Roll-tube Technique Research Articles

Roughage biodegradation by natural co-cultures of rumen fungi and methanogens from Qinghai yaks

Anaerobic fungus–methanogen co-cultures from rumen liquids and faeces can degrade lignocellulose efficiently. In this study, 31 fungus–methanogen co-cultures were first obtained from the rumen of yaks grazing in Qinghai Province, China, using the Hungate roll-tube technique. The fungi were identified according to morphological characteristics and internal transcribed spacer (ITS) sequences. The methanogens associated with each fungus were identified by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and 16S rRNA gene sequencing. They were five co-culture types: Neocallimastix frontalis + Methanobrevibacter ruminantium, Neocallimastix frontalis + Methanobrevibacter gottschalkii, Orpinomyces joyonii + Methanobrevibacter ruminantium, Caecomyces communis + Methanobrevibacter ruminantium, and Caecomyces communis + Methanobrevibacter millerae. Among the 31 co-cultures, during the 5-day incubation, the N. frontalis + M. gottschalkii co-culture YakQH5 degraded 59.0%–68.1% of the dry matter (DM) and 49.5%–59.7% of the neutral detergent fiber (NDF) of wheat straw, corn stalk, rice straw, oat straw and sorghum straw to produce CH4 (3.0–4.6 mmol/g DM) and acetate (7.3–8.6 mmol/g DM) as end-products. Ferulic acid (FA) released at 4.8 mg/g DM on corn stalk and p-coumaric acid (PCA) released at 11.7 mg/g DM on sorghum straw showed the highest values, with the following peak values of enzyme activities: xylanase at 12,910 mU/mL on wheat straw, ferulic acid esterase (FAE) at 10.5 mU/mL on corn stalk, and p-coumaric acid esterase (CAE) at 20.5 mU/mL on sorghum straw. The N. frontalis + M. gottschalkii co-culture YakQH5 from Qinghai yaks represents a new efficient combination for lignocellulose biodegradation, performing better than previously reported fungus–methanogen co-cultures from the digestive tract of ruminants.

Anaerobic Fungi Isolated From Bactrian Camel Rumen Contents Have Strong Lignocellulosic Bioconversion Potential.

This study aims to obtain anaerobic fungi from the rumen and fecal samples and investigates their potential for lignocellulosic bioconversion. Multiple anaerobic strains were isolated from rumen contents (CR1–CR21) and fecal samples (CF1–CF10) of Bactrian camel using the Hungate roll tube technique. After screening for fiber degradability, strains from rumen contents (Oontomyces sp. CR2) and feces (Piromyces sp. CF9) were compared with Pecoramyces sp. F1 (earlier isolated from goat rumen, having high CAZymes of GHs) for various fermentation and digestion parameters. The cultures were fermented with different substrates (reed, alfalfa stalk, Broussonetia papyrifera leaves, and Melilotus officinalis) at 39°C for 96 h. The Oontomyces sp. CR2 had the highest total gas and hydrogen production from most substrates in the in vitro rumen fermentation system and also had the highest digestion of dry matter, neutral detergent fiber, acid detergent fiber, and cellulose present in most substrates used. The isolated strains provided higher amounts of metabolites such as lactate, formate, acetate, and ethanol in the in vitro rumen fermentation system for use in various industrial applications. The results illustrated that anaerobic fungi isolated from Bactrian camel rumen contents (Oontomyces sp. CR2) have the highest lignocellulosic bioconversion potential, suggesting that the Bactrian camel rumen could be a good source for the isolation of anaerobic fungi for industrial applications.

Characterization of an Anaerobic, Thermophilic, Alkaliphilic, High Lignocellulosic Biomass-Degrading Bacterial Community, ISHI-3, Isolated from Biocompost

