutrient digestibility, ruminal pH and $NH_3$-N concentrations were not affected (p>0.05) by treatments. Concentrations of ruminal pH and $NH_3$-N were not affected by dietary treatments although the concentration of BUN varied significantly (p<0.05) between protein levels (p<0.05). The populations of rumen bacteria and fungal zoospores did not differ among treatments (p>0.05); however, the population of protozoa was decreased (p<0.05) when cows received Mago-pel supplementation. The composition of the population of bacteria, identified by real-time PCR technique, including total bacteria, methanogens, Fibrobacter succinogenes and Ruminococcus albus was similar (p>0.05) among dietary treatments (p>0.05); however, copy numbers of Ruminococcus flavefaciens was increased when protein level increased (p<0.05). Microbial protein synthesis, in terms of both quantity and efficiency, was enriched by Mago-pel supplementation. Milk yield was greatest in cows fed UTRS based diets with concentrate containing protein at 16% CP with Mago-pel, but were lowest without Mago-pel (p<0.05). In addition, protein level and supplementation of Mago-pel did not affect (p>0.05) milk composition except solids-not-fat which was higher in cows fed the diet with 19% CP. Therefore, feeding a concentrate containing 16% CP together with 300 g/hd/d Mago-pel supplementation results in changes in rumen fermentation and microbial population and improvements in milk production in lactating dairy crossbreds fed on UTRS.

• Asian-Australasian Journal of Animal Sciences
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• v.32 no.6
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• pp.808-814
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• 2019

Objective: The objective of this study was to investigate the effects of essential oil mixture (EOM) supplementation on rumen fermentation characteristics and microbial changes in an in vitro. Methods: Three experimental treatments were used: control (CON, no additive), EOM 0.1 (supplementation of 1 g EOM/kg of substrate), and EOM 0.2 (supplementation of 2 g EOM/kg of substrate). An in vitro fermentation experiment was carried out using strained rumen fluid for 12 and 24 h incubation periods. At each time point, in vitro dry matter digestibility (IVDMD), neutral detergent fiber digestibility (IVNDFD), pH, ammonia nitrogen ($NH_3-N$), and volatile fatty acid (VFA) concentrations, and relative microbial diversity were estimated. Results: After 24 h incubation, treatments involving EOM supplementation led to significantly higher IVDMD (treatments and quadratic effect; p = 0.019 and 0.008) and IVNDFD (linear effect; p = 0.068) than did the CON treatment. The EOM 0.2 supplementation group had the highest $NH_3-N$ concentration (treatments; p = 0.032). Both EOM supplementations did not affect total VFA concentration and the proportion of individual VFAs; however, total VFA tended to increase in EOM supplementation groups, after 12 h incubation (linear; p = 0.071). Relative protozoa abundance significantly increased following EOM supplementation (treatments, p<0.001). Selenomonas ruminantium and Ruminococcus albus (treatments; p<0.001 and p = 0.005), abundance was higher in the EOM 0.1 treatment group than in CON. The abundance of Butyrivibrio fibrisolvens, fungi and Ruminococcus flavefaciens (treatments; p<0.001, p<0.001, and p = 0.005) was higher following EOM 0.2 treatment. Conclusion: The addition of newly developed EOM increased IVDMD, IVNDFD, and tended to increase total VFA indicating that it may be used as a feed additive to improve rumen fermentation by modulating rumen microbial communities. Further studies would be required to investigate the detailed metabolic mechanism underlying the effects of EOM supplementation.

• Asian-Australasian Journal of Animal Sciences
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• v.27 no.2
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• pp.179-186
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• 2014

