P Weimer - Academia.edu (original) (raw)
Papers by P Weimer
Applied and Environmental Microbiology, 1997
Three predominant ruminal cellulolytic bacteria (Fibrobacter succinogenes S85, Ruminococcus flave... more Three predominant ruminal cellulolytic bacteria (Fibrobacter succinogenes S85, Ruminococcus flavefaciens FD-1, and Ruminococcus albus 7) were grown in different binary combinations to determine the outcome of competition in either cellulose-excess batch culture or in cellulose-limited continuous culture. Relative populations of each species were estimated by using signature membrane-associated fatty acids and/or 16S rRNA-targeted oligonucleotide probes. Both F. succinogenes and R. flavefaciens coexisted in cellulose-excess batch culture with similar population sizes (58 and 42%, respectively; standard error, 12%). By contrast, under cellulose limitation R. flavefaciens predominated (> 96% of total cell mass) in coculture with F. succinogenes, regardless of whether the two strains were inoculated simultaneously or whether R. flavefaciens was inoculated into an established culture of F. succinogenes. The predominance of R. flavefaciens over F. succinogenes under cellulose limitatio...
Applied and Environmental Microbiology, 1996
Growth of the ruminal bacteria Fibrobacter succinogenes S85, Ruminococcus flavefaciens FD-1, and ... more Growth of the ruminal bacteria Fibrobacter succinogenes S85, Ruminococcus flavefaciens FD-1, and R. albus 7 followed Monod kinetics with respect to concentrations of individual pure cellodextrins (cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose). Under the conditions tested, R. flavefaciens FD-1 possesses the greatest capacity to compete for low concentrations of these cellodextrins.
Biotechnology for Biofuels, 2018
Background: Cellulose is the most abundant biological polymer on earth, making it an attractive s... more Background: Cellulose is the most abundant biological polymer on earth, making it an attractive substrate for the production of next-generation biofuels and commodity chemicals. However, the economics of cellulose utilization are currently unfavorable due to a lack of efficient methods for its hydrolysis. Fibrobacter succinogenes strain S85, originally isolated from the bovine rumen, is among the most actively cellulolytic mesophilic bacteria known, producing succinate as its major fermentation product. In this study, we examined the transcriptome of F. succinogenes S85 grown in continuous culture at several dilution rates on cellulose, cellobiose, or glucose to gain a system-level understanding of cellulose degradation by this bacterium. Results: Several patterns of gene expression were observed for the major cellulases produced by F. succinogenes S85. A large proportion of cellulase genes were constitutively expressed, including the gene encoding for Cel51A, the major cellulose-binding endoglucanase produced by this bacterium. Moreover, other cellulase genes displayed elevated expression during growth on cellulose relative to growth on soluble sugars. Growth rate had a strong effect on global gene expression, particularly with regard to genes predicted to encode carbohydrate-binding modules and glycoside hydrolases implicated in hemicellulose degradation. Expression of hemicellulase genes was tightly regulated, with these genes displaying elevated expression only during slow growth on soluble sugars. Clear differences in gene expression were also observed between adherent and planktonic populations within continuous cultures growing on cellulose. Conclusions: This work emphasizes the complexity of the fiber-degrading system utilized by F. succinogenes S85, and reinforces the complementary role of hemicellulases for accessing cellulose by these bacteria. We report for the first time evidence of global differences in gene expression between adherent and planktonic populations of an anaerobic bacterium growing on cellulose at steady state during continuous cultivation. Finally, our results also highlight the importance of controlling for growth rate in investigations of gene expression.
Applied and Environmental Microbiology, 1995
When glucose or cellobiose was provided as an energy source for Fibrobacter succinogenes, there w... more When glucose or cellobiose was provided as an energy source for Fibrobacter succinogenes, there was a transient accumulation (as much as 0.4 mM hexose equivalent) of cellobiose or cellotriose, respectively, in the growth medium. Nongrowing cell suspensions converted cellobiose to cellotriose and longer-chain cellodextrins, and in this case the total cellodextrin concentration was as much as 20 mM (hexose equivalent). Because cell extracts of glucose- or cellobiose-grown cells cleaved cellobioise and cellotriose by phosphate-dependent reactions and glucose 1-phosphate was an end product, it appeared that cellodextrins were being produced by a reversible phosphorylase reaction. This conclusion was supported by the observation that the ratio of cellodextrins to cellodextrins with one greater hexose [n/(n + 1)] was approximately 4, a value similar to the equilibrium constant (Keq) of cellobiose phosphorylase (J. K. Alexander, J. Bacteriol. 81:903-910, 1961). When F. succinogenes was gro...
Applied and Environmental Microbiology, 1990
The digestion kinetics of a variety of pure celluloses were examined by using an in vitro assay e... more The digestion kinetics of a variety of pure celluloses were examined by using an in vitro assay employing mixed ruminal microflora and a modified detergent extraction procedure to recover residual cellulose. Digestion of all of the celluloses was described by a discontinuous first-order rate equation to yield digestion rate constants and discrete lag times. These kinetic parameters were compared with the relative crystallinity indices and estimated accessible surface areas of the celluloses. For type I celluloses having similar crystallinities and simple nonaggregating particle morphologies, the fermentation rate constants displayed a strong positive correlation (r2 = 0.978) with gross specific surface area; lag time exhibited a weaker, negative correlation (r2 = 0.930) with gross specific surface area. Crystallinity was shown to have a relatively minor effect on the digestion rate and lag time. Swelling of microcrystalline cellulose with 72 to 77% phosphoric acid yielded substrates...
