Our research involves the microbiology of fiber digestion by the anaerobic microflora of the rumen. The rumen is an ideal ecosystem for studying metabolic interactions owing to the wide physiological and biochemical diversity of its many microbial species. Our group is interested in elucidating strategies for cellulosic biomass conversion in the rumen as a model system for understanding "consolidated bioprocessing" (CBP) schemes that employ anaerobic bacteria to carry out all steps of the conversion (cellulase synthesis, microbial cell growth and end product formation) in the same bioreactor; CBP is the lead candidate platform for second-generation production of fuels and chemicals from cellulosic biomass. In addition, we are determining bacterial community composition and dynamics in cows under different feeding and management conditions, with the goal of establishing more favorable species mixtures to improve animal production.
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. 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 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. Several patterns of gene expression were observed for the major cellulases produced by 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. This work emphasizes the complexity of the fiber-degrading system utilized by 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.
Gastrointestinal tract (GIT) microorganisms play important roles in the health of ruminant livestock and impact 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 first lactation using Illumina amplicon sequencing of bacterial (V4 16S), archaeal (V6-8 16S), and fungal (ITS1) communities. Fecal and rumen 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 if early-life diets have long-term impacts. Significant diet-associated differences in total microbial communities and specific taxa were observed by weaning (8 weeks), but all animals reached an adult-like composition between weaning and 1-year. While some calf diet-driven differences were apparent in the microbiota of adult cows, these dissimilarities did not correlate with animal growth or milk production. This suggests that initial microbial community establishment is impacted by early-life diet, but post-weaning factors have a greater influence on adult communities and production outcomes. The gut microbiota is essential to the survival of many organisms, including ruminants that rely on microorganisms for nutrient acquisition from dietary inputs for the production of products like milk and meat. While alteration of the adult ruminant microbiota to improve production is possible, changes are often unstable and fail to persist. In contrast, the early-life microbiota may be more amenable to sustained modification. However, few studies have determined the impacts of early-life interventions on downstream production. Here, we investigated the impacts of agriculturally relevant calf diets, including calf starter and corn silage, on gut microbial communities, growth, and production through the first lactation cycle. Thus, this work serves to further our understanding of early-life microbiota acquisition as well as informs future practices in livestock management.
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 and , 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 spp., spp., and spp. The most abundant genus across treatments, , was reduced by high dietary EE levels, while and 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.
The rumen is a large bioreactor that enables dairy cattle to derive nutrition from otherwise indigestible plant polymers and compounds. Despite the direct contribution of the rumen's microbial community toward the nutrition of the dairy cow, only a general knowledge has been gained of the metabolic processes within the rumen, and less still is known about most of the individual microbial species that colonize the organ. What has been discovered is that the rumen contains a diverse community of microbial species from all of the major domains of life, and that the contents of the rumen can vary greatly among individual animals. Preliminary evidence also indicates that rumen microbial profiles are heritable and sustainable within an individual, and that rumen microbial community structure can revert to its original profile within a short period following substantial perturbation. Much progress has been made in recent years to identify the diversity of microbial species in the rumen; however, the most popular methods used to identify microbial species often lack the predictive power necessary to associate particular microbial profiles with rumen metabolic activity. This represents the most significant barrier to the design of models that can estimate the direct effects of rumen microbial content on downstream dairy production traits. If such challenges can be overcome, it is possible that rumen microbial content could be assessed as a new phenotypic trait in cattle. In the future, we may estimate dairy production using a "genotype × environment × microbial" interaction model that accurately combines all factors affecting milk production.
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 -8, -7, -5, -4, -1, 1, 2, 3, 7, 10, 14, 21, 28, 35, 42, and 56, relative to the exchange date. High-MPE cows had higher total ruminal VFA concentrations, higher molar percentages of propionate and valerate, and lower molar percentages of acetate and butyrate than did LE cows, and re-established these differences 1 d after contents exchange. Across all LE cows, MPE increased during 7 d postexchange but declined thereafter. Two of the 3 HE cows displayed decreases in MPE following introduction of the ruminal contents from the corresponding LE cow, but MPE increased in the third HE cow, which was determined to be an outlier. For all 6 cows, both liquid- and solids-associated BCC differed between individuals within a pair before contents exchange. Upon exchange, BCC of both phases in all 3 pairs was more similar to that of the donor inoculum than to preexchange host BCC. For 5 of 6 cows, the solids-associated community returned within 10 d to more resemble the preexchange community of that host than that of the donor community. Individual variability before the exchange was greater in liquids than in solids, as was the variability in response of bacterial communities to the exchange. Individual cows varied in their response, but generally moved toward re-establishment of their preexchange communities by 10 d after contents exchange. By contrast, ruminal pH and VFA profiles returned to preexchange levels within 1 d. Despite the small number of cows studied, the data suggest an apparent role for the ruminal bacterial community as a determinant of MPE.
Hemicelluloses are major components of plant biomass, but their fermentation in the rumens of cattle and other ruminants is poorly understood. We compared four species of the ruminally dominant genus Prevotella and the well-known hemicellulose utilizer, Butyrivibrio fibrisolvens, with respect to degradation of several isolated hemicelluloses (xylans, glucomannan, and xyloglucan). We also performed Illumina sequencing of the V3/V4 region of 16S rRNA genes to determine the relative proportions of Prevotella and Butyrivibrio in hemicellulose-fed enrichment cultures inoculated from ruminal contents of dairy cattle fed a total mixed ration (TMR) rich in hemicelluloses. Results confirmed the xylan fermentation and butyrate production abilities of B. fibrisolvens. Despite their reputation as generalist fermenters, the Prevotella strains poorly fermented these hemicelluloses but exhibited dramatic differences in fermentation end products. Prevotella was much less abundant in mixed bacterial enrichment cultures fed the same TMR than in the ruminal inoculum, yet Prevotella was again the most abundant genus in enrichment cultures fed xylans. By contrast, glucomannan fermentations were dominated by Streptococcus sp. Genera known for hemicellulose degradation (Butyrivibrio, Ruminococcus, and Fibrobacter) were not significantly enriched on these hemicelluloses. Substantial differences in fermentation end product distribution from the different hemicelluloses were observed, which would likely affect nutrient partitioning in the host animal. Differences in community composition between in vitro hemicellulose enrichments and inoculum samples emerged at every phylogenetic level, suggesting that in vitro conditions provide unique selective pressures on the bacterial community and also that ruminal bacteria exhibit specialization with respect to hemicellulose utilization.
To assess the effect of two additives on alfalfa silage and on in vitro ruminal fermentation when using ruminal inocula from high feed-efficient (HE) and low feed-efficient (LE) lactating cows. First- and second-cut alfalfa was harvested at 40% bloom stage, treated with control (no additive), Lactobacillus plantarum (LP) or formic acid (Formic), ensiled in 1·0 l minisilos, and fermented for 60 days. Fermented alfalfa was incubated in vitro for 24 h using ruminal inoculum from HE and LE lactating cows. The pH was lower in alfalfa silage treated with LP and Formic, and produced lower ammonia-N than did the control. In vitro true dry matter digestibility (IVTDMD) was higher with ruminal inoculum from HE than LE cows, but there was no consistent effect of treated alfalfa on microbial biomass yield and in vitro volatile fatty acids. The IVTDMD was numerically greater with ruminal inoculum from higher feed-efficient cows although statistical significance was only demonstrated with the first-cut alfalfa. However, treated alfalfa silage did not show the effect expected on in vitro microbial biomass yield. The feed efficiency of cows used as a source of ruminal inocula may affect IVTDMD and be a source of variation across in vitro runs. Differences in ruminal fermentation between cows of different feed efficiency could help to explain differences in milk yield and other parameters of dairy cattle performance.
No abstract available.
The ruminal microbiome rapidly converts plant biomass to short-chain fatty acids (SCFA) that nourish the ruminant animal host. Because of its high species diversity, functional redundancy, and ease of extraruminal cultivation, this mixed microbial community is a particularly accomplished practitioner of the carboxylate platform for producing fuels and chemical precursors. Unlike reactor microbiomes derived from anaerobic digesters or sediments, the ruminal community naturally produces high concentrations of SCFA, with only modest methane production owing to the absence of both proton-reducing acetogens and aceticlastic methanogens. The extraruminal fermentation can be improved by addition of ethanol or lactate product streams, particularly in concert with reverse β-oxidizing bacteria (e.g., Clostridium kluyveri or Megasphaera elsdenii) that facilitate production of valeric and caproic acids. Application of fundamental principles of thermodynamics allows identification of optimal conditions for SCFA chain elongation, as well as discovery of novel synthetic capabilities (e.g., medium-chain alcohol and alkane production) by this mixed culture system.
Megasphaera elsdenii is a lactate-utilizing bacterium whose ruminal abundance has been shown to be greatly elevated during milk fat depression (MFD). To further examine this association, a total of 23 cannulated multiparous Holstein cows were examined in 3 experiments in which strains of M. elsdenii were directly dosed into the rumen (~2 × 10(12) cells/dose); control cows were dosed with sterile lactate-free culture medium. Cows were fed a total mixed ration (292 g of starch/kg of dry matter) that contained primarily corn silage, alfalfa silage, finely ground high-moisture corn, supplemental protein, and corn oil (3 g/kg of dry matter). Experiments differed in stage of lactation of the cows (early or late), dosing events (single dose, or 4 doses over a 5-d period), timing of dose (prefeed or 4 h postfeed), and M. elsdenii strain (laboratory strain YI9 or 3 strains isolated from cows in the same herd). Dry matter intake and milk yield and composition were measured from 5 to 0 d before dosing and 1 to 7d after first dosing, plus later time points that varied by experiment. Milk yield and composition were not affected by dosing. Megasphaera elsdenii was quantified in the liquid phase of ruminal contents by automated ribosomal intergenic spacer analysis, or by PCR with relative quantification (M. elsdenii 16S rRNA gene copy number as a percentage of total bacterial 16S rRNA gene copies). Neither the M. elsdenii-dosed or control cows displayed MFD after dosing, and in almost all cases M. elsdenii populations returned to low baseline levels (<0.02% of 16S rRNA gene copy number) within 24 h of dosing. This rapid decline in M. elsdenii also occurred in several cows that were dosed with a strain of M. elsdenii that had been isolated from that particular cow during a previous bout of MFD. Ruminal pH and total millimolar volatile fatty acids and lactate did not differ between dosed and control cows, although acetate-to-propionate ratio declined in both groups and butyrate increased after dosing with M. elsdenii. The results confirm that establishing exogenously added bacterial strains in the rumen is difficult, even for strains previously isolated from the recipient cow. The potential role of M. elsdenii as an agent of MFD remains unclear in the absence of successful establishment of the dosed strains.
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 redox dyes to act as alternative electron carriers suggests that ruminal fermentation is inherently manipulable toward retaining a higher fraction of substrate energy in the form of VFA.