The generation of a complex microbial consortium is a promising approach for efficient biomass decomposition. An anaerobic thermophilic alkaliphilic microbial consortium with efficient degradation ability was screened from bovine manure compost using non-pretreated milling corn stover (CS) and rice straw (RS). A stable microbial consortium ISHI-3 with high degradation ability for CS and RS was isolated by the roll tube technique. ISHI-3 comprised Herbivorax saccincola and bacteria belonging to the classes Pelotomaculum, Tepidanaerobacter, and Tepidimicrobium, as determined by DGGE of the PCR-generated 16S rRNA genes. Furthermore, metagenomics analysis using a 16S rRNA library was carried out to determine the bacterial distribution during degradation of CS and RS. H. saccincola and bacteria belonging to Pelotomaculum were relatively abundant in the beginning to middle periods of culture with CS and RS whereas bacteria belonging to Tepidanaerobacter and Tepidimicrobium gradually increased in the population during the later stages. To understand the role of non-cellulolytic bacteria in the consortium, novel strains ET1 and GL4, which were most closely related to Tepidimicrobium ferriphilum and Tepidanaerobacter acetatoxydans, were isolated from ISHI-3. Based on their carbon source usage, morphology, and phylogenetic analysis, we propose that strains ET1 and GL4 should be classified as a novel genus or species. Bacteria ET1 and GL4 can utilize different organic compounds as carbon and energy sources such as organic acids, alcohols, sugars, and amino acids, showing a preference for organic acids and alcohols rather than sugars such as glucose and cellobiose. These results indicated that ET1 and GL4 help to accelerate efficient lignocellulose degradation of H. saccincola.

Isolation of Cellulolytic Bacteria from the Rumen.

To isolate strictly anaerobic rumen bacteria capable of degrading cellulose, environmental and nutritional conditions similar to the rumen environment should be simulated in vitro. One of the most useful techniques for isolating rumen bacteria is the roll-tube technique. In this chapter, the roll-tube technique for isolating cellulolytic rumen bacteria is briefly outlined.

Isolation, identification and fibrolytic characteristics of rumen fungi grown with indigenous methanogen from yaks (Bos grunniens) grazing on the Qinghai-Tibetan Plateau.

To obtain co-cultures of anaerobic fungi and their indigenously associated methanogens from the rumen of yaks grazing on the Qinghai-Tibetan Plateau and investigate their morphology features and ability to degrade lignocellulose. Twenty fungus-methanogen co-cultures were obtained by Hungate roll-tube technique. The fungi were identified as Orpinomyces, Neocallimastix and Piromyces genera based on the morphological characteristics and internal transcribed spacer 1 sequences analysis. All methanogens were identified as Methanobrevibacter sp. by 16S rRNA gene sequencing. There were four types of co-cultures: Neocallimastix with Methanobrevibacter ruminantium, Orpinomyces with M.ruminantium, Orpinomyces with Methanobrevibacter millerae and Piromyces with M.ruminantium among 20 co-cultures. In vitro studies with wheat straw as substrate showed that the Neocallimastix with M.ruminantium co-cultures and Piromyces with M.ruminantium co-cultures exhibited higher xylanase, filter paper cellulase (FPase), ferulic acid esterase, acetyl esterase activities, invitro dry matter digestibility, gas, CH4 , acetate production, ferulic acid and p-coumaric acid releases. The Neocallimastix frontalis Yak16 with M.ruminantium co-culture presented the strongest lignocellulose degradation ability among 20 co-cultures. Twenty fungus-methanogen co-cultures were obtained from the rumen of grazing yaks. The N.frontalis with M.ruminantium co-cultures were highly effective combination for developing a fermentative system that bioconverts lignocellulose to high activity fibre-degrading enzyme, CH4 and acetate. The N.frontalis with M.ruminantium co-cultures from yaks grazing on the Qinghai-Tibetan Plateau present great potential in lignocellulose biodegradation industry.

Characterization of exoelectrogenic bacteria enterobacter strains isolated from a microbial fuel cell exposed to copper shock load.

Microorganisms capable of generating electricity in microbial fuel cells (MFCs) have gained increasing interest. Here fourteen exoelectrogenic bacterial strains were isolated from the anodic biofilm in an MFC before and after copper (Cu) shock load by Hungate roll-tube technique with solid ferric (III) oxide as an electron acceptor and acetate as an electron donor. Phylogenetic analysis of the 16S rRNA gene sequences revealed that they were all closely related to Enterobacter ludwigii DSM 16688T within the Enterobacteriaceae family, although these isolated bacteria showed slightly different morphology before and after Cu shock load. Two representative strains R2B1 (before Cu shock load) and B4B2 (after Cu shock load) were chosen for further analysis. B4B2 is resistant to 200 mg L−1 of Cu(II) while R2B1 is not, which indicated the potential selection of the Cu shock load. Raman analysis revealed that both R2B1 and B4B2 contained c-type cytochromes. Cyclic voltammetry measurements revealed that strain R2B1 had the capacity to transfer electrons to electrodes. The experimental results demonstrated that strain R2B1 was capable of utilizing a wide range of substrates, including Luria-Bertani (LB) broth, cellulose, acetate, citrate, glucose, sucrose, glycerol and lactose to generate electricity, with the highest current density of 440 mA·m−2 generated from LB-fed MFC. Further experiments indicated that the bacterial cell density had potential correlation with the current density.