Applied and Environmental Microbiology, 1992
The ruminal cellulolytic bacterium Ruminococcus flavefaciens FD-1 was grown in cellulose-fed cont... more The ruminal cellulolytic bacterium Ruminococcus flavefaciens FD-1 was grown in cellulose-fed continuous culture with 20 different combinations of pH and dilution rate (D); the combinations were selected according to the physiological pH range of the organism (6.0 to 7.1) and growth rate of the organism on cellulose (0.017 to 0.10 h-1). A response surface analysis was used to characterize the effects of pH and D on the extent of cellulose consumption, growth yield, soluble sugar concentration, and yields of fermentation products. The response surfaces indicate that pH and D coordinately affect cellulose digestion and growth yield in this organism. As expected, the net cellulose consumption increased with increasing D while the fraction of added cellulose that was utilized decreased with increasing D. The effect of changes in pH within the physiological range on cellulose consumption was smaller than that of changes in D. Cellulose degradation was less sensitive to low pH than to high...
Applied and Environmental Microbiology, 1992
The ruminal cellulolytic bacterium Ruminococcus flavefaciens FD-1 was grown in cellulose-fed cont... more The ruminal cellulolytic bacterium Ruminococcus flavefaciens FD-1 was grown in cellulose-fed continuous culture with 20 different combinations of pH and dilution rate (D); the combinations were selected according to the physiological pH range of the organism (6.0 to 7.1) and growth rate of the organism on cellulose (0.017 to 0.10 h-1). A response surface analysis was used to characterize the effects of pH and D on the extent of cellulose consumption, growth yield, soluble sugar concentration, and yields of fermentation products. The response surfaces indicate that pH and D coordinately affect cellulose digestion and growth yield in this organism. As expected, the net cellulose consumption increased with increasing D while the fraction of added cellulose that was utilized decreased with increasing D. The effect of changes in pH within the physiological range on cellulose consumption was smaller than that of changes in D. Cellulose degradation was less sensitive to low pH than to high...
Applied Biochemistry and Biotechnology, 2010
Journal of Dairy Science, 2017
The objectives of this study were to determine if milk production efficiency (MPE) is altered by ... more The objectives of this study were to determine if milk production efficiency (MPE) is altered by near-total exchange of ruminal contents between high-(HE) and low-MPE (LE) cows and to characterize ruminal bacterial community composition (BCC) before exchange and over time postexchange. Three pairs of ruminally cannulated, third-lactation cows were selected whose MPE (energy-corrected milk per unit of dry matter intake) differed over their first 2 lactations. Approximately 95% of ruminal contents were exchanged between cows within each pair. Ruminal pH and volatile fatty acid (VFA) profiles, along with BCC (characterized by sequencing of the variable 4 region of 16S rRNA genes), were assessed just before feeding on d −
AMB Express, 2015
The fermentation system of mixed ruminal bacteria is capable of generating large amounts of short... more The fermentation system of mixed ruminal bacteria is capable of generating large amounts of short-chain volatile fatty acids (VFA) via the carboxylate platform in vitro. These VFAs are subject to elongation to larger, more energy-dense products through reverse β-oxidation, and the resulting products are useful as precursors for liquid fuels production. This study examined the effect of several redox mediators (neutral red, methyl viologen, safranin O, tannic acid) as alternative electron carriers for mixed ruminal bacteria during the fermentation of biomass (ground switchgrass not subjected to other pretreatments) and their potential to enhance elongation of end-products to medium-chain VFAs with no additional run-time. Neutral red (1 mM) in particular facilitated chain elongation, increasing average VFA chain length from 2.42 to 2.97 carbon atoms per molecule, while simultaneously inhibiting methane accumulation by over half yet maintaining total C in end products. The ability of r...
Journal of Dairy Science, 2010
The influence of pH dynamics on ruminal bacterial community composition was studied in 8 ruminall... more The influence of pH dynamics on ruminal bacterial community composition was studied in 8 ruminally cannulated Holstein cows fitted with indwelling electrodes that recorded pH at 10-min intervals over a 54-h period. Cows were fed a silage-based total mixed ration supplemented with monensin. Ruminal samples were collected each day just before feeding and at 3 and 6 h after feeding. Solid and liquid phases were separated at collection, and extracted DNA was subjected to PCR amplification followed by automated ribosomal intergenic spacer analysis (ARISA). Although cows displayed widely different pH profiles (mean pH = 6.11 to 6.51, diurnal pH range = 0.45 to 1.39), correspondence analysis of the ARISA profiles revealed that 6 of the 8 cows showed very similar bacterial community compositions. The 2 cows having substantially different community compositions had intermediate mean pH values (6.30 and 6.33) and intermediate diurnal pH ranges (averaging 0.89 and 0.81 pH units). Fortuitously, these 2 cows alone also displayed milk fat depression, along with markedly higher ruminal populations of 1 bacterial operational taxonomic unit (OTU) and reduced populations of another ARISA amplicon. Cloning and sequencing of the elevated OTU revealed phylogenetic similarity to Megasphaera elsdenii, a species reportedly associated with milk fat depression. The higher populations of both M. elsdenii and OTU246 in these 2 cows were confirmed using quantitative real-time PCR (qPCR) with species-specific primers, and the fraction of total bacterial rDNA copies contributed by these 2 taxa were very highly correlated within individual cows. By contrast, the fraction of total bacterial rDNA copies contributed by Streptococcus bovis and genus Ruminococcus, 2 taxa expected to respond to ruminal pH, did not differ among cows (mean = <0.01 and 10.6%, respectively, of rRNA gene copies, determined by qPCR). The data indicate that cows with widely differing pH profiles can have similar ruminal bacterial community compositions, and that milk fat depression can occur at intermediate ruminal pH. The results support recent reports that milk fat depression is associated with shifts in bacterial community composition in rumine and is specifically related to the relative abundance of Megasphaera elsdenii.