Fructans are an important nonfiber carbohydrate in cool season grasses. Their fermentation by ruminal microbes is not well described, though such information is needed to understand their nutritional value to ruminants. Our objective was to compare kinetics and product formation of orchardgrass fructan (phlein; PHL) to other nonfiber carbohydrates when fermented in vitro with mixed or pure culture ruminal microbes. Studies were carried out as randomized complete block designs. All rates given are first-order rate constants. With mixed ruminal microbes, rate of substrate disappearance tended to be greater for glucose (GLC) than for PHL and chicory fructan (inulin; INU), which tended to differ from each other (0.74, 0.62, and 0.33 h(-1), respectively). Disappearance of GLC had almost no lag time (0.04 h), whereas the fructans had lags of 1.4h. The maximum microbial N accumulation, a proxy for cell growth, tended to be 20% greater for PHL and INU than for GLC. The N accumulation rate for GLC (1.31h(-1)) was greater than for PHL (0.75 h(-1)) and INU (0.26 h(-1)), which also differed. More microbial glycogen (+57%) was accumulated from GLC than from PHL, though accumulation rates did not differ (1.95 and 1.44 h(-1), respectively); little glycogen accumulated from INU. Rates of organic acid formation were 0.80, 0.28, and 0.80 h(-1) for GLC, INU, and PHL, respectively, with PHL tending to be greater than INU. Lactic acid production was more than 7-fold greater for GLC than for the fructans. The ratio of microbial cell carbon to organic acid carbon tended to be greater for PHL (0.90) and INU (0.86) than for GLC (0.69), indicating a greater yield of cell mass per amount of substrate fermented with fructans. Reduced microbial yield for GLC may relate to the greater glycogen production that requires ATP, and lactate production that yields less ATP; together, these processes could have reduced ATP available for cell growth. Acetate molar proportion was less for GLC than for fructans, and less for PHL than for INU. In studies with pure cultures, all microbes evaluated showed differences in specific growth rate constants (μ) for GLC, fructose, sucrose, maltose, and PHL. Selenomonas ruminantium and Streptococcus bovis showed the highest μ for PHL (0.55 and 0.67 h(-1), respectively), which were 50 to 60% of the μ achieved for GLC. The 10 other species tested had μ between 0.01 and 0.11h(-1) with PHL. Ruminal microbes use PHL differently than they do GLC or INU.
Symbiotic microbial communities are critical to the function and survival of animals. This relationship is obligatory for herbivores that engage gut microorganisms for the conversion of dietary plant materials into nutrients such as short-chain organic acids (SCOAs). The constraint on body size imposed by their arboreal lifestyle is thought to make this symbiosis especially important for sloths. Here, we use next-generation sequencing to identify the bacteria present in the fore and distal guts of wild two- and three-toed sloths, and correlate these communities with both diet and SCOAs. We show that, unlike other mammalian herbivores, sloth gut communities are dominated by the bacterial phyla Proteobacteria and Firmicutes. Specifically, three-toed sloths possess a highly conserved, low-diversity foregut community with a highly abundant Neisseria species associated with foregut lactate. In contrast, two-toed sloths have a more variable and diverse foregut microbiota correlated with a variety of SCOAs. These differences support the hypothesis that feeding behaviour selects for specific gut bacterial communities, as three-toed sloths subsist primarily on Cecropia tree leaves while two-toed sloths have a more generalist diet. The less diverse diet and gut microbiota of three-toed sloths may render them more susceptible to habitat loss and other diet-altering conditions.
Fourteen Holstein cows of similar ages were monitored through their first two lactation cycles, during which ruminal solids and liquids, milk samples, production data, and feed consumption data were collected for each cow during early (76 to 82 days in milk [DIM]), middle (151 to 157 DIM), and late (251 to 257 DIM) lactation periods. The bacterial community of each ruminal sample was determined by sequencing the region from V6 to V8 of the 16S rRNA gene using 454 pyrosequencing. Gross feed efficiency (GFE) for each cow was calculated by dividing her energy-corrected milk by dry matter intake (ECM/DMI) for each period of both lactation cycles. Four pairs of cows were identified that differed in milk production efficiency, as defined by residual feed intake (RFI), at the same level of ECM production. The most abundant phyla detected for all cows were Bacteroidetes (49.42%), Firmicutes (39.32%), Proteobacteria (5.67%), and Tenericutes (2.17%), and the most abundant genera included Prevotella (40.15%), Butyrivibrio (2.38%), Ruminococcus (2.35%), Coprococcus (2.29%), and Succiniclasticum (2.28%). The bacterial microbiota between the first and second lactation cycles were highly similar, but with a significant correlation between total community composition by ruminal phase and specific bacteria whose relative sequence abundances displayed significant positive or negative correlation with GFE or RFI. These data suggest that the ruminal bacterial community is dynamic in terms of membership and diversity and that specific members are associated with high and low milk production efficiency over two lactation cycles.
The ruminal microbial community is remarkably diverse, containing 100s of different bacterial and archaeal species, plus many species of fungi and protozoa. Molecular studies have identified a "core microbiome" dominated by phyla Firmicutes and Bacteroidetes, but also containing many other taxa. The rumen provides an ideal laboratory for studies on microbial ecology and the demonstration of ecological principles. In particular, the microbial community demonstrates both redundancy (overlap of function among multiple species) and resilience (resistance to, and capacity to recover from, perturbation). These twin properties provide remarkable stability that maintains digestive function for the host across a range of feeding and management conditions, but they also provide a challenge to engineering the rumen for improved function (e.g., improved fiber utilization or decreased methane production). Direct ruminal dosing or feeding of probiotic strains often fails to establish the added strains, due to intensive competition and amensalism from the indigenous residents that are well-adapted to the historical conditions within each rumen. Known exceptions include introduced strains that can fill otherwise unoccupied niches, as in the case of specialist bacteria that degrade phytotoxins such as mimosine or fluoroacetate. An additional complicating factor in manipulating the ruminal fermentation is the individuality or host specificity of the microbiota, in which individual animals contain a particular community whose species composition is capable of reconstituting itself, even following a near-total exchange of ruminal contents from another herd mate maintained on the same diet. Elucidation of the interactions between the microbial community and the individual host that establish and maintain this specificity may provide insights into why individual hosts vary in production metrics (e.g., feed efficiency or milk fat synthesis), and how to improve herd performance.
Mixed bacterial communities from the rumen ferment cellulosic biomass primarily to C2-C4 volatile fatty acids, and perform only limited chain extension to produce C5 (valeric) and C6 (caproic) acids. The aim of this study was to increase production of caproate and valerate in short-term in vitro incubations. Co-culture of mixed ruminal microbes with a rumen-derived strain of the bacterium Clostridium kluyveri converted cellulosic biomass (alfalfa stems or switchgrass herbage) plus ethanol to VFA mixtures that include valeric and caproic acids as the major fermentation products over a 48-72h run time. Concentrations of caproate reached 6.1gL(-1), similar to or greater than those reported in most conventional carboxylate fermentations that employ substantially longer run times.
Bacteria in the genus Ruminococcus are ubiquitous members of the mammalian gastrointestinal tract. In particular, they are important in ruminants where they digest a wide range of plant cell wall polysaccharides. For example, Ruminococcus albus 7 is a primary cellulose degrader that produces acetate usable by its bovine host. Moreover, it is one of the few organisms that ferments cellulose to form ethanol at mesophilic temperatures in vitro. The mechanism of cellulose degradation by R. albus 7 is not well-defined and is thought to involve pilin-like proteins, unique carbohydrate-binding domains, a glycocalyx, and cellulosomes. Here, we used a combination of comparative genomics, fermentation analyses, and transcriptomics to further clarify the cellulolytic and fermentative potential of R. albus 7. A comparison of the R. albus 7 genome sequence against the genome sequences of related bacteria that either encode or do not encode cellulosomes revealed that R. albus 7 does not encode for most canonical cellulosomal components. Fermentation analysis of R. albus 7 revealed the ability to produce ethanol and acetate on a wide range of fibrous substrates in vitro. Global transcriptomic analysis of R. albus 7 grown at identical dilution rates on cellulose and cellobiose in a chemostat showed that this bacterium, when growing on cellulose, utilizes a carbohydrate-degrading strategy that involves increased transcription of the rare carbohydrate-binding module (CBM) family 37 domain and the tryptophan biosynthetic operon. Our data suggest that R. albus 7 does not use canonical cellulosomal components to degrade cellulose, but rather up-regulates the expression of CBM37-containing enzymes and tryptophan biosynthesis. This study contributes to a revised model of carbohydrate degradation by this key member of the rumen ecosystem.
The rich and diverse microbiota of the rumen provides ruminant animals the capacity to utilize highly fibrous feedstuffs as their energy source, but there is surprisingly little information on the composition of the microbiome of ruminants fed all-forage diets, despite the importance of such agricultural production systems worldwide. In three 28-day periods, three ruminally-cannulated Holstein heifers sequentially grazed orchardgrass pasture (OP), then were fed orchardgrass hay (OH), then returned to OP. These heifers displayed greater shifts in ruminal bacterial community composition (determined by automated ribosomal intergenic spacer analysis and by pyrotag sequencing of 16S rRNA genes) than did two other heifers maintained 84 d on the same OP. Phyla Firmicutes and Bacteroidetes dominated all ruminal samples, and quantitative PCR indicated that members of the genus Prevotella averaged 23% of the 16S rRNA gene copies, well below levels previously reported with cows fed total mixed rations. Differences in bacterial community composition and ruminal volatile fatty acid (VFA) profiles were observed between the OP and OH despite similarities in gross chemical composition. Compared to OP, feeding OH increased the molar proportion of ruminal acetate (P = 0.02) and decreased the proportion of ruminal butyrate (P < 0.01), branched-chain VFA (P < 0.01) and the relative population size of the abundant genus Butyrivibrio (P < 0.001), as determined by pyrotag sequencing. Despite the low numbers of animals examined, the observed changes in VFA profile in the rumens of heifers on OP vs. OH are consistent with the shifts in Butyrivibrio abundance and its known physiology as a butyrate producer that ferments both carbohydrates and proteins.
The concept of co-production of liquid fuel (ethanol) along with animal feed on farm was proposed, and the strategy of using ambient-temperature acid pretreatment, ensiling and washing to improve ethanol production from alfalfa stems was investigated. Alfalfa stems were separated and pretreated with sulfuric acid at ambient-temperature after harvest, and following ensiling, after which the ensiled stems were subjected to simultaneous saccharification and fermentation (SSF) for ethanol production. Ethanol yield was improved by ambient-temperature sulfuric acid pretreatment before ensiling, and by washing before SSF. It was theorized that the acid pretreatment at ambient temperature partially degraded hemicellulose, and altered cell wall structure, resulted in improved cellulose accessibility, whereas washing removed soluble ash in substrates which could inhibit the SSF. The pH of stored alfalfa stems can be used to predict the ethanol yield, with a correlation coefficient of +0.83 for washed alfalfa stems.
Arboreal herbivory is rare among mammals. The few species with this lifestyle possess unique adaptions to overcome size-related constraints on nutritional energetics. Sloths are folivores that spend most of their time resting or eating in the forest canopy. A three-toed sloth will, however, descend its tree weekly to defecate, which is risky, energetically costly and, until now, inexplicable. We hypothesized that this behaviour sustains an ecosystem in the fur of sloths, which confers cryptic nutritional benefits to sloths. We found that the more specialized three-toed sloths harboured more phoretic moths, greater concentrations of inorganic nitrogen and higher algal biomass than the generalist two-toed sloths. Moth density was positively related to inorganic nitrogen concentration and algal biomass in the fur. We discovered that sloths consumed algae from their fur, which was highly digestible and lipid-rich. By descending a tree to defecate, sloths transport moths to their oviposition sites in sloth dung, which facilitates moth colonization of sloth fur. Moths are portals for nutrients, increasing nitrogen levels in sloth fur, which fuels algal growth. Sloths consume these algae-gardens, presumably to augment their limited diet. These linked mutualisms between moths, sloths and algae appear to aid the sloth in overcoming a highly constrained lifestyle.