Effects of supplementation of Simmental steers with 2-methylbutyrate on rumen microflora, enzyme activities and methane production

The objective of this study was to evaluate the effects of 2-methylbutyrate supplementation on rumen microflora, enzyme activities and methane production in Simmental steers consuming a diet consisting of 600g/kg corn stover and 400g/kg concentrate (dry matter [DM] basis). Eight ruminally cannulated Simmental steers were used in a replicated 4×4 Latin square design. The treatments were: control (without 2-methylbutyrate), low 2-methylbutyrate (LMB), medium 2-methylbutyrate (MMB) and high 2-methylbutyrate (HMB) with 0, 8.4, 16.8 and 25.2g 2-methylbutyrate per steer and per day, respectively. Dry matter intake (averaged 9kg/day) was restricted to a maximum of 0.9 of ad libitum intake. Whether direct counts, roll-tube technique or real-time PCR quantification were used, populations of total bacteria, cellulolytic bacteria and anaerobic fungi linearly increased (P<0.04) and were higher (P<0.05) for MMB and HMB than for control and LMB, whereas those of protozoa and total methanogens linearly reduced and were lower for MMB and HMB than for control and LMB. With increasing 2-methylbutyrate supplementation, real-time PCR quantification of populations of Ruminococcus albus, Ruminococcus flavefaciens, Bufyrivibrio fibrisolvens and Fibrobacter succinogenes linearly increased (P<0.05) and was higher for MMB and HMB than for control (P<0.05). Activities of CMCase, xylanase and β-glucosidase linearly increased (P<0.05) and were higher (P<0.05) for MMB and HMB than for control and LMB, whereas that of protease linearly reduced (P<0.05) and was lower for MMB and HMB than for control and LMB (P<0.05). Activities of α-amylase and α-glucosidase tended to be higher (P<0.08) for MMB and HMB than for control and LMB, but activity of urease tended to be lower for MMB and HMB than for control and LMB. Methane production linearly decreased (P<0.01) and was lower for MMB and HMB than for control (P<0.05) with increasing 2-methylbutyrate supplementation. Effective degradabilities of cellulose and hemicellulose of corn stover linearly increased (P<0.01) and were higher for MMB and HMB than for control (P<0.05), whereas that of crude protein in concentrate linearly decreased (P<0.01) and was lower for MMB and HMB than for control and LMB (P<0.05) with increasing 2-methylbutyrate supplementation. The present results indicate that supplementation of diet with 2-methylbutyrate altered ruminal microbial populations and improved ruminal enzyme activities in a dose-dependent manner in steers fed high-roughage diet. In the experimental conditions of this trial, the optimum daily dose of 2-methylbutyrate at 16.8g per steer is suggested.

Effects of isovalerate supplementation on microbial status and rumen enzyme profile in steers fed on corn stover based diet

The objective of this study was to evaluate the effects of isovalerate supplementation on microbial status and ruminal enzyme activities in steers fed on corn stover based diet with a low proein. Eight ruminally cannulated Simmental steers were used in a replicated 4×4 Latin square experiment. The treatments were control (without isovalerate), LIV, MIV and HIV with 8.4, 16.8 and 25.2g isovalerate per steer per day, respectively. Isovalerate was hand- mixed into the concentrate portion. Diet consisted of 60% corn stover and 40% concentrate (dry matter [DM] basis). Dry matter intake (averaged 9kg/day) was restricted to a maximum of 90% of ad libitum intake. Whether direct counts, cultured using a roll-tube technique or real-time PCR quantification, population of total bacteria, cellulolytic bacteria and anaerobic fungi were linearly increased, whereas that of protozoa and total methanogens was linearly reduced with increasing isovalerate supplementation. Real-time PCR quantification of population of R. albus, R. Flavefaciens, B. Fibrisolvens and F. succinogenes was linearly increased (p<0.04) with increasing isovalerate supplementation. Activities of CMCase, xylanase and β-glucosidase were linearly increased (p<0.05), whereas that of protease was linearly reduced (p=0.005) with increasing isovalerate supplementation. Methane production was linearly decreased (p<0.001) with increasing isovalerate supplementation. Effective degradabilities of cellulose and hemicellulose of corn stover was linearly increased (p<0.001), whereas that of crude protein in diet was linearly decreased (p<0.001) with increasing isovalerate supplementation. The present results indicate that supplementation of diet with isovalerate improved microbial status and ruminal enzyme activities in steers. It was suggested that the isovalerate stimulated the digestive microorganisms or enzymes in a dose-dependent manner based on corn stover diet with a low protein. In the experimental conditions of this trial, the optimum isovalerate dose was about 16.8g isovalerate per steer per day.