When glucose or cellobiose was provided as an energy source for Fibrobacter succinogenes, there w... more When glucose or cellobiose was provided as an energy source for Fibrobacter succinogenes, there was a transient accumulation (as much as 0.4 mM hexose equivalent) of cellobiose or cellotriose, respectively, in the growth medium. Nongrowing cell suspensions converted cellobiose to cellotriose and longer-chain cellodex- trins, and in this case the total cellodextrin concentration was as much as 20 mM (hexose equivalent). Because cell extracts of glucose- or cellobiose-grown cells cleaved cellobiose and cellotriose by phosphate-dependent reactions and glucose 1-phosphate was an end product, it appeared that cellodextrins were being produced by a reversible phosphorylase reaction. This conclusion was supported by the observation that the ratio of cellodextrins to cellodextrins with one greater hexose (n/(n 11)) was approximately 4, a value similar to the equilibrium constant (Keq) of cellobiose phosphorylase (J. K. Alexander, J. Bacteriol. 81:903-910, 1961). When F. succinogeneswas grow...
Journal of Dairy Science, 1999
The effects of four contrasting diets were determined on populations of three species of ruminal ... more The effects of four contrasting diets were determined on populations of three species of ruminal cellulolytic bacteria (Ruminococcus albus, Ruminococcus flavefaciens, and Fibrobacter succinogenes) using oligonucleotide probes to rRNA. Diets based on alfalfa silage or corn silage as the primary fiber source were formulated to contain either 24 or 32% neutral detergent fiber measured after a-amylase treatment. The diets were fed twice daily to four ruminally fistulated, lactating Holstein cows in a trial using a Latin square design. The cows fed the alfalfa silage diets had higher dry matter intakes and milk production and smaller pH fluctuations than did cows fed the corn silage diets (0.3 vs. 0.8 units). The total populations of the three cellulolytic species at 3 h after feeding ranged from 0.3 to 3.9% of the bacterial domain; R. albus was generally the most abundant of the three species. The data are in general agreement with population assessments obtained by some traditional methods of culture enumeration. Although diet and individual cows had major effects on ruminal pH and volatile fatty acid concentrations and on milk production and composition, differences in cellulolytic populations that were attributable to individual cows were larger than those attributable to diet, suggesting that each cow maintained a unique assemblage of cellulolytic species.
The characterization of microbial communities by metagenomic approaches has been enhanced by rece... more The characterization of microbial communities by metagenomic approaches has been enhanced by recent improvements in short-read sequencing efficiency and assembly algorithms. We describe the results of adding long-read sequencing to the mix of technologies used to assemble a highly complex cattle rumen microbial community, and compare the assembly to current short read-based methods applied to the same sample. Contigs in the long-read assembly were 7-fold longer on average, and contained 7-fold more complete open reading frames (ORF), than the short read assembly, despite having three-fold lower sequence depth. The linkages between long-read contigs, provided by proximity ligation data, supported identification of 188 novel viral-host associations in the rumen microbial community that suggest cross-species infectivity of specific viral strains. The improved contiguity of the long-read assembly also identified 94 antimicrobial resistance genes, compared to only seven alleles identifie...
Frontiers in Microbiology
This experiment aimed to determine the effects of camelina seed (CS) supplementation at different... more This experiment aimed to determine the effects of camelina seed (CS) supplementation at different dietary fat levels on ruminal bacterial community composition and how it relates to changes in ruminal fermentation in a dual-flow continuous culture system. Diets were randomly assigned to 8 fermenters (1,200-1,250 mL) in a 2 × 2 factorial arrangement of treatments in a replicated 4 × 4 Latin square with four 10-day experimental periods that consisted of 7 days for diet adaptation and 3 days for sample collection. Treatments were: (1) no CS at 5% ether extract (EE, NCS5); (2) no CS at 8% EE (NCS8); (3) 7.7% CS at 5% EE (CS5); and (4) 17.7% CS at 8% EE (CS8). Megalac was used as a control to adjust EE levels. Diets contained 55% orchardgrass hay and 45% concentrate, and fermenters were equally fed a total of 72 g/day (DM basis) twice daily. The bacterial community was determined by sequencing the V4 region of the 16S rRNA gene using the Illumina MiSeq platform. Sequencing data were analyzed using mothur and statistical analyses were performed in R and SAS. The most abundant phyla across treatments were the Bacteroidetes and Firmicutes, accounting for 49 and 39% of the total sequences, respectively. The bacterial community composition in both liquid and solid fractions of the effluent digesta changed with CS supplementation but not by dietary EE. Including CS in the diets decreased the relative abundances of Ruminococcus spp., Fibrobacter spp., and Butyrivibrio spp. The most abundant genus across treatments, Prevotella, was reduced by high dietary EE levels, while Megasphaera and Succinivibrio were increased by CS supplementation in the liquid fraction. Correlatively, the concentration of acetate was decreased while propionate increased; C18:0 was decreased and polyunsaturated fatty acids, especially C18:2 n-6 and C18:3 n-3, were increased by CS supplementation. Based on the correlation analysis between genera and fermentation end products, this study revealed that CS supplementation could be energetically beneficial to dairy cows by increasing propionate-producing bacteria and suppressing ruminal bacteria associated with biohydrogenation. However, attention should be given to avoid the effects of CS supplementation on suppressing cellulolytic bacteria.