Sixty samples of 'ForagePlus' oat were selected from a previous plot study for analysis of in vitro gas production (IVGP) on the basis of 2 factors: (1) high (n=29) or low (n=31) neutral detergent fiber (NDF; 62.7±2.61 and 45.1±3.91%, respectively); and (2) the range of water-soluble carbohydrates (WSC) within the high- and low-NDF groups. For the WSC selection factor, concentrations ranged from 4.7 to 13.4% (mean=7.9±2.06%) and from 3.5 to 19.4% (mean=9.7±4.57%) within high- and low-NDF forages, respectively. Our objectives were to assess the relationships between IVGP and various agronomic or nutritional characteristics for high- and low-NDF fall-oat forages. Cumulative IVGP was fitted to a single-pool nonlinear regression model: Y=MAX × (1 - e ([-)(K)(× (t - lag)])), where Y=cumulative gas produced (mL), MAX=maximum cumulative gas produced with infinite incubation time (mL), K=rate constant, t=incubation time (h), and lag=discrete lag time (h). Generally, cumulative IVGP after 12, 24, 36, or 48h within high-NDF fall-oat forages was negatively correlated with NDF, hemicellulose, lignin, and ash, but positively correlated with WSC, nonfiber carbohydrate (NFC), and total digestible nutrients (TDN). For low-NDF fall-grown oat forages, IVGP was positively correlated with growth stage, canopy height, WSC, NFC, and TDN; negative correlations were observed with ash and crude protein (CP) but not generally with fiber components. These responses were also reflected in multiple regression analysis for high- and low-NDF forages. After 12, 24, or 36h of incubation, cumulative IVGP within high-NDF fall-oat forages was explained by complex regression equations utilizing (lignin:NDF)(2), lignin:NDF, hemicellulose, lignin, and TDN(2) as independent variables (R(2)≥0.43). Within low-NDF fall-grown oat forages, cumulative IVGP at these incubation intervals was explained by positive linear relationships with NFC that also exhibited high coefficients of determination (R(2)≥0.75). Gas production was accelerated at early incubation times within low-NDF forages, specifically in response to large pools of WSC that were most likely to be present as forages approached boot stage by late-fall.
The effect of dietary nitrate supplementation on rumen bacterial community composition was examined in beef steers fed either a nitrate-N diet or urea-N diet. An automated method of ribosomal intergenic spacer analysis was applied to solid and liquid fractions of ruminal contents to allow comparison of bacterial communities. Supplemental N source affected relative population size of four amplicon lengths (ALs) in the liquid fraction and three ALs in the solid fraction. Five ALs were more prevalent after adaptation to nitrate. Correspondence analysis indicated that feeding the steers the nitrate-N diet versus urea-N diet changed the bacterial community composition in the liquid but not in the solid fraction. This led to an investigation of the relative sizes of potential nitrate-reducing populations. Mannheimia succiniciproducens, Veillonella parvula, and Campylobacter fetus were obtained from nitrate enrichment culture and quantified by real-time PCR based on 16S rRNA sequence. Nitrate supplementation increased the percentage of C. fetus in the liquid and solid phases, and in solid phase, the percentage of M. succiniciproducens increased. No change in species prevalence was observed for V. parvula. However, even after adaptation to dietary nitrate, the relative population sizes for all three putative nitrate-reducing species were very low (<0.06 % of 16S rRNA gene copy number). The liquid-associated bacterial community composition changed due to nitrate supplementation, and at least part of this change reflects an increase in the species prevalence of C. fetus, a species which is not typically regarded as a ruminal inhabitant.
"Green juice", obtained by squeezing fresh alfalfa leaves inoculated with lactic acid bacteria, was fermented at room temperature for 7-21 d to obtain 12-47 g lactic acid L(-1). Inoculation of green juice with Streptococcus bovis and incubation at 39°C reduced fermentation time to 8-12h. The resulting "brown juice" from either fermentation had a pH of ∼4.5 and a protein precipitate. Upon adjustment to pH 5.2-6.8 and inoculation with Megasphaera elsdenii, brown juice was fermented within 48 h to up to 18 g of mixed volatile fatty acids (VFA) L(-1). Single-stage fermentation of green juice by both species in coculture typically resulted in overgrowth of S. bovis and acid inhibition of M. elsdenii, inhibiting VFA production. Because the juice fermentations are conducted without sterilization or supplemental nutrients, they can potentially contribute to an integrated process featuring protein recovery and fermentation of fractionated solids to VFA and other products.
Megasphaera elsdenii T81 grew on either DL-lactate or D-glucose at similar rates (0.85 h(-1)) but displayed major differences in the fermentation of these substrates. Lactate was fermented at up to 210-mM concentration to yield acetic, propionic, butyric, and valeric acids. The bacterium was able to grow at much higher concentrations of D-glucose (500 mM), but never removed more than 80 mM of glucose from the medium, and nearly 60 % the glucose removed was sequestered as intracellular glycogen, with low yields of even-carbon acids (acetate, butyrate, caproate). In the presence of both substrates, glucose was not used until lactate was nearly exhausted, even by cells pregrown on glucose. Glucose-grown cultures maintained only low extracellular concentrations of acetate, and addition of exogenous acetate increased yields of butyrate, but not caproate. By contrast, exogenous acetate had little effect on lactate fermentation. At pH 6.6, growth rate was halved by exogenous addition of 60 mM propionate, 69 mM butyrate, 44 mM valerate, or 33 mM caproate; at pH 5.9, these values were reduced to 49, 49, 18, and 22 mM, respectively. The results are consistent with this species' role as an effective ruminal lactate consumer and suggest that this organism may be useful for industrial production of volatile fatty acids from lactate if product tolerance could be improved. The poor fermentation of glucose and sensitivity to caproate suggests that this strain is not practical for industrial caproate production.
Although clinical benefits of dietary fiber supplementation seem to depend partially on the extent of fiber degradation and fermentation by colonic bacteria, little is known about the effect of supplemental fiber type on bacterial metabolism. In an experiment using a nonadapted human bacterial population from three normal subjects, the extent of in vitro fermentation was greater for gum arabic (GA) than for psyllium (PSY), which was greater than that for carboxymethylcellulose (CMC). In a separate experiment, in vitro incubation with feces from 52 subjects with fecal incontinence, before and after random assignment to and consumption of one of three fiber (GA, PSY, or CMC) supplements or a placebo for 20-21 days, indicated that prior consumption of a specific fiber source did not increase its degradation by fecal bacteria. Results suggest that the colonic microbial community enriched on a particular fiber substrate can rapidly adapt to the presentation of a new fiber substrate. Clinical implications of the findings are that intake of a fiber source by humans is not expected to result in bacterial adaptation that would require continually larger and eventually intolerable amounts of fiber to achieve therapeutic benefits.
The purpose of this study was to investigate variability among individual cows in their severity of ruminal acidosis (RA) pre- and postpartum, and determine whether this variability was related to differences in their ruminal bacterial community composition (BCC). Variability in the severity of RA among individual cows was characterized based on ruminal fermentation variables. Effects of prepartum dietary treatment on the severity of RA were also examined. Fourteen Holstein heifers paired by expected calving date and BCS were allotted to 1 of 2 prepartum dietary treatments: low-concentrate or high-concentrate diets. All cows received the same lactation diet postpartum. Microbial DNA extracted from 58 ruminal digesta samples in total collected prepartum (d -50, -31, and -14; 27 samples) and postpartum (d +14 and +52; 31 samples) and amplified by PCR were subjected to automated ribosomal intergenic spacer analysis. Changes in ruminal variables over time [pH, volatile fatty acids (VFA), and acidosis indicators, including duration and area under the rumen pH curve below 5.8, 5.5, and 5.2, measured on d -54, -35, -14, -3, +3, +17, +37, and +58] were analyzed using principal components analysis. Based on the shift (defined as the distance of the mean loadings) between the prepartum and postpartum period for each cow, the 14 cows were classified into 3 groups: least acidotic (n=5), most acidotic (n=5), and intermediate (n=4). Cows in the most acidotic group had greater severity of RA (measured as duration of total RA, mild RA, moderate RA, and acute RA; area under the pH curve for total RA, mild RA, and moderate RA) postpartum than prepartum, and this difference between periods was greater than for the least acidotic cows. Similarly, the RA index (total area of pH <5.8 normalized to intake) showed an interaction between severity of RA and period. The variation in the severity of RA was independent of intake, total VFA concentration, and individual VFA proportions. Production variables (milk yield, fat percentage, fat yield, fat-corrected milk, and efficiency of milk production) were not influenced by the severity of RA. Ruminal BCC was not influenced by dietary treatment or period. However, some cows experienced greater shift in BCC than other cows across the periods. Based on the magnitude of the shift in BCC (distance between mean ordination values across the periods for each cow), cows were grouped into 3 BCC profile categories: stable (5 cows with lesser shift), unstable (5 cows with greater shift), and intermediate (4 cows with average shift). Cows demonstrating a greater shift in BCC were not necessarily those in the most acidotic group and vice versa. The shift in ruminal fermentation variables (principal components analysis rankings) and the shift in BCC (automated ribosomal intergenic spacer analysis rankings) between pre- and postpartum were not related (n=14; R(2)=0.00). It was concluded that not all cows are equally susceptible to RA and postpartum shifts in BCC appear to be independent of the differences in the severity of RA postpartum.
A strain of Clostridium kluyveri was isolated from the bovine rumen in a medium containing ethanol as an electron donor and acetate and succinate (common products of rumen fermentation) as electron acceptors. The isolate displayed a narrow substrate range but wide temperature and pH ranges atypical of ruminal bacteria and a maximum specific growth rate near the typical liquid dilution rate of the rumen. Quantitative real-time PCR revealed that C. kluyveri was widespread among bovine ruminal samples but was present at only very low levels (0.00002% to 0.0002% of bacterial 16S rRNA gene copy number). However, the species was present in much higher levels (0.26% of bacterial 16S rRNA gene copy number) in lucerne silage (but not maize silage) that comprised much of the cows' diet. While C. kluyveri may account for several observations regarding ethanol utilization and volatile fatty acid production in the rumen, its population size and growth characteristics suggest that it is not a significant contributor to ruminal metabolism in typical dairy cattle, although it may be a significant contributor to silage fermentation. The ability of unadapted cultures to produce substantial levels (12.8 g L(-1)) of caproic (hexanoic) acid in vitro suggests that this strain may have potential for industrial production of caproic acid.
Some silage inoculants help to improve silage quality and promote an increase in milk production, possibly through altering the rumen microflora. We hypothesized that rumen bacterial community composition (BCC) would be different in cows fed alfalfa ensiled with the inoculant Lactobacillus plantarum MTD/1 (LP) compared with those fed alfalfa ensiled without the inoculant (Ctrl). Eight ruminally cannulated Holstein cows were allotted to 2 diets (Ctrl or LP) in a double crossover design with four 28-d periods. Diets were formulated to contain (% dry matter basis) 28.0% neutral detergent fiber and 16.2% crude protein, and contained alfalfa silage, 50.9; corn silage, 20.6; high-moisture shelled corn, 21.4; soy hulls, 4.7; plus minerals and vitamins, 2.4. Ruminal digesta were collected just before feeding on 3 consecutive days near the end of each period, and were separated into solid and liquid phases. Microbial DNA was extracted from each phase, amplified by PCR using domain-level bacterial primers, and subjected to automated ribosomal intergenic spacer analysis. The pH was 4.56 and 4.86 and the lactate-to-acetate ratio 9.8 and 4.4, respectively, for the treated and untreated alfalfa silages. Dry matter intakes and milk production data were not influenced by diets but showed a cow effect. Total volatile fatty acids (mM) tended to be greater for LP compared with Ctrl. Individual volatile fatty acids were not influenced by diets but showed a significant cow effect. Ruminal acetate (mol/100 mol) and acetate-to-propionate ratio were lower and propionate (mol/100 mol) greater for the 2 milk fat-depressed (MFD; <3.2% fat content) cows compared with the other 6 cows. Correspondence analysis of the 265 peaks in the automated ribosomal intergenic spacer analysis profile across the 188 samples revealed that the first 2 components contributed 7.1 and 3.8% to the total variation in the profile. The ordination points representing the liquid and solid phases clustered separately, indicating that these phases differed in BCC. The analysis of similarity data showed differences between Ctrl and LP. The lactic acid bacterial counts (log(10) cfu/g of wet silage) were 3.94 and 4.53 for the untreated and treated silage, respectively, at ensiling. The relative population size (RPS) of L. plantarum, determined by real-time PCR of 16S rRNA gene copies, was greater in LP compared with Ctrl. The ordination points corresponding to certain individual cows clustered separately, and the most distinctive bacterial communities were those associated with MFD cows. The RPS of Megasphaera elsdenii was greater in 1 of the 2 MFD cows, although mean RPS of M. elsdenii did not differ between the treatments. In addition to the differences in rumen BCC between LP and Ctrl, MFD cows also displayed differences in BCC compared with cows with normal milk fat yield.