Rapid enrichment of (homo)acetogenic consortia from animal feces using a high mass-transfer gas-lift reactor fed with syngas

A gas-lift reactor having a high mass transfer coefficient (k(L)a = 80.28 h(-1)) for a relatively insoluble gas (carbon monoxide; CO) was used to enrich (homo)acetogens from animal feces. Samples of fecal matter from cow, rabbit, chicken, and goat were used as sources of inoculum for the enrichment of CO and H(2) utilizing microbial consortia. To confirm the successful enrichment, the Hungate roll tube technique was employed to count and then isolate putative CO utilizers. The results of this work showed that CO and H(2) utilizing consortia were established for each inoculum source after 8 days. The number of colony-forming units in cow, rabbit, chicken, and goat fecal samples were 3.83 × 10(9), 1.03 × 10(9), 8.3 × 10(8), and 3.25 × 10(8) cells/ml, respectively. Forty-two colonies from the animal fecal samples were screened for the ability to utilize CO/H(2). Ten of these 42 colonies were capable of utilizing CO/H(2). Five isolates from cow feces (samples 5, 6, 8, 16, and 22) were highly similar to previously unknown (homo)acetogen, while cow-7 has shown 99 % similarity with Acetobacterium sp. as acetogens. On the other hand, four isolates from chicken feces (samples 3, 8, 10, and 11) have also shown high CO/H(2) utilizing activity. Hence, it is expected that this research could be used as the basis for the rapid enrichment of (homo)acetogenic consortia from various environmental sources.

Decolourisation of Turquoise HGN by anaerobic bacterial isolates from rumen enrichments

Anaerobic bacteria capable of decolourising Turquoise HGN dye were isolated by roll tube technique from enrichments inoculated with rumen fluid and were tested for their decolourisation efficiency under different conditions. The presence of glucose in the culture medium favoured the decolourisation and specific dye reduction activity (equivalent to the reduction of 1 mg dye. hr−1.mg−1 of protein), but both were inhibited in the presence of yeast extract. The maximum growth and dye reduction activity was recorded at pH 7.0 and 30°C, and were in the range of 36.4–97.4 U and 68–82 U, respectively with glucose as co-metabolite.

The modification of glucose levels and N source in the Hungate's medium to stimulate the production of fibrolytic enzymes of Anaeromyces sp. YQ3 grown on corn stalks

Hungate's method is a well-accepted protocol for the isolation or incubation of anaerobes with a roll tube technique. The aim of this study was to stimulate fungal enzyme production by optimizing the components of Hungate's medium for the growth of a rumen fungus Anaeromyces sp. YQ3. The organism was grown on corn stalks and incubated for 10 days in defined media with two glucose levels (G+, glucose in the Hungate's medium as a glucose control; G−, glucose removed in a modified Hungate's medium) and four N sources (N1: yeast extract+tryptone+(NH4)2SO4 in Hungate's medium (control); N2: yeast extract+(NH4)2SO4; N3: tryptone+(NH4)2SO4; and N4: tryptone+yeast extract). In the G− media, the recovered activities of feruloyl esterase (FAE) (P<0.0001), acetyl esterase (AE) (P=0.0065) and xylanase (P<0.0001) were decreased, while the G+ media with N1 nitrogen stimulated the production of FAE and xylanase (P<0.0001). The G− medium with N2 nitrogen increased the recovered activities of carboxymethyl cellulase (P=0.0001) and avicelase (P<0.0001), while the N3 and N4 media increased the recovered activity of AE (P=0.0015). The N4 medium was comparable to the N1 medium in stimulating the amount of recovered xylanase activity. The activities of FAE (P<0.0001), AE (P<0.0001), and xylanase (P<0.0001) showed a time-dependent increase and reached their peaks at day 10, while the avicelase activity peaked at day 8 (P=0.0071). The esterase activities (FAE and AE) were positively correlated with the enzyme activities of xylanase and carboxymethyl cellulase (r>0.48, P<0.05). After a 10-day incubation, the glucose in the Hungate's media contributed to an increase in organic matter disappearance (P<0.0001) and volatile fatty acid (VFA) concentration (P<0.0001), except for molar acetate proportions. The N4 treatment increased organic matter disappearance and total VFA concentration (P=0.0002). The change in N source did not alter molar proportions of acetate, propionate and valerate, while the N2 treatment increased molar butyrate proportion (P<0.0035), and both N2 and N3 increased the molar proportion of branched chain VFAs (P<0.0041). In summary, the glucose in the Hungate's medium is beneficial for stimulating the production of esterases and xylanase, thereby promoting fungal growth. Amending the N source in Hungate's medium brings about different yields of rumen fungal esterases and polysaccharide hydrolases that have important nutritional impacts on fibre degradation in ruminant animals.