Applied and Environmental Microbiology
Gastrointestinal tract (GIT) microorganisms play important roles in the health of ruminant livest... more Gastrointestinal tract (GIT) microorganisms play important roles in the health of ruminant livestock and affect the production of agriculturally relevant products, including milk and meat. Despite this link, interventions to alter the adult microbiota to improve production have proven ineffective, as established microbial communities are resilient to change. In contrast, developing communities in young animals may be more easily altered but are less well studied. Here, we measured the GIT-associated microbiota of 45 Holstein dairy cows from 2 weeks to the first lactation cycle, using Illumina amplicon sequencing of bacterial (16S rRNA V4), archaeal (16S rRNA V6 to V8), and fungal (internal transcribed region 1 [ITS1]) communities. Fecal and ruminal microbiota of cows raised on calf starter grains and/or corn silage were correlated to lifetime growth as well as milk production during the first lactation cycle, in order to determine whether early-life diets have long-term impacts. Sig...
Microbiology
The anaerobic fermentation of glucose by Bacillus macerans ATCC 7068 was studied in batch culture... more The anaerobic fermentation of glucose by Bacillus macerans ATCC 7068 was studied in batch culture with and without pH control. The fermentation was characterized by two distinct metabolic phases. In the primary growth phase, the concentrations of ethanol and acetic acid increased exponentially, and formate was detected as a minor product. The secondary phase was marked by a slowing and eventual cessation of growth, along with the disappearance of formate and acetate, and the appearance of H2, C02, and acetone. Exogenously-added substrates were converted with stoichiometries of 1 formate-+ 1 H2 + 1 C o t , and approximately 2 acetate + 1 acetone. Consumption of > 8 g glucose 1required exogenous pH control, and acetone formation was strongly pH-dependent. Glucose was fermented by the Embden-Meyerhof pathway. Cell extracts contained pyruvate formate-lyase and formate dehydrogenase activities, but only low pyruvate dehydrogenase activity. A balanced fermentation pathway is presented which is consistent with reaction stoichiometries and [ 14C]glucose labelling data in whole cultures, and with enzyme activities in extracts. The pathway is compared with those of other facultative anaerobes and the acetone-producing Clostridium acetobutylicum. METHODS Materials. All chemicals were reagent grade, obtained from either Sigma or Fisher (Fair Lawn, N.J., U.S.A.). Enzymes used for coupled enzymic assays were obtained from Sigma. Anaerobic grade N 2 was purchased from Union Carbide-Linde Division (Somerset, N .J., U.S.A.). [ l J C ] G l u c~~e specifically labelled in the C-1, C-3,4 or C-6 positions was obtained from New England Nuclear. Organism and growth conditions. Bacillus nracerans ATCC 7068 was obtained from the American Type Culture Collection. According to Gordon etal. (1973), this strain is identical to 'B. acetoethylicus'described by Northrop et a/. (1919). The organism was grown in CM5 medium which contained (I-'
Anaerobe
For the growth of several strictly anaerobic microorganisms, cysteine is the preferred reducing a... more For the growth of several strictly anaerobic microorganisms, cysteine is the preferred reducing agent because of its low toxicity, but cysteine reduces media only slowly, making it inconvenient if the medium is needed immediately. A culture medium employed to cultivate ruminal anaerobic bacteria supplied with cysteine achieved the desired degree of anaerobiosis (measured spectrophotometrically by the bleaching of the indicator, resazurin) in considerably less time when exposed to high light intensity from a halogen lamp. The effect of light intensity on time to reduce media was quadratic; tubes kept under no illumination (dark) took more than 12 h to become colorless while tubes kept under normal laboratory light, took more than 2 h. On the other hand, media exposed to high light intensity (equivalent to a regular 100 W bulb lamp) became completely bleached in less than 20 min. Separate experiments demonstrated a direct correlation between the photocatalytic effect on medium reduction and a decrease in dissolved O 2 content. However, media gassing with CO 2 is still required, as the photocatalytic reaction requires semi-reduced conditions. A comparison of growth parameters for five species of ruminal bacteria cultivated in media prepared under high light vs normal light conditions showed no differences, indicating no adverse affect of cysteine catalysed medium reduction on growth. Aside from this benefit, other procedures such as extensive heating, cooling, evacuation and degassing with O 2-free gases can be eliminated, thereby further reducing the time required for media preparation. Thus, use of high light intensity on cysteine/resazurin containing media greatly accelerated the establishment of anaerobic conditions, so that the media can be used almost immediately.