Ruminococcus albus 7 is a highly cellulolytic ruminal bacterium that is a member of the phylum Firmicutes. Here, we describe the complete genome of this microbe. This genome will be useful for rumen microbiology and cellulosome biology and in biofuel production, as one of its major fermentation products is ethanol.
Fibrobacter succinogenes is an important member of the rumen microbial community that converts plant biomass into nutrients usable by its host. This bacterium, which is also one of only two cultivated species in its phylum, is an efficient and prolific degrader of cellulose. Specifically, it has a particularly high activity against crystalline cellulose that requires close physical contact with this substrate. However, unlike other known cellulolytic microbes, it does not degrade cellulose using a cellulosome or by producing high extracellular titers of cellulase enzymes. To better understand the biology of F. succinogenes, we sequenced the genome of the type strain S85 to completion. A total of 3,085 open reading frames were predicted from its 3.84 Mbp genome. Analysis of sequences predicted to encode for carbohydrate-degrading enzymes revealed an unusually high number of genes that were classified into 49 different families of glycoside hydrolases, carbohydrate binding modules (CBMs), carbohydrate esterases, and polysaccharide lyases. Of the 31 identified cellulases, none contain CBMs in families 1, 2, and 3, typically associated with crystalline cellulose degradation. Polysaccharide hydrolysis and utilization assays showed that F. succinogenes was able to hydrolyze a number of polysaccharides, but could only utilize the hydrolytic products of cellulose. This suggests that F. succinogenes uses its array of hemicellulose-degrading enzymes to remove hemicelluloses to gain access to cellulose. This is reflected in its genome, as F. succinogenes lacks many of the genes necessary to transport and metabolize the hydrolytic products of non-cellulose polysaccharides. The F. succinogenes genome reveals a bacterium that specializes in cellulose as its sole energy source, and provides insight into a novel strategy for cellulose degradation.
"Extraruminal" fermentations employing in vitro incubation of mixed ruminal bacterial consortia, are capable of converting a complex array of biomass materials to mixtures of volatile fatty acids (VFA), methane, and carbon dioxide. Most of the potential energy in the biomass feedstock is retained in the VFA products, which are potential reactants for electrochemical conversion to hydrocarbon fuels. Quantitative data on VFA yields and proportions from biomass components are necessary for determining industrial feasibility, but such measurements have not been systematically reported. VFA yields and proportions were determined for a variety of carbohydrates, proteins and nucleic acids. Carbohydrates yielded primarily acetic and propionic acids, while proteins also yielded a more favorable product mix (longer average chain length and branched chain VFAs). Addition of certain co-substrates (e.g., glycerol) favorably improved the VFA product mix. The results have implications for hydrocarbon fuel generation from biomass materials by hybrid fermentation/chemical processes.
The purpose of this study was to examine the stability and host specificity of a cow's ruminal bacterial community following massive challenge with ruminal microflora from another cow. In each of 2 experiments, 1 pair of cows was selected on the basis of differences in ruminal bacterial community composition (BCC), determined by automated ribosomal intergenic spacer analysis (ARISA), a culture-independent "community fingerprinting" technique. Each pair of cows was then subjected to a 1-time exchange of >95% of ruminal contents without changing the composition of a corn silage/alfalfa haylage-based TMR. In experiment 1, the 2 cows differed (P<0.01) in prefeed ruminal pH (mean = 6.88 vs. 6.14) and prefeed total VFA concentration (mean = 57 vs. 77 mM), averaged over 3 d. Following exchange of ruminal contents, ruminal pH and total VFA concentration in both cows returned to their preexchange values within 24h. Ruminal BCC also returned to near its original profile, but this change required 14 d for 1 cow and 61 d for the other cow. In experiment 2, the 2 other cows differed in prefeed ruminal pH (mean = 6.69 vs. 6.20) and total VFA concentration (mean = 101 vs. 136 mM). Following exchange of ruminal contents, the first cow returned to its preexchange pH and VFA values within 24h; the second cow's rumen rapidly stabilized to a higher prefeed pH (mean = 6.47) and lower prefeed VFA concentration (mean = 120 mM) that was retained over the 62-d test period. Both cows reached somewhat different BCC than before the exchange. However, the BCC of both cows remained distinct and were ultimately more similar to that of the preexchange BCC than of the donor animal BCC. The data indicate that the host animal can quickly reestablish its characteristic ruminal pH and VFA concentration despite dramatic perturbation of its ruminal microbial community. The data also suggest that ruminal BCC displays substantial host specificity that can reestablish itself with varying success when challenged with a microbial community optimally adapted to ruminal conditions of a different host animal.
Herbivores can gain indirect access to recalcitrant carbon present in plant cell walls through symbiotic associations with lignocellulolytic microbes. A paradigmatic example is the leaf-cutter ant (Tribe: Attini), which uses fresh leaves to cultivate a fungus for food in specialized gardens. Using a combination of sugar composition analyses, metagenomics, and whole-genome sequencing, we reveal that the fungus garden microbiome of leaf-cutter ants is composed of a diverse community of bacteria with high plant biomass-degrading capacity. Comparison of this microbiome's predicted carbohydrate-degrading enzyme profile with other metagenomes shows closest similarity to the bovine rumen, indicating evolutionary convergence of plant biomass degrading potential between two important herbivorous animals. Genomic and physiological characterization of two dominant bacteria in the fungus garden microbiome provides evidence of their capacity to degrade cellulose. Given the recent interest in cellulosic biofuels, understanding how large-scale and rapid plant biomass degradation occurs in a highly evolved insect herbivore is of particular relevance for bioenergy.
Switchgrass (Panicum virgatum L.) and reed canarygrass (Phalaris arundinacea L.) were pretreated under ambient temperature and pressure with sulfuric acid and calcium hydroxide in separate experiments. Chemical loadings from 0 to 100g (kg DM)(-1) and durations of anaerobic storage from 0 to 180days were investigated by way of a central composite design at two moisture contents (40% or 60% w.b.). Pretreated and untreated samples were fermented to ethanol by Saccharomyces cerevisiae D5A in the presence of a commercially available cellulase (Celluclast 1.5L) and beta-glucosidase (Novozyme 188). Xylose levels were also measured following fermentation because xylose is not metabolized by S. cerevisiae. After sulfuric acid pretreatment and anaerobic storage, conversion of cell wall glucose to ethanol for reed canarygrass ranged from 22% to 83% whereas switchgrass conversions ranged from 16% to 46%. Pretreatment duration had a positive effect on conversion but was mitigated with increased chemical loadings. Conversions after calcium hydroxide pretreatment and anaerobic storage ranged from 21% to 55% and 18% to 54% for reed canarygrass and switchgrass, respectively. The efficacy of lime pretreatment was found to be highly dependent on moisture content. Moreover, pretreatment duration was only found to be significant for reed canarygrass. Although significant levels of acetate and lactate were observed in the biomass after storage, S. cerevisiae was not found to be inhibited at a 10% solids loading.
The performance of two pretreatment methods, sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL) and dilute acid (DA), was compared in pretreating softwood (spruce) for fuel ethanol production at 180 degrees Celsius for 30 min with a sulfuric acid loading of 5% on oven-dry wood and a 5:1 liquor-to-wood ratio. SPORL was supplemented with 9% sodium sulfite (w/w of wood). The recoveries of total saccharides (hexoses and pentoses) were 87.9% (SPORL) and 56.7% (DA), while those of cellulose were 92.5% (SPORL) and 77.7% (DA). The total of known inhibitors (furfural, 5-hydroxymethylfurfural, and formic, acetic and levulinic acids) formed in SPORL were only 35% of those formed in DA pretreatment. SPORL pretreatment dissolved approximately 32% of the lignin as lignosulfonate, which is a potential high-value co-product. With an enzyme loading of 15 FPU (filter paper units) per gram of cellulose, the cellulose-to-glucose conversion yields were 91% at 24h for the SPORL substrate and 55% at 48 h for the DA substrate, respectively.
The bovine rumen undergoes substantial changes in environmental conditions during the animal's feeding cycle, but the effects of these changes on microbial populations have not been examined systematically. Two dairy cows fed a mixed forage/concentrate ration at 12 h intervals over 4 feeding cycles displayed substantial changes in ruminal pH and volatile fatty acid (VFA) concentrations. Automated ribosomal intergenic spacer analysis (ARISA) of solid- and liquid-associated bacterial populations in samples collected at 2, 4, 6, 9, and 12 h after feeding revealed a high degree of bacterial diversity. A total of 155 different amplicon lengths (ALs) were detected across all 83 samples, and 11-74 detected per sample. A substantial proportion (11%) of the ALs was detected in one cow but not in the other. The proportions of ALs that were detected only in the liquid phase or the solid phase were 13.5% and 1.9%, respectively. Correspondence analysis indicated that bacterial community composition differed between cows and between solid or liquid phases, but overall the solid-associated population displayed less change in composition within and across feeding cycles. The data support the notion that cows fed the same diets can have substantial differences in bacterial community composition, and that the solids-associated (biofilm) communities display greater stability than do associated planktonic communities.
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 6h 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.
Eighteen ruminally cannulated dairy cattle were fed a series of diets (in 28-d periods) designed to elicit different degrees of milk fat depression (MFD) for the purpose of relating MFD to ruminal bacterial populations. Cows were fed a TMR containing 25% starch (DM basis) supplied as corn silage, a slowly fermented starch (SFS treatment, period 1), then switched to a TMR containing 27% starch, much of it supplied as ground high-moisture corn, a rapidly fermented starch (RFS treatment, period 2). In period 3, the RFS diet was amended with 13.6 mg of monensin/kg of DM (RFS/Mon treatment), and in period 4, the cows were returned to the RFS diet without monensin (RFS/Post treatment). Effect of both starch source and monensin on milk fat percentage varied by cow, and cluster analysis identified 4 pairs of cows having distinct milk fat patterns. Archived ruminal liquors and solids from the 4 pairs were processed to isolate bacterial DNA, which was subjected to automated ribosomal intergenic spacer analysis followed by correspondence analysis to visualize bacterial community composition (BCC). One pair of cows (S-responsive) showed MFD on RFS feeding, but displayed no additional MFD upon monensin feeding and a fat rebound upon monensin withdrawal. The second pair of cows (M-responsive) showed no MFD upon switch from the SFS diet to the RFS diet, but displayed strong MFD upon monensin feeding and no recovery after monensin withdrawal. Both groups displayed major shifts in BCC upon dietary shifts, including dietary shifts that both did and did not change milk fat production. The third pair of cows (SM-responsive) displayed reduction of milk fat on both RFS and RFS/Mon diets, and fat returned to the levels on the RFS diet upon monensin withdrawal; these cows showed a more gradual shift in BCC in response to both starch source and monensin. The fourth pair of cows (nonresponsive) did not display changes in milk fat percentage with dietary treatment and showed only minor shifts in BCC with dietary treatment. Regardless of milk fat response, BCC did not reassemble its original state upon monensin withdrawal, though the difference was strongest in M-responsive cows. One amplicon length (representing a single bacterial species) was elevated in most, but not all, MFD-susceptible (S-, M-, or SM-responsive) cows relative to milk fat-nonresponsive cows, whereas 2 amplicon lengths displayed reduced abundance under MFD conditions. Overall, this study demonstrates an association between MFD and wholesale shifts of microbial communities in the rumen.