This study aimed to investigate the diversity of the Butyrivibrio group bacteria in goat rumen and its response to garlic oil (GO) supplementation as revealed by molecular analysis of cloned 16S rRNA genes. Six wethers fitted with ruminal fistulas were assigned to two groups for a cross-over design with 28-d experimental period and 14-d interval. Goats were fed a basal diet without (control) or with GO ruminal infusion (0.8 g/d). Ruminal contents were used for DNA extraction collected before morning feeding on d 28. A total bacterial clone library was firstly constructed by nearly full-length 16S rRNA gene cloned sequences using universal primers. The resulting plasmids selected by Butyrivibrio-specific primers were used to construct a Butyrivibrio group-specific bacterial clone library. Butyrivibrio group represented 12.98% and 10.95% of total bacteria in control and GO group, respectively. In libraries, clones were classified to the genus Pseudobutyrivibrio, Butyrivibrio and others within the family Lachnospiraceae. Additionally, some specific clones were observed in GO group, being classified to the genus Ruminococcus and others within the family Ruminococcaceae. Based on the criterion that the similarity was 97% or greater with database sequences, there were 29.73% and 18.42% of clones identified as known isolates (i.e. B. proteoclasticus and Ps. ruminis) in control and GO groups, respectively. Further clones identified as B. fibrisolvens (5.41%) and R. flavefaciens (7.89%) were specifically found in control and GO groups, respectively. The majority of clones resembled Ps. ruminis (98% to 99% similarity), except for Lachnospiraceae bacteria (87% to 92% similarity) in the two libraries. The two clone libraries also appeared different in Shannon diversity index (control 2.47 and GO group 2.91). Our results indicated that the Butyrivibrio group bacteria had a complex community with considerable unknown species in the goat rumen.

• Asian-Australasian Journal of Animal Sciences
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• v.24 no.6
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• pp.810-817
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• 2011

A feeding trial was conducted to study the effect of tannin rich Pakar (Ficus infectoria) leaves on microbial profile, rumen fermentation and nutrient utilization in goats. Eight goats divided in two groups were fed pakar leaves (experimental group) and green oats (control group) as sole roughage source along with a fixed quantity of concentrate mixture for a period of 3 months. Two metabolic trials of six days duration were conducted after 30 and 90 days of experimental feeding. The dry matter intake was significantly higher (p<0.05) and digestibility's of DM, OM, CP, EE, NDF and ADF were reduced in experimental as compared with the control group. The TDN intake was similar (236.52 vs. 240.39 g/d) in both the groups. All the animals were in positive nitrogen balance. The concentration of ammonia nitrogen, TVFA, lactic acid and activities of xylanase and protease were reduced in pakar leaves fed goats. The rumen microbial profile as obtained by MPN technique showed no change in total bacterial population but total fungi and cellulolytic bacteria were reduced (p<0.05), whereas, tannin degrading/tolerant bacteria increased with the feeding of pakar leaves. Real time PCR data revealed a decrease in Ruminococcus flavefaciens, an increase in methanogens and no change in the Fibrobacter succinogenes population by feeding of pakar leaves.

• Asian-Australasian Journal of Animal Sciences
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• v.26 no.1
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• pp.72-81
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• 2013

Four Luxi beef cattle ($400{\pm}10$ kg) fitted with ruminal, duodenal and ileal cannulas were used in a $4{\times}4$ Latin square to assess the effects of soybean small peptide (SSP) infusion on rumen fermentation, diet digestion and flow of nutrient in the gastrointestinal tract. The ruminal infusion of SSP was 0 (control), 100, 200 and 300 g/d. Ruminal SSP infusion linearly (p<0.01) and quadratically (p<0.01) increased microbial protein synthesis and rumen ammonia-N concentration. Concentrations of total volatile fatty acid were linearly increased (p = 0.029) by infusion SSP. Rumen samples were obtained for analysis of microbial ecology by real-time PCR. Populations of rumen Butyrivibrio fibrisolvens, Streptococcus bovis, Ciliate protozoa, Ruminococcus flavefaciens, and Prevotella ruminicola were expressed as a proportion of total Rumen bacterial 16S ribosomal deoxyribonucleic acid (rDNA). Butyrivibrio fibrisolvens populations which related to total bacterial 16S rDNA were increased (p<0.05), while Streptococcus bovis populations were linearly (p = 0.049) and quadratically (p = 0.020) decreased by infusion of SSP. Apparent rumen digestibility of DM and NDF were (Q, p<0.05; L, p<0.05) increased with infusion SSP. Total tract digestion of DM, OM and NDF were linearly (p<0.01) and quadratically (p<0.01) increased by infusing SSP. The flow of total amino acids (AA), essential amino acids (EAA) and individual amino acids were linearly (p<0.01) and quadratically (p<0.01) increased with infusion SSP. The digestibility of Lysine was quadratically (p = 0.033) increased and apparent degradability of Arginine was linearly (p = 0.032) and quadratically (p = 0.042) increased with infusion SSP. The results indicated that infusion SSP could improve nutrient digestion, ruminal fermentation and AA availability.