Applied Microbiology and Biotechnology
Applied and Environmental Microbiology, 1997
Three predominant ruminal cellulolytic bacteria (Fibrobacter succinogenes S85, Ruminococcus flave... more Three predominant ruminal cellulolytic bacteria (Fibrobacter succinogenes S85, Ruminococcus flavefaciens FD-1, and Ruminococcus albus 7) were grown in different binary combinations to determine the outcome of competition in either cellulose-excess batch culture or in cellulose-limited continuous culture. Relative populations of each species were estimated by using signature membrane-associated fatty acids and/or 16S rRNA-targeted oligonucleotide probes. Both F. succinogenes and R. flavefaciens coexisted in cellulose-excess batch culture with similar population sizes (58 and 42%, respectively; standard error, 12%). By contrast, under cellulose limitation R. flavefaciens predominated (> 96% of total cell mass) in coculture with F. succinogenes, regardless of whether the two strains were inoculated simultaneously or whether R. flavefaciens was inoculated into an established culture of F. succinogenes. The predominance of R. flavefaciens over F. succinogenes under cellulose limitatio...
Applied and Environmental Microbiology, 1996
Growth of the ruminal bacteria Fibrobacter succinogenes S85, Ruminococcus flavefaciens FD-1, and ... more Growth of the ruminal bacteria Fibrobacter succinogenes S85, Ruminococcus flavefaciens FD-1, and R. albus 7 followed Monod kinetics with respect to concentrations of individual pure cellodextrins (cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose). Under the conditions tested, R. flavefaciens FD-1 possesses the greatest capacity to compete for low concentrations of these cellodextrins.
Biotechnology for Biofuels, 2018
Background: Cellulose is the most abundant biological polymer on earth, making it an attractive s... more Background: Cellulose is the most abundant biological polymer on earth, making it an attractive substrate for the production of next-generation biofuels and commodity chemicals. However, the economics of cellulose utilization are currently unfavorable due to a lack of efficient methods for its hydrolysis. Fibrobacter succinogenes strain S85, originally isolated from the bovine rumen, is among the most actively cellulolytic mesophilic bacteria known, producing succinate as its major fermentation product. In this study, we examined the transcriptome of F. succinogenes S85 grown in continuous culture at several dilution rates on cellulose, cellobiose, or glucose to gain a system-level understanding of cellulose degradation by this bacterium. Results: Several patterns of gene expression were observed for the major cellulases produced by F. succinogenes S85. A large proportion of cellulase genes were constitutively expressed, including the gene encoding for Cel51A, the major cellulose-binding endoglucanase produced by this bacterium. Moreover, other cellulase genes displayed elevated expression during growth on cellulose relative to growth on soluble sugars. Growth rate had a strong effect on global gene expression, particularly with regard to genes predicted to encode carbohydrate-binding modules and glycoside hydrolases implicated in hemicellulose degradation. Expression of hemicellulase genes was tightly regulated, with these genes displaying elevated expression only during slow growth on soluble sugars. Clear differences in gene expression were also observed between adherent and planktonic populations within continuous cultures growing on cellulose. Conclusions: This work emphasizes the complexity of the fiber-degrading system utilized by F. succinogenes S85, and reinforces the complementary role of hemicellulases for accessing cellulose by these bacteria. We report for the first time evidence of global differences in gene expression between adherent and planktonic populations of an anaerobic bacterium growing on cellulose at steady state during continuous cultivation. Finally, our results also highlight the importance of controlling for growth rate in investigations of gene expression.
Applied and Environmental Microbiology, 1995
When glucose or cellobiose was provided as an energy source for Fibrobacter succinogenes, there w... more When glucose or cellobiose was provided as an energy source for Fibrobacter succinogenes, there was a transient accumulation (as much as 0.4 mM hexose equivalent) of cellobiose or cellotriose, respectively, in the growth medium. Nongrowing cell suspensions converted cellobiose to cellotriose and longer-chain cellodextrins, and in this case the total cellodextrin concentration was as much as 20 mM (hexose equivalent). Because cell extracts of glucose- or cellobiose-grown cells cleaved cellobioise and cellotriose by phosphate-dependent reactions and glucose 1-phosphate was an end product, it appeared that cellodextrins were being produced by a reversible phosphorylase reaction. This conclusion was supported by the observation that the ratio of cellodextrins to cellodextrins with one greater hexose [n/(n + 1)] was approximately 4, a value similar to the equilibrium constant (Keq) of cellobiose phosphorylase (J. K. Alexander, J. Bacteriol. 81:903-910, 1961). When F. succinogenes was gro...
Applied and Environmental Microbiology, 1990
The digestion kinetics of a variety of pure celluloses were examined by using an in vitro assay e... more The digestion kinetics of a variety of pure celluloses were examined by using an in vitro assay employing mixed ruminal microflora and a modified detergent extraction procedure to recover residual cellulose. Digestion of all of the celluloses was described by a discontinuous first-order rate equation to yield digestion rate constants and discrete lag times. These kinetic parameters were compared with the relative crystallinity indices and estimated accessible surface areas of the celluloses. For type I celluloses having similar crystallinities and simple nonaggregating particle morphologies, the fermentation rate constants displayed a strong positive correlation (r2 = 0.978) with gross specific surface area; lag time exhibited a weaker, negative correlation (r2 = 0.930) with gross specific surface area. Crystallinity was shown to have a relatively minor effect on the digestion rate and lag time. Swelling of microcrystalline cellulose with 72 to 77% phosphoric acid yielded substrates...