Marginal croplands, such as those in the Conservation Reserve Program (CRP), have been suggested as a source of biomass for biofuel production. However, little is known about the composition of plant species on these conservation grasslands or their potential for ethanol production. Our objective was to assess the potential of CRP and other conservation grasslands for biofuel production, describing the relationships of plant species richness and tall native C4 prairie grass abundance with plant chemical composition and the resulting potential ethanol yield. We determined plant species composition and diversity at multiple scales with the modified Whittaker plot technique, aboveground biomass, plant chemical composition, and potential ethanol yield at 34 sites across the major ecological regions of the northeastern USA. Conservation grasslands with higher numbers of plant species had lower biomass yields and a lower ethanol yield per unit biomass compared with sites with fewer species. Thus, biofuel yield per unit land area decreased by 77% as plant species richness increased from 3 to 12.8 species per m2. We found that, as tall native C4 prairie grass abundance increased from 1.7% to 81.6%, the number of plant species decreased and aboveground biomass per unit land area and ethanol yield per unit biomass increased resulting in a 500% increased biofuel yield per unit land area. Plant species richness and composition are key determinants of biomass and ethanol yields from conservation grasslands and have implications for low-input high-diversity systems. Designing systems to include a large proportion of species with undesirable fermentation characteristics could reduce ethanol yields.
Consolidated bioprocessing (CBP) of cellulosic biomass is a promising source of ethanol. This process uses anaerobic bacteria, their own cellulolytic enzymes and fermentation pathways that convert the products of cellulose hydrolysis to ethanol in a single reactor. However, the engineering and economics of the process remain questionable. The ruminal fermentation is a very highly developed natural cellulose-degrading system. We propose that breakthroughs developed by cattle and other ruminant animals in cellulosic biomass conversion can guide future improvements in engineered CBP systems. These breakthroughs include, among others, an elegant and effective physical pretreatment; operation at high solids loading under non-aseptic conditions; minimal nutrient requirements beyond the plant biomass itself; efficient fermentation of nearly all plant components; efficient recovery of primary fermentation end-products; and production of useful co-products. Ruminal fermentation does not produce significant amounts of ethanol, but it produces volatile fatty acids and methane at a rapid rate. Because these alternative products have a high energy content, efforts should be made to recover these products and convert them to other organic compounds, particularly transportation fuels.
Bacteria-mediated acquisition of atmospheric N2 serves as a critical source of nitrogen in terrestrial ecosystems. Here we reveal that symbiotic nitrogen fixation facilitates the cultivation of specialized fungal crops by leaf-cutter ants. By using acetylene reduction and stable isotope experiments, we demonstrated that N2 fixation occurred in the fungus gardens of eight leaf-cutter ant species and, further, that this fixed nitrogen was incorporated into ant biomass. Symbiotic N2-fixing bacteria were consistently isolated from the fungus gardens of 80 leaf-cutter ant colonies collected in Argentina, Costa Rica, and Panama. The discovery of N2 fixation within the leaf-cutter ant-microbe symbiosis reveals a previously unrecognized nitrogen source in neotropical ecosystems.
Bacteria-mediated acquisition of atmospheric N2 serves as a critical source of nitrogen in terrestrial ecosystems. Here we reveal that symbiotic nitrogen fixation facilitates the cultivation of specialized fungal crops by leaf-cutter ants. By using acetylene reduction and stable isotope experiments, we demonstrated that N2 fixation occurred in the fungus gardens of eight leaf-cutter ant species and, further, that this fixed nitrogen was incorporated into ant biomass. Symbiotic N2-fixing bacteria were consistently isolated from the fungus gardens of 80 leaf-cutter ant colonies collected in Argentina, Costa Rica, and Panama. The discovery of N2 fixation within the leaf-cutter ant-microbe symbiosis reveals a previously unrecognized nitrogen source in neotropical ecosystems.
Improvement of biofeedstock quality for cellulosic ethanol production will be facilitated by inexpensive and rapid methods of evaluation, such as those already employed in the field of ruminant nutrition. Our objective was to evaluate whether forage quality and compositional measurements could be used to estimate ethanol yield of maize stover as measured by a simplified pretreatment and simultaneous saccharification and fermentation assay. Twelve maize varieties selected to be diverse for stover digestibility and composition were evaluated. Variation in ethanol yield was driven by glucan convertibility rather than by glucan content. Convertibility was highly correlated with ruminal digestibility and lignin content. There was no relationship between structural carbohydrate content (glucan and neutral detergent fiber) and ethanol yield. However, when these variables were included in multiple regression equations including convertibility or neutral detergent fiber digestibility, their partial regression coefficients were significant and positive. A regression model including both neutral detergent fiber and its ruminal digestibility explained 95% of the variation in ethanol yield. Forage quality and composition measurements may be used to predict cellulosic ethanol yield to guide biofeedstock improvement through agronomic research and plant breeding.
Ruminant animals digest cellulose via a symbiotic relationship with ruminal microorganisms. Because feedstuffs only remain in the rumen for a short time, the rate of cellulose digestion must be very rapid. This speed is facilitated by rumination, a process that returns food to the mouth to be rechewed. By decreasing particle size, the cellulose surface area can be increased by up to 10(6)-fold. The amount of cellulose digested is then a function of two competing rates, namely the digestion rate (K(d)) and the rate of passage of solids from the rumen (K(p)). Estimation of bacterial growth on cellulose is complicated by several factors: (1) energy must be expended for maintenance and growth of the cells, (2) only adherent cells are capable of degrading cellulose and (3) adherent cells can provide nonadherent cells with cellodextrins. Additionally, when ruminants are fed large amounts of cereal grain along with fiber, ruminal pH can decrease to a point where cellulolytic bacteria no longer grow. A dynamic model based on STELLA software is presented. This model evaluates all of the major aspects of ruminal cellulose degradation: (1) ingestion, digestion and passage of feed particles, (2) maintenance and growth of cellulolytic bacteria and (3) pH effects.
Real-time polymerase chain reaction (PCR) was used to quantify 16 procaryotic taxa in the rumina of two lactating dairy cows following supply and subsequent withdrawal of the feed additive monensin (13.9 mg/kg of diet dry matter) in a high-starch, silage-based ration. PCR was conducted on DNA from rumen samples collected 6 h post feeding on two successive days before monensin supplementation, after 30 days of monensin supplementation, and at six weekly intervals after monensin withdrawal. Mean values of relative population size (RPS, the percent of bacterial 16S rRNA copy number) for genus Prevotella increased (P < 0.05) from 41.8% without monensin to 49.2% with monensin and declined to 42.5% after monensin withdrawal. Mean RPS values for two biohydrogenating species (Megasphaera elsdenii and Butyrivibrio fibrisolvens) were low (<0.4%) and declined several-fold in response to monensin. Mean RPS values for the biohydrogenating species Eubacterium ruminantium, four cellulolytic species, four starch- or dextrin-fermenting species, and Domain Archaea were not altered (P > 0.10) upon monensin feeding or withdrawal. The data suggest that monensin in high-starch diets does not suppress populations of classical ruminal Gram-positive bacteria or the availability of H2, though it may affect bacteria involved in biohydrogenation of lipids that regulate bovine mammary lipogenesis.
Effects of sucrose (Suc) concentration on fermentation kinetics and products were evaluated using 3 concentrations of Suc, with 1 concentration of isolated NDF from Bermudagrass fermented together in batch culture in vitro with rumen inoculum. Fixed amounts of medium and inoculum were the protein sources, so protein:Suc decreased with increasing Suc. Kinetics were calculated from gas production over 48 h in a randomized complete block design (n = 28), and product yield was evaluated with sampling every 4 h for 24 h in a split-split plot in time design (n = 84). Fermentation vial was the experimental unit. Increasing Suc increased the lag time of rapidly (P < 0.01) and slowly fermented (P < 0.01) fractions and tended to decrease the rate of gas production from the rapid fraction (P = 0.07). Gas production from the slow fraction decreased linearly with increasing Suc (P = 0.02), suggesting a decrease in NDF fermentation. Sucrose was the predominant substrate at </=8 h of fermentation. Maxima for microbial CP (MCP) production were detected at </=8 h of fermentation. At detected MCP maxima, MCP production increased linearly (P = 0.02) and total organic acids (sum of lactate, acetate, propionate, and butyrate; mmol) tended to increase linearly (P = 0.07) with increasing Suc. Maximum lactate production at 0 and 4 h increased (P = 0.01), and yield of lactate from Suc tended to increase, linearly (P = 0.09) with increasing Suc. At detected MCP maxima, yield of C in products (total organic acids, MCP, CO(2), CH(4), glycogen) from utilized Suc C declined linearly for total products (P = 0.01) and organic acids (P = 0.01) and tended to decline for MCP (P = 0.12) as Suc increased. This may be a function of increased catabolic inefficiency of microbes with increasing Suc, as evidenced by increasing yields of lactate, or the use of C for products not measured. Product C yields were 1.28, 0.98, and 0.81 from lowest to greatest Suc inclusion, respectively. Values >1 indicate incorporation of C from the medium, likely from AA and peptides. The results support the premises that direct effects of Suc concentration and perhaps protein:Suc alter yields of fermentation products. That substrate concentration altered fermentation products and kinetics, possibly due to interactions with the run conditions, advises the clear definition of substrates and fermentation conditions to determine how the results integrate into our knowledge of ruminant nutrition.
Relative quantification real-time PCR was used to quantify several bacterial species in ruminal samples from two lactating cows, each sampled 3 h after feeding on two successive days. Abundance of each target taxon was calculated as a fraction of the total 16S rRNA gene copies in the samples, using taxon-specific and eubacterial domain-level primers. Bacterial populations showed a clear predominance of members of the genus Prevotella, which comprised 42% to 60% of the bacterial rRNA gene copies in the samples. However, only 2% to 4% of the bacterial rRNA gene copies were represented by the classical ruminal Prevotella species Prevotella bryantii, Prevotella ruminicola and Prevotella brevis. The proportion of rRNA gene copies attributable to Fibrobacter succinogenes, Ruminococcus flavefaciens, Selenomonas ruminantium and Succinivibrio dextrinosolvens were each generally in the 0.5% to 1% range. Proportions for Ruminobacter amylophilus and Eubacterium ruminantium were lower (0.1% to 0.2%), while Butyrivibrio fibrisolvens, Streptococcus bovis, Ruminococcus albus and Megasphaera elsdenii were even less abundant, each comprising <0.03% of the bacterial rRNA gene copies. The data suggest that the aggregate abundance of the most intensively studied ruminal bacterial species is relatively low and that a large fraction of the uncultured population represents a single bacterial genus.
Plant biomass has attracted interest as a feedstock for biofuels production, but much of this work has been focused on relatively few plant species. In this study, three relatively-unstudied species of warm-season perennial grasses, grown at multiple locations in the eastern and central US and harvested over a three year period, were examined for fermentability via in vitro ruminal gas production and dry matter digestibility assays, and near-infrared reflectance calibrations were developed for these fermentation parameters. Big bluestem (Andropogon gerardii Vitman) displayed greater fermentability than did sand bluestem (Andropogon hallii Hack) or eastern gamagrass [Tripsacum dactyloides (L.) L.], but displayed lower biomass yields. The bluestems also displayed lower N contents and less variation in fermentability over different growth environments (geographic locations and harvest years), suggesting a more consistent biomass quality than for eastern gamagrass. Thus, in addition to their use as forage for ruminant animals, bluestems may be of particular interest as feedstocks for bioconversion to ethanol and other products via direct microbial fermentation (consolidated bioprocessing) schemes, and thus merit additional efforts to enhance biomass yield potential.