Isolation of natural cultures of anaerobic fungi and indigenously associated methanogens from herbivores and their bioconversion of lignocellulosic materials to methane

This study aimed to obtain natural cultures of anaerobic fungi and their indigenously associated methanogens from herbivores and investigate their ability to degrade lignocelluloses to methane. Eight natural cultures were obtained by Hungate roll tube technique. The fungi were identified as belonging to Piromyces, Anaeromyces and Neocallimastix respectively by microscopy, and the methanogens as Methanobrevibacter spp. by 16S rRNA gene sequencing. In vitro studies with rice straw showed that these cultures degraded 33.5–48.3% substrate and produced 0.33–0.84mmol/(100ml culture) methane. Two cultures were further selected for their ability to degrade different lignocellulosic materials and could produce 0.38–1.27mmol/(100ml culture) methane. When methanogens were inhibited, the lignocellulose-degrading ability of cultures significantly reduced. In conclusion, natural cultures of anaerobic fungi with indigenously associated methanogens with high fiber degradation ability were obtained, and these cultures may have the potential in industrial use in lignocelluloses degradation and methane production.

Evaluation of the microbial population in ruminal fluid using real time PCR in steers treated with virginiamycin

The objective of the present study was to investigate the effects of virginiamycin (VM ) supplementation on ruminal fermentation and microbial populations in steers. Four ruminally cannulated Chinese Luxi steers (BW 559.4 &amp;plusmn; 30.1 kg) were used in a crossover design experiment with an experimental period of 28 days. The forage to concentrate ratio of the basal diet was 35:65 on dry matter basis. The experiment consisted of control treatment and treatment with control diet plus VM at a dose of 30 mg/kg concentrate (DM basis). Rumen fluid was collected at 07:30 prefeeding, at 11:30 and 17:30 postfeeding on day 27 and 28. A part of the pooled sample from rumen fluid was transferred to anaerobic culture by a roll-tube technique and analysed for species-specific real-time PCR quantification. The remaining pooled rumen fluid sample was analyzed for pH, VFA, ammonia N and l-lactic acid. The results showed that VM increased the ruminal pH (6.70 vs. 6.63; P &amp;lt; 0.05), but it decreased ammonia nitrogen (4.94 vs. 6.19 mg/100 ml; P &amp;lt; 0.01) and mean counts of amylolytic bacteria and proteolytic bacteria (P &amp;lt; 0.01) as compared to the control. The additive VM did not affect the l-lactic acid concentration (1.39 vs. 1.26 mmol/l) in rumen fluid. Compared to the control, the steers receiving VM have altered a trend of quantification of Selenomonas ruminantium, Anaerovibrio lipolytica, Ruminococcus albus and Streptococcus bovis in rumen fluid (0.05&amp;lt;p&amp;lt;0.1) as compared to the control. However, VM had no significant effect on Lactobacillus spp. (P = 0.41), Butyrivibrio fibrisolvens (P = 0.35), on the genus Ruminococcus (P = 0.25), Ruminococcus flavefaciens (P = 0.52), Prevotella ruminicola (P = 0.54), on the genus Prevotella (P = 0.67) and Megasphaera elsdenii (P = 0.97). In this study, we found that VM had selective effects on ruminal bacteria and influenced ruminal fermentation by changing a part of the specific ruminal bacteria populations.