Applied and Environmental Microbiology, 1992
The ruminal cellulolytic bacterium Ruminococcus flavefaciens FD-1 was grown in cellulose-fed cont... more The ruminal cellulolytic bacterium Ruminococcus flavefaciens FD-1 was grown in cellulose-fed continuous culture with 20 different combinations of pH and dilution rate (D); the combinations were selected according to the physiological pH range of the organism (6.0 to 7.1) and growth rate of the organism on cellulose (0.017 to 0.10 h-1). A response surface analysis was used to characterize the effects of pH and D on the extent of cellulose consumption, growth yield, soluble sugar concentration, and yields of fermentation products. The response surfaces indicate that pH and D coordinately affect cellulose digestion and growth yield in this organism. As expected, the net cellulose consumption increased with increasing D while the fraction of added cellulose that was utilized decreased with increasing D. The effect of changes in pH within the physiological range on cellulose consumption was smaller than that of changes in D. Cellulose degradation was less sensitive to low pH than to high...
Applied and Environmental Microbiology, 1992
The ruminal cellulolytic bacterium Ruminococcus flavefaciens FD-1 was grown in cellulose-fed cont... more The ruminal cellulolytic bacterium Ruminococcus flavefaciens FD-1 was grown in cellulose-fed continuous culture with 20 different combinations of pH and dilution rate (D); the combinations were selected according to the physiological pH range of the organism (6.0 to 7.1) and growth rate of the organism on cellulose (0.017 to 0.10 h-1). A response surface analysis was used to characterize the effects of pH and D on the extent of cellulose consumption, growth yield, soluble sugar concentration, and yields of fermentation products. The response surfaces indicate that pH and D coordinately affect cellulose digestion and growth yield in this organism. As expected, the net cellulose consumption increased with increasing D while the fraction of added cellulose that was utilized decreased with increasing D. The effect of changes in pH within the physiological range on cellulose consumption was smaller than that of changes in D. Cellulose degradation was less sensitive to low pH than to high...
Applied Biochemistry and Biotechnology, 2010
Journal of Dairy Science, 2017
The objectives of this study were to determine if milk production efficiency (MPE) is altered by ... more The objectives of this study were to determine if milk production efficiency (MPE) is altered by near-total exchange of ruminal contents between high-(HE) and low-MPE (LE) cows and to characterize ruminal bacterial community composition (BCC) before exchange and over time postexchange. Three pairs of ruminally cannulated, third-lactation cows were selected whose MPE (energy-corrected milk per unit of dry matter intake) differed over their first 2 lactations. Approximately 95% of ruminal contents were exchanged between cows within each pair. Ruminal pH and volatile fatty acid (VFA) profiles, along with BCC (characterized by sequencing of the variable 4 region of 16S rRNA genes), were assessed just before feeding on d −
AMB Express, 2015
The fermentation system of mixed ruminal bacteria is capable of generating large amounts of short... more The fermentation system of mixed ruminal bacteria is capable of generating large amounts of short-chain volatile fatty acids (VFA) via the carboxylate platform in vitro. These VFAs are subject to elongation to larger, more energy-dense products through reverse β-oxidation, and the resulting products are useful as precursors for liquid fuels production. This study examined the effect of several redox mediators (neutral red, methyl viologen, safranin O, tannic acid) as alternative electron carriers for mixed ruminal bacteria during the fermentation of biomass (ground switchgrass not subjected to other pretreatments) and their potential to enhance elongation of end-products to medium-chain VFAs with no additional run-time. Neutral red (1 mM) in particular facilitated chain elongation, increasing average VFA chain length from 2.42 to 2.97 carbon atoms per molecule, while simultaneously inhibiting methane accumulation by over half yet maintaining total C in end products. The ability of r...
Journal of Dairy Science, 2010
The influence of pH dynamics on ruminal bacterial community composition was studied in 8 ruminall... more The influence of pH dynamics on ruminal bacterial community composition was studied in 8 ruminally cannulated Holstein cows fitted with indwelling electrodes that recorded pH at 10-min intervals over a 54-h period. Cows were fed a silage-based total mixed ration supplemented with monensin. Ruminal samples were collected each day just before feeding and at 3 and 6 h after feeding. Solid and liquid phases were separated at collection, and extracted DNA was subjected to PCR amplification followed by automated ribosomal intergenic spacer analysis (ARISA). Although cows displayed widely different pH profiles (mean pH = 6.11 to 6.51, diurnal pH range = 0.45 to 1.39), correspondence analysis of the ARISA profiles revealed that 6 of the 8 cows showed very similar bacterial community compositions. The 2 cows having substantially different community compositions had intermediate mean pH values (6.30 and 6.33) and intermediate diurnal pH ranges (averaging 0.89 and 0.81 pH units). Fortuitously, these 2 cows alone also displayed milk fat depression, along with markedly higher ruminal populations of 1 bacterial operational taxonomic unit (OTU) and reduced populations of another ARISA amplicon. Cloning and sequencing of the elevated OTU revealed phylogenetic similarity to Megasphaera elsdenii, a species reportedly associated with milk fat depression. The higher populations of both M. elsdenii and OTU246 in these 2 cows were confirmed using quantitative real-time PCR (qPCR) with species-specific primers, and the fraction of total bacterial rDNA copies contributed by these 2 taxa were very highly correlated within individual cows. By contrast, the fraction of total bacterial rDNA copies contributed by Streptococcus bovis and genus Ruminococcus, 2 taxa expected to respond to ruminal pH, did not differ among cows (mean = <0.01 and 10.6%, respectively, of rRNA gene copies, determined by qPCR). The data indicate that cows with widely differing pH profiles can have similar ruminal bacterial community compositions, and that milk fat depression can occur at intermediate ruminal pH. The results support recent reports that milk fat depression is associated with shifts in bacterial community composition in rumine and is specifically related to the relative abundance of Megasphaera elsdenii.