Anaerobic cellulolytic bacteria are thought to adhere to cellulose via several mechanisms, including production of a glycocalyx containing extracellular polymeric substances (EPS). As the compositions and structures of these glycocalyces have not been elucidated, variable-pressure scanning electron microscopy (VP-SEM) and chemical analysis were used to characterize the glycocalyx of the ruminal bacterium Ruminococcus albus strain 7. VP-SEM revealed that growth of this strain was accompanied by the formation of thin cellular extensions that allowed the bacterium to adhere to cellulose, followed by formation of a ramifying network that interconnected individual cells to one another and to the unraveling cellulose microfibrils. Extraction of 48-h-old whole-culture pellets (bacterial cells plus glycocalyx [G] plus residual cellulose [C]) with 0.1 N NaOH released carbohydrate and protein in a ratio of 1:5. Boiling of the cellulose fermentation residue in a neutral detergent solution removed almost all of the adherent cells and protein while retaining a residual network of adhering noncellular material. Trifluoroacetic acid hydrolysis of this residue (G plus C) released primarily glucose, along with substantial amounts of xylose and mannose, but only traces of galactose, the most abundant sugar in most characterized bacterial exopolysaccharides. Linkage analysis and characterization by nuclear magnetic resonance suggested that most of the glucosyl units were not present as partially degraded cellulose. Calculations suggested that the energy demand for synthesis of the nonprotein fraction of EPS by this organism represents only a small fraction (<4%) of the anabolic ATP expenditure of the bacterium.
Real-time polymerase chain reaction (RT-PCR) was used to quantify seven species of lactic acid bacteria (LAB) in alfalfa silage prepared in the presence or absence of four commercial inoculants and in uninoculated corn stover harvested and stored under a variety of field conditions. Species-specific PCR primers were designed based on recA gene sequences. Commercial inoculants improved the quality of alfalfa silage, but species corresponding to those in the inoculants displayed variations in persistence over the next 96 h. Lactobacillus brevis was the most abundant LAB (12 to 32% of total sample DNA) in all of the alfalfa silages by 96 h. Modest populations (up to 10%) of Lactobacillus plantarum were also observed in inoculated silages. Pediococcus pentosaceus populations increased over time but did not exceed 2% of the total. Small populations (0.1 to 1%) of Lactobacillus buchneri and Lactococcus lactis were observed in all silages, while Lactobacillus pentosus and Enterococcus faecium were near or below detection limits. Corn stover generally displayed higher populations of L. plantarum and L. brevis and lower populations of other LAB species. The data illustrate the utility of RT-PCR for quantifying individual species of LAB in conserved forages prepared under a wide variety of conditions.
Cattle generally avoid grazing leafy spurge (LS; Euphorbia esula), whereas sheep and goats will often eat it. Understanding metabolism of toxic phytochemicals in LS by bovine rumen microflora may help explain why cattle often develop aversions to LS after initially eating it. Toxicity of LS compounds after in vitro fermentation with normal vs. antibiotic-modified bovine rumen digesta was evaluated at different lengths of fermentation. Levels of toxic and aversion-inducing ingenols were determined for fermented and nonfermented mixtures of LS and bovine rumen digesta, and the toxicity of an aversion-inducing extract of LS to rumen microbial species that are common in cattle, sheep, and goats was evaluated. Fermentation of LS with bovine digesta increased the toxicity of extracted compounds. Introduction of neomycin (an antibiotic that preferentially inhibits gram-negative bacteria) into the LS and bovine rumen digesta mixtures did not appear to affect toxicities regardless of fermentation length. Levels of ingenol were observed in LS and bovine digesta mixtures (both fermented and nonfermented) that were consistent with levels of ingenols reported for LS. Finally, a toxic extract of LS had little or no negative effect on the growth of several common species of rumen bacteria. The results indicate that LS is not generally toxic to the ruminal bacteria, but that microbial activity in the rumen may be responsible for enhancing LS toxicity to cattle.
Because archaea are generally associated with extreme environments, detection of nonthermophilic members belonging to the archaeal division Crenarchaeota over the last decade was unexpected; they are surprisingly ubiquitous and abundant in nonextreme marine and terrestrial habitats. Metabolic characterization of these nonthermophilic crenarchaeotes has been impeded by their intractability toward isolation and growth in culture. From studies employing a combination of cultivation and molecular phylogenetic techniques (PCR-single-strand conformation polymorphism, sequence analysis of 16S rRNA genes, fluorescence in situ hybridization, and real-time PCR), we present evidence here that one of the two dominant phylotypes of Crenarchaeota that colonizes the roots of tomato plants grown in soil from a Wisconsin field is selectively enriched in mixed cultures amended with root extract. Clones recovered from enrichment cultures were found to group phylogenetically with sequences from clade C1b.A1. This work corroborates and extends our recent findings, indicating that the diversity of the crenarchaeal soil assemblage is influenced by the rhizosphere and that mesophilic soil crenarchaeotes are found associated with plant roots, and provides the first evidence for growth of nonthermophilic crenarchaeotes in culture.
Clostridium thermocellum is a thermophilic, anaerobic, cellulolytic bacterium that produces ethanol and acetic acid as major fermentation end products. The effect of growth conditions on gene expression in C. thermocellum ATCC 27405 was studied using cells grown in continuous culture under cellobiose or cellulose limitation over a approximately 10-fold range of dilution rates (0.013 to 0.16 h(-1)). Fermentation product distribution displayed similar patterns in cellobiose- or cellulose-grown cultures, including substantial shifts in the proportion of ethanol and acetic acid with changes in growth rate. Expression of 17 genes involved or potentially involved in cellulose degradation, intracellular phosphorylation, catabolite repression, and fermentation end product formation was quantified by real-time PCR, with normalization to two calibrator genes (recA and the 16S rRNA gene) to determine relative expression. Thirteen genes displayed modest (fivefold or less) differences in expression with growth rate or substrate type: sdbA (cellulosomal scaffoldin-dockerin binding protein), cdp (cellodextrin phosphorylase), cbp (cellobiose phosphorylase), hydA (hydrogenase), ldh (lactate dehydrogenase), ack (acetate kinase), one putative type IV alcohol dehydrogenase, two putative cyclic AMP binding proteins, three putative Hpr-like proteins, and a putative Hpr serine kinase. By contrast, four genes displayed >10-fold-reduced levels of expression when grown on cellobiose at dilution rates of >0.05 h(-1): cipA (cellulosomal scaffolding protein), celS (exoglucanase), manA (mannanase), and a second type IV alcohol dehydrogenase. The data suggest that at least some cellulosomal components are transcriptionally regulated but that differences in expression with growth rate or among substrates do not directly account for observed changes in fermentation end product distribution.
Current methods for measuring ethanol yields from lignocellulosic biomass are relatively slow and are not well geared for analyzing large numbers of samples generated by feedstock management and breeding research. The objective of this study was to determine if an in vitro ruminal fermentation assay used in forage quality research was predictive of results obtained using a conventional biomass-to-ethanol conversion assay. In the conventional assay, herbaceous biomass samples were converted to ethanol by Saccharomyces cerevisiae cultures in the presence of cellulase enzymes. Cultures were grown in sealed serum bottles and gas production monitored by measuring increasing head space pressure. Gas accumulation as calculated from the pressure measurements was highly correlated (r(2)>0.9) with ethanol production measured by gas chromatography at 24 h or 7 days. The same feedstocks were also analyzed by in vitro ruminal digestion, as also measured by gas accumulation. Good correlations (r(2) approximately 0.63-0.82) were observed between ethanol production during simultaneous saccharification and fermentation and gas accumulation in parallel in vitro ruminal fermentations. Because the in vitro ruminal fermentation assay can be performed without sterilization of the medium and does not require aseptic conditions, this assay may be useful for biomass feedstock agronomic and breeding research.
Fermentation residues (consisting of incompletely fermented fiber, adherent bacterial cells, and a glycocalyx material that enhanced bacterial adherence) were obtained by growing the anaerobic cellulolytic bacteria Ruminococcus albus 7 or Clostridium thermocellum ATCC 27405 on a fibrous fraction derived from lucerne (Medicago sativa L.). The dried residue was able to serve as an effective co-adhesive for phenol-formaldehyde (PF) bonding of aspen veneer sheets to one another. Testing of the resulting plywood panels revealed that the adhesive, formulated to contain 30% of its total dry weight as fermentation residue, displayed shear strength and wood failure values under both wet and dry conditions that were comparable with those of industry standards for PF that contained much smaller amounts of fillers or extenders. By contrast, PF adhesives prepared with 30% of dry weight as either unfermented lucerne fiber or conventional fillers or extenders rather than as fermentation residues, displayed poor performance, particularly under wet conditions.
Many studies have shown the beneficial effects on ruminant performance of feeding them with silages inoculated with lactic acid bacteria (LAB). These benefits might derive from probiotic effects. The purpose of the current study was to determine whether LAB included in inoculants for silage can survive in rumen fluid (RF), as the first step in studying their probiotic effects. Experiments were conducted in the United States and Israel with clarified (CRF) and strained RF (SRF) that were inoculated at 10(6)-10(8) microorganisms/mL with and without glucose at 5 g/L. RF with no inoculants served as control. Ten commercial inoculants were used. The RF was incubated at 39 degrees C and sampled in duplicates at 6, 12, 24, 48, 72, and 96 h for pH and LAB counts. The results indicate that with glucose the pH of the RF decreased during the incubation period. In the SRF, the pH of the inoculated samples was higher than that of the controls in most cases. This might be a clue to the mechanism by which LAB elicit the enhancement in animal performance. LAB counts revealed that the inoculants survived in the RF during the incubation period. The addition of glucose resulted in higher LAB counts.
An approximately 32-kDa protein (albusin B) that inhibited growth of Ruminococcus flavefaciens FD-1 was isolated from culture supernatants of Ruminococcus albus 7. Traditional cloning and gene-walking PCR techniques revealed an open reading frame (albB) encoding a protein with a predicted molecular mass of 32,168 Da. A BLAST search revealed two homologs of AlbB from the unfinished genome of R. albus 8 and moderate similarity to LlpA, a recently described 30-kDa bacteriocin from Pseudomonas sp. strain BW11M1.
Residues from the fermentation of cellulose by the anaerobic bacteria Ruminococcus albus (strain 7) or Ruminococcus flavefaciens (strains FD-1 or B34b) containing residual cellulose, bacterial cells and their associated adhesins, were examined for their ability to serve as components of adhesives for plywood fabrication. The residues contained differing amounts of protein (0.4-4.2% of dry weight), but the ratios of monosaccharides recovered following two-stage treatment of the residue with detergent (pH 7) and TFA were similar for all three strains (0.71 glucose:0.18 xylose:0.08 mannose:0.02 galactose), suggesting similarities in exopolysaccharide composition. Three-ply aspen panels prepared with fermentation residues (FR) displayed better shear strength and wood failure under dry conditions than following a vacuum/pressure/soak/dry treatment, but adhesive properties were inferior to those prepared with conventional phenol-formaldehyde (PF) adhesives. However, panels prepared by incorporating the R. albus 7 FR into PF formulation, at 73% by weight of the total adhesive, exhibited shear strength and wood failure similar to that obtained with PF adhesive alone. Use of residues from fermentations by these bacteria as components of adhesives may add value to biomass fermentations aimed primarily at producing ethanol and other chemical products.