Effects of crude feruloyl and acetyl esterase solutions of Neocallimastix sp. YQ1 and Anaeromyces sp. YQ3 isolated from Holstein steers on hydrolysis of Chinese wildrye grass hay, wheat bran, maize bran, wheat straw and corn stalks

Neocallimastix sp. YQ1 and Anaeromyces sp. YQ3 were isolated from rumen fluids of Holstein steers using a modification of the roll-tube technique of Hungate (1969). After a 9-day incubation for YQ1, and a 4-day incubation for YQ3, with optimal carbon and N supply in cultures, crude enzyme solutions of both fungi were prepared by centrifuging cultures for 10 min at 1000 × g at 4 °C. Enzymological characteristics of ferulic acid esterase and acetyl esterase were measured in the crude enzyme solution. The Michaelis constants ( K m ) and maximum velocities ( V max ) of ferulic acid esterase against methyl ferulate at pH 6.0 and 39 °C were 30 μM and 8.05 mU for YQ1 and 129 μM and 3.15 mU for YQ3, respectively. With substrate of p-nitrophenyl acetate, the K m and V max of AE at pH 6.0 and 39 °C were 0.21 mM and 336 mU for YQ1 and 5.26 mM and 854 mU for YQ3. Enzymatic release of reducing sugar, ferulic acid, and p-coumaric acid from complex fibre-rich feedstuffs was evaluated, and YQ3 released more reducing sugars from wheat straw, but less from the other plant materials especially maize bran and corn stalks. YQ3 liberated much more hydroxycinnamic acids from corn stalks, and YQ1 liberated much more hydroxycinnamic acids from maize bran. Anaerobic rumen fungi showed species diversity of enzymological characteristics of ferulic acid esterase and acetyl ester esterase, and exhibited a strong capability to release ferulic acid and p-coumaric acid from complex fibre-rich feedstuffs. Therefore anaerobic fungi, especially their FAE, should not be neglected in studies on biodegradation of plant cell walls in the rumen as well as on industrial processes developed to release ferulic acid and p-coumaric acid from agricultural by-products with potential commercial industrial applications, such as precursors of natural vanillin, natural antioxidants, food preservative agents, anti-inflammatory agents and photoprotectants.

Ruminal ecology of swamp buffalo as influenced by dietary sources

Four, 3-year old, rumen fistulated swamp buffalo bulls were randomly assigned in a 2 × 2 factorial arrangement in a 4 × 4 Latin square design to received four dietary treatments; factor A = 2 sources of energy (cassava chip and corn cobs), factor B = 2 levels of urea in concentrate mixture (15 and 30 g/kg). During the experiment, concentrate was offered at 5 g/kg BW while 50 g/kg urea-treated rice straw was given on ad libitum basis. Feeds, rumen fluid and rectal feces were collected for chemical analyses during each experimental period (21 days). In addition, rumen fluid was analyzed for, pH, temperature and NH 3-N concentration and microbial population and diversity by using direct count technique, roll-tube technique and molecular techniques including PCR-DGGE and real-time PCR technique. It was found that energy sources and urea levels had no effects on rumen pH, temperature, NH 3-N and total VFA concentrations as well as proportion of individual VFA. Energy sources especially cassava chip had a higher protozoal population than those of corn cob while urea had no effect. The two factors, energy sources and urea levels resulted in similar rumen bacterial and fungal zoospore population. Similarly with nutrient digestion coefficients, there were no differences among treatments, while cassava chip gave a slightly higher intake of urea-treated rice straw than corn cob fed group. Methanogenic and cellulolytic bacteria were variable among treatments and were highest in corn cob with 15 g/kg urea fed group which were higher than those in other groups. In addition, the use of PCR-DGGE has found diversity of rumen microorganisms particularly methanogenic and cellulolytic bacteria. Moreover, using of real-time PCR technique provided that population of Fibrobacter succinogenes had relatively higher than those Ruminococus flavefacieus and Ruminococus albus. Based on this study, corn cob and urea at 15 g/kg could be efficiently utilized in the rumen and thus, could provide good fermentation end-products and improve rumen ecology for the host swamp buffaloes.