When glucose or cellobiose was provided as an energy source for Fibrobacter succinogenes, there w... more When glucose or cellobiose was provided as an energy source for Fibrobacter succinogenes, there was a transient accumulation (as much as 0.4 mM hexose equivalent) of cellobiose or cellotriose, respectively, in the growth medium. Nongrowing cell suspensions converted cellobiose to cellotriose and longer-chain cellodex- trins, and in this case the total cellodextrin concentration was as much as 20 mM (hexose equivalent). Because cell extracts of glucose- or cellobiose-grown cells cleaved cellobiose and cellotriose by phosphate-dependent reactions and glucose 1-phosphate was an end product, it appeared that cellodextrins were being produced by a reversible phosphorylase reaction. This conclusion was supported by the observation that the ratio of cellodextrins to cellodextrins with one greater hexose (n/(n 11)) was approximately 4, a value similar to the equilibrium constant (Keq) of cellobiose phosphorylase (J. K. Alexander, J. Bacteriol. 81:903-910, 1961). When F. succinogeneswas grow...
Journal of Dairy Science, 1999
The effects of four contrasting diets were determined on populations of three species of ruminal ... more The effects of four contrasting diets were determined on populations of three species of ruminal cellulolytic bacteria (Ruminococcus albus, Ruminococcus flavefaciens, and Fibrobacter succinogenes) using oligonucleotide probes to rRNA. Diets based on alfalfa silage or corn silage as the primary fiber source were formulated to contain either 24 or 32% neutral detergent fiber measured after a-amylase treatment. The diets were fed twice daily to four ruminally fistulated, lactating Holstein cows in a trial using a Latin square design. The cows fed the alfalfa silage diets had higher dry matter intakes and milk production and smaller pH fluctuations than did cows fed the corn silage diets (0.3 vs. 0.8 units). The total populations of the three cellulolytic species at 3 h after feeding ranged from 0.3 to 3.9% of the bacterial domain; R. albus was generally the most abundant of the three species. The data are in general agreement with population assessments obtained by some traditional methods of culture enumeration. Although diet and individual cows had major effects on ruminal pH and volatile fatty acid concentrations and on milk production and composition, differences in cellulolytic populations that were attributable to individual cows were larger than those attributable to diet, suggesting that each cow maintained a unique assemblage of cellulolytic species.
The characterization of microbial communities by metagenomic approaches has been enhanced by rece... more The characterization of microbial communities by metagenomic approaches has been enhanced by recent improvements in short-read sequencing efficiency and assembly algorithms. We describe the results of adding long-read sequencing to the mix of technologies used to assemble a highly complex cattle rumen microbial community, and compare the assembly to current short read-based methods applied to the same sample. Contigs in the long-read assembly were 7-fold longer on average, and contained 7-fold more complete open reading frames (ORF), than the short read assembly, despite having three-fold lower sequence depth. The linkages between long-read contigs, provided by proximity ligation data, supported identification of 188 novel viral-host associations in the rumen microbial community that suggest cross-species infectivity of specific viral strains. The improved contiguity of the long-read assembly also identified 94 antimicrobial resistance genes, compared to only seven alleles identifie...
Frontiers in Microbiology
This experiment aimed to determine the effects of camelina seed (CS) supplementation at different... more This experiment aimed to determine the effects of camelina seed (CS) supplementation at different dietary fat levels on ruminal bacterial community composition and how it relates to changes in ruminal fermentation in a dual-flow continuous culture system. Diets were randomly assigned to 8 fermenters (1,200-1,250 mL) in a 2 × 2 factorial arrangement of treatments in a replicated 4 × 4 Latin square with four 10-day experimental periods that consisted of 7 days for diet adaptation and 3 days for sample collection. Treatments were: (1) no CS at 5% ether extract (EE, NCS5); (2) no CS at 8% EE (NCS8); (3) 7.7% CS at 5% EE (CS5); and (4) 17.7% CS at 8% EE (CS8). Megalac was used as a control to adjust EE levels. Diets contained 55% orchardgrass hay and 45% concentrate, and fermenters were equally fed a total of 72 g/day (DM basis) twice daily. The bacterial community was determined by sequencing the V4 region of the 16S rRNA gene using the Illumina MiSeq platform. Sequencing data were analyzed using mothur and statistical analyses were performed in R and SAS. The most abundant phyla across treatments were the Bacteroidetes and Firmicutes, accounting for 49 and 39% of the total sequences, respectively. The bacterial community composition in both liquid and solid fractions of the effluent digesta changed with CS supplementation but not by dietary EE. Including CS in the diets decreased the relative abundances of Ruminococcus spp., Fibrobacter spp., and Butyrivibrio spp. The most abundant genus across treatments, Prevotella, was reduced by high dietary EE levels, while Megasphaera and Succinivibrio were increased by CS supplementation in the liquid fraction. Correlatively, the concentration of acetate was decreased while propionate increased; C18:0 was decreased and polyunsaturated fatty acids, especially C18:2 n-6 and C18:3 n-3, were increased by CS supplementation. Based on the correlation analysis between genera and fermentation end products, this study revealed that CS supplementation could be energetically beneficial to dairy cows by increasing propionate-producing bacteria and suppressing ruminal bacteria associated with biohydrogenation. However, attention should be given to avoid the effects of CS supplementation on suppressing cellulolytic bacteria.