To determine whether lactic acid bacteria (LAB) used in inoculants for silage can survive in rumen fluid (RF), and to identify those that survive best. Twelve commercial silage inoculants were added at 107 CFU ml-1 to strained RF (SRF) taken from dairy cows, with and without 5 g l-1 glucose and incubated in vitro at 39 degrees C. Changes in pH, LAB numbers and fermentation products were monitored for 72 h. In the inoculated RF with glucose, the pH decreased and numbers of LAB increased. The inoculants varied with regard to their effect on pH change and growth. In the SRF, both with and without glucose, the pH values of the inoculated samples were generally higher than those of the uninoculated controls throughout most of the incubation period. This may suggest a positive effect on the rumen environment. LAB used in silage inoculants can survive in RF in vitro. This is the first step in studying the probiotic potential of silage LAB inoculants for dairy cattle. The survival of these LAB in RF may enable them to interact with rumen microorganisms and to affect rumen functionality.
The direct fermentation of cellulosic biomass to ethanol has long been a desired goal. To this end, we screened the environment for fungal strains capable of this conversion when grown on minimal medium. One strain, identified as a member of the genus Trichoderma and designated strain A10, was isolated from cow dung and initially produced about 0.4 g ethanol l(-1). This strain cannot grow on any substrate under anaerobic conditions, but can ferment microcrystalline cellulose or several sugars to ethanol. Ethanol accumulation was eventually increased, by selection and the use of a vented fermentation flask, to 2 g l(-1) when the fermentation was carried out in submerged culture in minimal medium. The highest levels of ethanol, >5.0 g l(-1), were obtained by the fermentation of glucose. Little ethanol was produced by the fermentation of xylose, although other fermentation products such as succinate and acetate were observed. Strain A10 was also found to utilize (aerobically) a wide range of carbon sources. In addition, auxotrophic mutants were generated and used to demonstrate parasexuality by complementation between auxotrophs and between morphological mutants. The ability of this strain to use a wide variety of carbohydrates (including crystalline cellulose) combined with its minimal nutrient requirements and the availability of a genetic system suggests that the strain merits further investigation of its ability to convert biomass to ethanol.
Fundamental features of microbial cellulose utilization are examined at successively higher levels of aggregation encompassing the structure and composition of cellulosic biomass, taxonomic diversity, cellulase enzyme systems, molecular biology of cellulase enzymes, physiology of cellulolytic microorganisms, ecological aspects of cellulase-degrading communities, and rate-limiting factors in nature. The methodological basis for studying microbial cellulose utilization is considered relative to quantification of cells and enzymes in the presence of solid substrates as well as apparatus and analysis for cellulose-grown continuous cultures. Quantitative description of cellulose hydrolysis is addressed with respect to adsorption of cellulase enzymes, rates of enzymatic hydrolysis, bioenergetics of microbial cellulose utilization, kinetics of microbial cellulose utilization, and contrasting features compared to soluble substrate kinetics. A biological perspective on processing cellulosic biomass is presented, including features of pretreated substrates and alternative process configurations. Organism development is considered for "consolidated bioprocessing" (CBP), in which the production of cellulolytic enzymes, hydrolysis of biomass, and fermentation of resulting sugars to desired products occur in one step. Two organism development strategies for CBP are examined: (i) improve product yield and tolerance in microorganisms able to utilize cellulose, or (ii) express a heterologous system for cellulose hydrolysis and utilization in microorganisms that exhibit high product yield and tolerance. A concluding discussion identifies unresolved issues pertaining to microbial cellulose utilization, suggests approaches by which such issues might be resolved, and contrasts a microbially oriented cellulose hydrolysis paradigm to the more conventional enzymatically oriented paradigm in both fundamental and applied contexts.
In vitro fermentations of pure cellulose by mixed ruminal microorganisms were conducted under conditions in which pH declined within ranges similar to those observed in the rumen. At low cellulose concentrations (12.5 g/L), the first-order rate constants (k) of cellulose disappearance were successively lower at initial pH values of 6.86, 6.56, and 6.02, but in each case the value of k was maintained over a pH range of 0.3 to 1.2 units, as the fermentation progressed. Plots of k versus initial pH were linear, and k displayed a relative decrease of approximately 7% per 0.1 unit decrease in pH. At high cellulose concentration (50 g/L) and an initial pH of 6.8, cellulose digestion was initially zero order, the absolute rate of digestion declined with pH and digestion essentially ceased at pH 5.3 after only 30% of the added cellulose was digested. Further incubation resulted in a loss of bound N and P, suggesting that at low pH cells lysed or detached from the undigested fibers. Pure cultures of ruminal cellulolytic bacteria also were able to ferment cellulose to a minimum pH of 5.1 to 5.3, but the extent of fermentation was increased by coculture with noncellulolytic bacteria. A model is proposed in which the first-order rate constant of cellulose digestion is determined by the pH at which the fermentation is initiated, and end product ratios reflect greater activity of the noncellulolytic population as pH declines.
Growth of the cellulose-synthesizing bacterium Acetobacter xylinum ATCC 53524 in media supplemented with 5% (w/v) glucose and 0.2% (w/v) of a water-soluble, nearly linear xylan from tobacco stalks resulted in the synthesis of a highly crystalline composite having a xylose/glucose ratio ranging from 0.06 to 0.24. The digestion of one composite (88% cellulose/12% xylan) by mixed ruminal microflora displayed kinetics of gas production similar to those of an unassociated mixture of the two components added in a xylan/cellulose ratio similar to that of the composite. The data suggest that intimate association of xylan and cellulose, as is typically found in secondary plant cell walls, does not inhibit the rate of digestion of the component polysaccharides.
An accurate method for estimation of lignin concentration is important for prediction of the digestible energy content of livestock feeds. The accuracy of lignin concentration estimates based on the Klason lignin and acid detergent lignin methods was compared. Ten diverse forage samples were analyzed for protein, carbohydrates, lipids, organic acids, ash, lignin (by both methods), and gross energy. The accuracy of the two lignin concentration estimates was examined by comparing the measured forage gross energy to a gross energy value calculated from the compositional analysis. Use of the acid detergent lignin estimate in this gross energy calculation accounted for 68-84% of the forage gross energy compared to 85-97% of the gross energy using Klason lignin. These results indicate that while Klason lignin estimates are substantially higher than acid detergent lignin estimates, Klason lignin is the more accurate lignin method and does not overestimate lignin because gross energy recoveries were less than 100%.
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 alpha-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 essential role of ruminal microflora in ruminant nutrition provides the potential for improvement in animal production via altering the numbers or activities of specific classes of microorganisms. Successful alterations will be facilitated by an understanding of the microbial ecology of the rumen based on its mechanistic underpinnings. Demonstrated improvements in ruminal fermentation can be traced to their consonance with well-established principles of microbial ecology (niche occupancy, selective pressure, adaptation, and interactions) and the thermodynamics and kinetics of substrate utilization. Application of these principles to several proposed alterations of the ruminal bacterial population allows a prediction of their relative feasibility. Improving fiber digestion, decreasing protein degradation, and detoxifying feed components that are present in low concentrations will be difficult to achieve in the rumen and are best approached by altering the feed, either genetically or with postharvest treatment. By contrast, the detoxification of feed components present in high concentration, and redirection of electron disposal away from methanogenesis, are more productive targets for microbiological research.
A pathway for conversion of the metabolic intermediate phosphoenolpyruvate (PEP) and the formation of acetate, succinate, formate, and H2 in the anaerobic cellulolytic bacterium Ruminococcus flavefaciens FD-1 was constructed on the basis of enzyme activities detected in extracts of cells grown in cellulose- or cellobiose-limited continuous culture. PEP was converted to acetate and CO2 (via pyruvate kinase, pyruvate dehydrogenase, and acetate kinase) or carboxylated to form succinate (via PEP carboxykinase, malate dehydrogenase, fumarase, and fumarate reductase). Lactate was not formed even during rapid growth (batch culture, mu = 0.35/h). H2 was formed by a hydrogenase rather than by cleavage of formate, and 13C-NMR and 14C-exchange reaction data indicated that formate was produced by CO2 reduction, not by a cleavage of pyruvate. The distribution of PEP into the acetate and succinate pathways was not affected by changing extracellular pH and growth rates within the normal growth range. However, increasing growth rate from 0.017/h to 0.244/h resulted in a shift toward formate production, presumably at the expense of H2. This shift suggested that reducing equivalents could be balanced through formate or H2 production without affecting the yields of the major carbon-containing fermentation endproducts.
Phosphoenolpyruvate (PEP) carboxykinase was purified 42-fold with a 25% yield from cell extracts of Ruminococcus flavefaciens by ammonium sulfate precipitation, preparative isoelectric focusing, and removal of carrier ampholytes by chromatography. The enzyme had a subunit molecular mass of approximately 66.3 kDa (determined by mass spectrometry), but was retained by a filter having a 100-kDa nominal molecular mass cutoff. Optimal activity required activation of the enzyme by Mn2+ and stabilization of the nucleotide substrate by Mg2+. GDP was a more effective phosphoryl acceptor than ADP, while IDP was not utilized. Under optimal conditions the measured activity in the direction of PEP carboxylation was 17.2 micromol min-1 (mg enzyme)-1. The apparent Km values for PEP (0.3 mM) and GDP (2.0 mM) were 9- and 14-fold lower than the apparent Km values for the substrates of the back reaction (oxaloacetate and GTP, respectively). The data are consistent with the involvement of PEP carboxykinase as the primary carboxylation enzyme in the fermentation of cellulose to succinate by this bacterium.
The ruminal cellulolytic bacteria Ruminococcus flavefaciens FD-1 and Fibrobacter succinogenes S85 coexisted in substrate-excess coculture with about equal population size, but R. flavefaciens outcompeted F. succinogenes for cellobiose in the substrate-limited cocultures whether the two strains were coinoculated or a steady-state culture of F. succinogenes was challenged by R. flavefaciens. This outcome of competition between these two strains is due to a classical pure and simple competition mechanism based on affinity for cellobiose. Although the population size of F. succinogenes was much higher (> 70%) than that of another cellulolytic species, Ruminococcus albus 7 in substrate-excess coculture, F. succinogenes was replaced by a population of R. albus in the substrate-limited coculture in both coinoculation and challenge experiments. R albus outcompeted F. succinogenes, apparently due to selection in the chemostat of a population of R. albus with a higher affinity for cellobiose. R. albus also outcompeted R. flavefaciens under substrate-limited conditions.
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 limitation is in accord with the former's more rapid adherence to cellulose and its higher affinity for cellodextrin products of cellulose hydrolysis. In batch cocultures of F. succinogenes and R. albus, the populations of the two species were similar. However, under cellulose limitation, F. succinogenes was the predominant strain (approximately 80% of cell mass) in cultures simultaneously coinoculated with R. albus. The results from batch cocultures of R. flavefaciens and R. albus were not consistent within or among trials: some experiments yielded monocultures of R. albus (suggesting production of an inhibitory agent by R. albus), while others contained substantial populations of both species. Under cellulose limitation, R. flavefaciens predominated over R. albus (85 and 15%, respectively), as would be expected by the former's greater adherence to cellulose. The retention of R. albus in the cellulose-limited coculture may result from a combination of its ability to utilize glucose (which is not utilizable by R. flavefaciens), its demonstrated ability to adapt under selective pressure in the chemostat to utilization of lower concentrations of cellobiose, a major product of cellulose hydrolysis, and its possible production of an inhibitory agent.