Soybean oil and linseed oil supplementation affect profiles of ruminal microorganisms in dairy cows

The objective of this study was to evaluate changes in ruminal microorganisms and fermentation parameters due to dietary supplementation of soybean and linseed oil alone or in combination. Four dietary treatments were tested in a Latin square designed experiment using four primiparous rumen-cannulated dairy cows. Treatments were control (C, 60 : 40 forage to concentrate) or C with 4% soybean oil (S), 4% linseed oil (L) or 2% soybean oil plus 2% linseed oil (SL) in a 4 × 4 Latin square with four periods of 21 days. Forage and concentrate mixtures were fed at 0800 and 2000 h daily. Ruminal fluid was collected every 2 h over a 12-h period on day 19 of each experimental period and pH was measured immediately. Samples were prepared for analyses of concentrations of volatile fatty acids (VFA) by GLC and ammonia. Counts of total and individual bacterial groups (cellulolytic, proteolytic, amylolytic bacteria and total viable bacteria) were performed using the roll-tube technique, and protozoa counts were measured via microscopy in ruminal fluid collected at 0, 4 and 8 h after the morning feeding. Content of ruminal digesta was obtained via the rumen cannula before the morning feeding and used immediately for DNA extraction and quantity of specific bacterial species was obtained using real- time PCR. Ruminal pH did not differ but total VFA (110 v. 105 mmol/l) were lower (P < 0.05) with oil supplementation compared with C. Concentration of ruminal NH3-N (4.4 v. 5.6 mmol/l) was greater (P < 0.05) due to oil compared with C. Compared with C, oil supplementation resulted in lower (P < 0.05) cellulolytic bacteria (3.25 × 108v. 4.66 × 108 colony-forming units (CFU)/ml) and protozoa (9.04 × 104v. 12.92 × 104 cell/ml) colony counts. Proteolytic bacteria (7.01 × 108v. 6.08 × 108 CFU/ml) counts, however, were greater in response to oil compared with C (P < 0.05). Among oil treatments, the amount of Butyrivibrio fibrisolvens, Fibrobacter succinogenes and Ruminococcus flavefaciens in ruminal fluid was substantially lower (P < 0.05) when L was included. Compared to C, the amount of Ruminococcus albus decreased by an average of 40% regardless of oil level or type. Overall, the results indicate that some ruminal microorganisms, except proteolytic bacteria, are highly susceptible to dietary unsaturated fatty acids supplementation, particularly when linolenic acid rich oils were fed. Dietary oil effects on ruminal fermentation parameters seemed associated with the profile of ruminal microorganisms.

The use of molecular techniques based on ribosomal RNA and DNA for rumen microbial ecosystem studies: a review

This paper analyses the research progress in the use of molecular techniques based on ribosomal RNA and DNA (rRNA/rDNA) for rumen microbial ecosystem since first literature by Stahl et al. (1988). Because rumen microbial populations could be under-estimated by adopting the traditional techniques such as roll-tube technique or most-probable-number estimates, modern molecular techniques based on 16S/18S rRNA/rDNA can be used to more accurately provide molecular characterization, microbe populations and classification scheme than traditional methods. Phylogenetic-group-specific probes can be used to hybridize samples for detecting and quantifying of rumen microbes. But, competitive-PCR and real-time PCR can more sensitively quantify rumen microbes than hybridization. Molecular fingerprinting techniques including both denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE) and restriction fragment length polymorphisms (RFLP) can used to explore diversity of bacteria, protozoa and fungi in the rumen ecosystem. By constructing clone libraries of 16S/18S rRNA/rDNA of rumen microbes, more new microbes can be discovered and identified. For fungi, internal transcribed spacers (ITS) of fungi are better than 18S rRNA/rDNA for discriminating operational taxonomic units. In conclusion, 16S/18S rRNA/rDNA procedures have been used with success in rumen microbes and are quickly gaining acceptance for studying rumen microbial ecosystem, and will become useful methods for rumen ecology research. However, molecular techniques based on 16S/18S rRNA/rDNA don't preclude classical and traditional microbiological techniques. It should used together to acquire accurate and satisfactory results.