Applied and Environmental Microbiology
Gastrointestinal tract (GIT) microorganisms play important roles in the health of ruminant livest... more Gastrointestinal tract (GIT) microorganisms play important roles in the health of ruminant livestock and affect the production of agriculturally relevant products, including milk and meat. Despite this link, interventions to alter the adult microbiota to improve production have proven ineffective, as established microbial communities are resilient to change. In contrast, developing communities in young animals may be more easily altered but are less well studied. Here, we measured the GIT-associated microbiota of 45 Holstein dairy cows from 2 weeks to the first lactation cycle, using Illumina amplicon sequencing of bacterial (16S rRNA V4), archaeal (16S rRNA V6 to V8), and fungal (internal transcribed region 1 [ITS1]) communities. Fecal and ruminal microbiota of cows raised on calf starter grains and/or corn silage were correlated to lifetime growth as well as milk production during the first lactation cycle, in order to determine whether early-life diets have long-term impacts. Sig...
Microbiology
The anaerobic fermentation of glucose by Bacillus macerans ATCC 7068 was studied in batch culture... more The anaerobic fermentation of glucose by Bacillus macerans ATCC 7068 was studied in batch culture with and without pH control. The fermentation was characterized by two distinct metabolic phases. In the primary growth phase, the concentrations of ethanol and acetic acid increased exponentially, and formate was detected as a minor product. The secondary phase was marked by a slowing and eventual cessation of growth, along with the disappearance of formate and acetate, and the appearance of H2, C02, and acetone. Exogenously-added substrates were converted with stoichiometries of 1 formate-+ 1 H2 + 1 C o t , and approximately 2 acetate + 1 acetone. Consumption of > 8 g glucose 1required exogenous pH control, and acetone formation was strongly pH-dependent. Glucose was fermented by the Embden-Meyerhof pathway. Cell extracts contained pyruvate formate-lyase and formate dehydrogenase activities, but only low pyruvate dehydrogenase activity. A balanced fermentation pathway is presented which is consistent with reaction stoichiometries and [ 14C]glucose labelling data in whole cultures, and with enzyme activities in extracts. The pathway is compared with those of other facultative anaerobes and the acetone-producing Clostridium acetobutylicum. METHODS Materials. All chemicals were reagent grade, obtained from either Sigma or Fisher (Fair Lawn, N.J., U.S.A.). Enzymes used for coupled enzymic assays were obtained from Sigma. Anaerobic grade N 2 was purchased from Union Carbide-Linde Division (Somerset, N .J., U.S.A.). [ l J C ] G l u c~~e specifically labelled in the C-1, C-3,4 or C-6 positions was obtained from New England Nuclear. Organism and growth conditions. Bacillus nracerans ATCC 7068 was obtained from the American Type Culture Collection. According to Gordon etal. (1973), this strain is identical to 'B. acetoethylicus'described by Northrop et a/. (1919). The organism was grown in CM5 medium which contained (I-'
Anaerobe
For the growth of several strictly anaerobic microorganisms, cysteine is the preferred reducing a... more For the growth of several strictly anaerobic microorganisms, cysteine is the preferred reducing agent because of its low toxicity, but cysteine reduces media only slowly, making it inconvenient if the medium is needed immediately. A culture medium employed to cultivate ruminal anaerobic bacteria supplied with cysteine achieved the desired degree of anaerobiosis (measured spectrophotometrically by the bleaching of the indicator, resazurin) in considerably less time when exposed to high light intensity from a halogen lamp. The effect of light intensity on time to reduce media was quadratic; tubes kept under no illumination (dark) took more than 12 h to become colorless while tubes kept under normal laboratory light, took more than 2 h. On the other hand, media exposed to high light intensity (equivalent to a regular 100 W bulb lamp) became completely bleached in less than 20 min. Separate experiments demonstrated a direct correlation between the photocatalytic effect on medium reduction and a decrease in dissolved O 2 content. However, media gassing with CO 2 is still required, as the photocatalytic reaction requires semi-reduced conditions. A comparison of growth parameters for five species of ruminal bacteria cultivated in media prepared under high light vs normal light conditions showed no differences, indicating no adverse affect of cysteine catalysed medium reduction on growth. Aside from this benefit, other procedures such as extensive heating, cooling, evacuation and degassing with O 2-free gases can be eliminated, thereby further reducing the time required for media preparation. Thus, use of high light intensity on cysteine/resazurin containing media greatly accelerated the establishment of anaerobic conditions, so that the media can be used almost immediately.
Applied Microbiology and Biotechnology