The bacteria Fibrobacter succinogenes, Ruminococcus flavefaciens, and Ruminococcus albus generally are regarded as the predominant cellulolytic microbes in the rumen. Comparison of available data from the literature reveals that these bacteria are the most actively cellulolytic of all mesophilic organisms described to date from any habitat. In light of numerous proposals to improve microbial cellulose digestion in ruminants, it is instructive to examine the characteristics of these species that contribute to their superior cellulolytic capabilities and to identify the factors that prevent them from digesting cellulose even more rapidly. As a group, these species have extreme nutritional specialization. They are able to utilize cellulose (or in some cases xylan) and its hydrolytic products as their nearly sole energy sources for growth. Moreover, each species apparently has evolved to similar maximum rates of cellulose digestion (first-order rate constants of 0.05 to 0.08 h-1). Active cellulose digestion involves adherence of cells to the fibers via a glycoprotein glycocalyx, which protects cells from protozoal grazing and cellulolytic enzymes from degradation by ruminal proteases while it retains-at least temporarily-the cellodextrin products for use by the cellulolytic bacteria. These properties result in different ecological roles for the adherent and nonadherent populations of each species, but overall provide an enormous selective advantage to these cellulolytic bacteria in the ruminal environment. However, major constraints to cellulose digestion are caused by cell-wall structure of the plant (matrix interactions among wall biopolymers and low substrate surface area) and by limited penetration of the nonmotile cellulolytic microbes into the cell lumen. Because of these constraints and the highly adapted nature of cellulose digestion by the predominant cellulolytic bacteria in the rumen, transfer of cellulolytic capabilities to noncellulolytic ruminal bacteria (e.g., by genetic engineering) that display other desirable properties offers limited opportunities to improve ruminal digestion of cellulose.
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.
Ruminal cellulolytic bacteria (Fibrobacter succinogenes S85 or Ruminococcus flavefaciens FD-1) were combined with the non-ruminal bacterium Clostridium kluyveri and grown together on cellulose and ethanol. Succinate and acetate produced by the cellulolytic organisms were converted to butyrate and caproate only when the culture medium was supplemented with ethanol. Ethanol (244 mM) and butyrate (30 mM at pH 6.8) did not inhibit cellulose digestion or product formation by S85 or FD-1; however caproate (30 mM at pH 6.8) was moderately inhibitory to FD-1. Succinate consumption and caproate production were sensitive to culture pH, with more caproic acid being produced when the culture was controlled at a pH near neutrality. In a representative experiment under conditions of controlled pH (at 6.8) 6.0 g cellulose l-1 and 4.4 g ethanol l-1 were converted to 2.6 g butyrate l-1 and 4.6 g caproate l-1. The results suggest that bacteria that efficiently produce low levels of ethanol and acetate or succinate from cellulose should be useful in cocultures for the production of caproic acid, a potentially useful industrial chemical and bio-fuel precursor.
Chemical treatments similar to those routinely used to extract cellulose from plant biomass caused significant increases in the relative crystallinity index (RCI) of Sig-macell 100 (a commercial cellulose of moderate crystallinity), as measured by x-ray powder diffraction in both the reflectance and transmittance modes. In general, the largest increases in RCI were observed following higher (rather than lower) temperature treatments. Substantial increases in crystalliity were also observed upon resuspension in water prior to drying, with higher temperatures again resulting in the greatest increases in RCI. Measurement of the RCIs of wetted Sigmacell 100 samples by acid hydrolysis kinetics revealed that most of the increased crystallinity occurred rapidly upon contact with water. In contrast to Sigmacell 100, a cellulose of higher initial crystallinity (the microcrystalline cellulose Sigmacell 50) showed little change in crystallinity following the above treatments. The results provide a partial explanation for the inconsistent relationships reported between cellulose crystallinity and cellulose biodegradation. (c) 1995 John Wiley & Sons, Inc.
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 grown in a cellobiose-limited chemostat, cellobiose and cellotriose could both be detected, and the ratio of cellotriose to cellobiose was approximately 1 to 4. On the basis of these results, cellodextrin production is an equilibrium (mass action) function and not just an artifact of energy-rich cultural conditions. Cellodextrins could not be detected in low-dilution-rate, cellulose-limited continuous cultures, but these cultures had a large number of nonadherent cells. Because the nonadherent cells had a large reserve of polysaccharide and were observed at all stages of cell division, it appeared that they were utilizing cellodextrins as an energy source for growth.(ABSTRACT TRUNCATED AT 250 WORDS)
The ruminal cellulolytic bacterium Fibrobacter succinogenes S85 was grown in cellulose-fed continuous culture at 22 different combinations of dilution rate (D, 0.014-0.076 h-1) and extracellular pH (6.11-6.84). Effects of pH and D on the fermentation were determined by subjecting data on cellulose consumption, cell yield, product yield (succinate, acetate, formate), and soluble sugar concentration to response surface analysis. The extent of cellulose conversion decreased with increasing D. First-order rate constants at rapid growth rates were estimated as 0.07-0.11 h-1, and decreased with decreasing pH. Apparent decreases in the rate constant with increasing D was not due to inadequate mixing or preferential utilization of the more amorphous regions of the cellulose. Significant quantities of soluble sugars (0.04-0.18 g/l, primarily glucose) were detected in all cultures, suggesting that glucose uptake was rather inefficient. Cell yields (0.11-0.24 g cells/g cellulose consumed) increased with increasing D. Pirt plots of the predicted yield data were used to determine that maintenance coefficient (0.04-0.06 g cellulose/g cells.h) and true growth yield (0.23-0.25 g cells/g cellulose consumed) varied slightly with pH. Yields of succinate, the major fermentation endproduct, were as high as 1.15 mol/mol anhydroglucose fermented, and were slightly affected by dilution rate but were not affected by pH. Comparison of the fermentation data with that of other ruminal cellulolytic bacteria indicates that F. succinogenes S85 is capable of rapid hydrolysis of crystalline cellulose and efficient growth, despite a lower mu max on microcrystalline cellulose.
Cicer milkvetch (Astragalus cicer L.) is a perennial legume used as a pasture or rangeland plant for ruminants. A study was undertaken to determine whether reported variations in its ruminal digestibility may be related to the presence of an antinutritive material. In vitro fermentation of neutral detergent fiber (NDF) of cicer milkvetch by mixed rumen microflora was poorer than was the fermentation of NDF in alfalfa (Medicago sativa L.). Fermentation of cicer milkvetch NDF was improved by preextraction of the ground herbage with water for 3 h at 39 degrees C. Such water extracts selectively inhibited in vitro fermentation of pure cellulose by mixed ruminal microflora and by pure cultures of the ruminal bacteria Ruminococcus flavefaciens FD-1 and Fibrobacter succinogenes S85. Inhibition of the cellulose fermentation by mixed ruminal microflora was dependent upon the concentration of cicer milkvetch extract and was overcome upon prolonged incubation. Pure cultures exposed to the extract did not recover from inhibition, even after long incubation times, unless the inhibitory agent was removed (viz., by dilution of inhibited cultures into fresh medium). The extract did not affect the fermentation of cellobiose by R. flavefaciens but did cause some inhibition of cellobiose fermentation by F. succinogenes. Moreover, the extracts did not inhibit hydrolysis of crystalline cellulose, carboxymethyl cellulose, or p-nitrophenylcellobioside by supernatants of these pure cultures of cellulolytic bacteria or by a commercial cellulase preparation from the fungus Trichoderma reesei. The agent caused cellulose-adherent cells to detach from cellulose fibers, suggesting that the agent may act, at least in part, by disrupting the glycocalyx necessary for adherence to, and rapid digestion of, cellulose.
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 pH. At low Ds (longer retention times), cellulose degradation did not follow first-order kinetics. This decreased rate of cellulose digestion was not due to poor mixing, limitation by other medium components, or preferential utilization of the more amorphous fraction of the cellulose. The cell yield increased from 0.13 to 0.18 mg of cells per mg of cellulose with increasing Ds from 0.02 to 0.06 h-1 and decreased when the pH was shifted from the optimum of 6.5 to 6.8. The effect of pH on cell yield increased with increasing D. The reduced cell yield at low pH appears to be due to both an increase in maintenance energy requirements and a decrease in true growth yield.
In addition to its usual native crystalline form (cellulose I), cellulose can exist in a variety of alternative crystalline forms (allomorphs) which differ in their unit cell dimensions, chain packing schemes, and hydrogen bonding relationships. We prepared, by various chemical treatments, four different alternative allomorphs, along with an amorphous (noncrystalline) cellulose which retained its original molecular weight. We then examined the kinetics of degradation of these materials by two species of ruminal bacteria and by inocula from two bovine rumens. Ruminococcus flavefaciens FD-1 and Fibrobacter succinogenes S85 were similar to one another in their relative rates of digestion of the different celluloses, which proceeded in the following order: amorphous > III(I) > IV(I) > III(II) > I > II. Unlike F. succinogenes, R. flavefaciens did not degrade cellulose II, even after an incubation of 3 weeks. Comparisons of the structural features of these allomorphs with their digestion kinetics suggest that degradation is enhanced by skewing of adjacent sheets in the microfibril, but is inhibited by intersheet hydrogen bonding and by antiparallelism in adjacent sheets. Mixed microflora from the bovine rumens showed in vitro digestion rates quite different from one another and from those of both of the two pure bacterial cultures, suggesting that R. flavefaciens and F. succinogenes (purportedly among the most active of the cellulolytic bacteria in the rumen) either behave differently in the ruminal ecosystem from the way they do in pure culture or did not play a major role in cellulose digestion in these ruminal samples.
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 which were fermented slightly more rapidly than the original material. However, treatment with higher concentrations of phosphoric acid resulted in a more slowly fermented substrate, despite a decrease in crystallinity and an increase in pore volume. This reduced fermentation rate was apparently due to the partial conversion of the cellulose from the type I to the type II allomorph, since mercerized (type II) cellulose was also fermented more slowly, and only after a much longer lag period. The results are consistent with earlier evidence for the cell-associated nature of cellulolytic enzymes of ruminal bacteria and suggest that ruminal microflora do not rapidly adapt to utilization of celluloses with altered unit cell structures.
A field isolate of Desulfovibrio desulfuricans was grown in defined medium in a two-stage continuous culture apparatus with different concentrations of phosphate in the feed medium. The first state (V1) was operated as a conventional chemostat (D = 0.045 h) that was limited in energy source (lactate) or phosphate. The second stage (V2) received effluent from V1 but no additional nutrients, and contained a healthy population of transiently starved or resting cells. An increase in the concentration of phosphate in the medium fed to V1 resulted in increased corrosion rates of carbon steel in both V1 and V2. Despite the more rapid corrosion observed in growing cultures relative to that in resting cultures, corrosion products that were isolated under strictly anaerobic conditions from the two culture modes had similar bulk compositions which varied with the phosphate content of the medium. Crystalline mackinawite (Fe(9)S(8)), vivianite [Fe(3)(PO(4))(2) . 8H(2)O], and goethite [FeO(OH)] were detected in amounts which varied with the culture conditions. Chemical analyses indicated that the S in the corrosion product was almost exclusively in the form of sulfides, while the P was present both as phosphate and as unidentified components, possibly reduced P species. Some differential localization of S and P was observed in intact corrosion products. Cells from lactate-limited, but not from phosphate-limited, cultures contained intracellular granules that were enriched in P and Fe. The results are discussed in terms of several proposed mechanisms of microbiologically influenced corrosion.
Previous studies of anaerobic biocorrosion have suggested that microbial sulfur and phosphorus products as well as cathodic hydrogen consumption may accelerate anaerobic metal oxidation. Methanogenic bacteria, which normally use molecular hydrogen (H(2)) and carbon dioxide (CO(2)) to produce methane (CH(4)) and which are major inhabitants of most anaerobic ecosystems, use either pure elemental iron (Fe(0)) or iron in mild steel as a source of electrons in the reduction of CO(2) to CH(4). These bacteria use Fe(0) oxidation for energy generation and growth. The mechanism of Fe(0) oxidation is cathodic depolarization, in which electrons from Fe(0) and H(+) from water produce H(2), which is then released for use by the methanogens; thermodynamic calculations show that significant Fe(0) oxidation will not occur in the absence of H(2) consumption by the methanogens. The data suggest that methanogens can be significant contributors to the corrosion of iron-containing materials in anaerobic environments.