Criopreservación de aislados nativos de la bacteria ruminal Fibrobacter succinogenes

En el presente trabajo se estandarizó una técnica de criopreservación en nitrógeno líquido, usando como crioprotector dimetil sulfóxido al 5%, para la preservación de aislados nativos bacterianos de Fibrobacter succinogenes. El respectivo protocolo de criopreservación se evaluó determinando la viabilidad bacteriana por el transcurso de un año. La escala de MacFarland, ampliamente conocida en la estimación de poblaciones bacterianas aerobias, fue validada como técnica de estimación de bacterias anaerobias, usando la técnica de conteo bacteriano por roll-tube (R2 = 0.95). Se utilizaron dos aislados nativos, C7 y C9, de Fibrobacter succinogenes, obtenidos del rumen de bovinos en pastoreo de Brachiaria decumbens, en los Llanos Orientales de Colombia. Quince días después de la exposición de los cultivos al proceso de criopreservación, se apreció una reducción en el número de bacterias viables con relación a la población inicial de cada uno de los aislados (C7 = 9.25 x 108 UFCml-1 vs. 6.15 x 108 UFCml-1 y C9 = 12.51 x 108 UFCml-1 vs. 9.26 x 108 UFCml-1). Sin embargo, no se presentaron diferencias en la población de bacterias viables en los muestreos posteriores (C7 = 6.13 – 6.16 x 108; C9 = 9.26 – 9.35 x 108 UFCml-1). Esta técnica permite mantener la viabilidad bacteriana y puede considerarse como un procedimiento eficiente y de fácil aplicación para la preservación de bacterias ruminales. Además, constituye una herramienta fundamental para el establecimiento de bancos de germoplasma de microorganismos anaerobios ruminales en Colombia. El uso conjunto de las técnicas de criopreservación y la escala de MacFarland, posee ventajas sobre los métodos de preservación y estimación de poblaciones por cultivos activos, por su confiabilidad, eficiencia y bajo costo.

Improved Recovery of Stressed Bifidobacterium from Water and Frozen Yogurt

A roll-tube repair-detection procedure was developed to enumerate injured and noninjured cells of Bifidobacterium species from water and food samples. This procedure combined the Virginia Polytechnic Institute and State University's anaerobic roll-tube procedure and the repair-detection technique for detecting stressed cells. Mara and Oragui's human bifid sorbitol agar medium was modified for use in the roll-tube procedure by replacing the indicator bromocresol purple with phenyl red (0.027 g/l), adding 0.0006g of methylene blue per 1, increasing the agar content to 25 g/l and adjusting the pH of the medium to 7.1 ± 0.1. The repair-detection roll-tube technique was shown to recover Bifidobacterium cells significantly (P < 0.01) better than pour plates, using the same medium incubated in anaerobe jars, even when a repair-detection system was used. Most repair in the roll tubes occurred in the first hour. B. adolescentis had a poor survival rate after 96 hours in water. Glucose was substituted for sorbitol in the medium used for enumeration of B. longum added to frozen yogurt, because this organism cannot utilize sorbitol. This medium, when used as part of a repair detection system, significantly (P < 0.01) recovered more cells than anaerobic pour plate techniques and was able to separate Bifidobacterium species and Lactobacillus acidophilus by colony morphology. Bifidobacterium cells were 1mm or larger, round and yellow, while the L. acidophilus colonies were so small (< 1/4 mm) their detection and enumeration was difficult.

Populations of Anaerobic Phototrophic Bacteria in a Spartina alterniflora Salt Marsh

Habitat-simulating media were used with the Hungate anaerobic roll tube technique to enumerate culturable anaerobic photosynthetic bacteria in sediment, tidal waters, and Spartina alterniflora plant samples collected from the salt marsh at Sapelo Island, Ga. No phototrophs were detected in samples of creekside (low marsh) sediment or in tidal waters in creekside regions. In the high marsh region, 90% of anaerobic phototrophic bacteria occurred in the top 5 mm of sediment and none were detected below 6 mm. There was a seasonal variation, with maximal populations occurring in summer and fall (mean, 4.4 x 10 phototrophs g of dry sediment) and minimal numbers occurring in winter (mean, 3.9 x 10 phototrophs g of dry sediment). During winter and late spring, phototrophs had a patchy distribution over the high marsh sediment surface. In contrast, during late summer they had a random uniform distribution. Tidal water collected over high marsh sediment contained an average of 8.7 x 10 phototrophs ml, with no significant seasonal variation. Anaerobic phototrophic bacteria were also cultured from the lower stem tissue of S. alterniflora growing in both the high (4.3 x 10 phototrophs g of dry tissue) and creekside (4.9 x 10 phototrophs g of dry tissue) marsh regions. Chromatium buderi, Chromatium vinosum, Thiospirillum sanguineum, Rhodospirillum molischianum, and Chlorobium phaeobacteroides were the predominant anaerobic phototrophic species cultured from high marsh sediment. The two Chromatium species were dominant.