Lactate utilization mitigates rumen acidosis and is associated with decreased methane production in the rumen. While several lactate utilization pathways exist across different microbial species in the rumen, how they are metabolically differentiated remains unclear. Here, we show that the key lactate-utilizing species Megasphaera hexanoica and Megasphaera elsdenii display distinct growth strategies based on their fermentative end products. This allows them to co-exist and play distinct metabolic roles, which appear particularly relevant in the early stages of rumen development, as both species are highly enriched in the calf. Specifically, M. hexanoica is more strongly associated with rumen microbiome states that involve increased lactate utilization and preferentially runs reverse beta-oxidation (termed chain elongation) to produce butyrate and medium-chain fatty acids from lactate. As M. elsdenii instead utilizes lactate via the acrylate pathway to produce propionate, we leverage Enzyme Cost Minimization to predict how this pathway relates to a distinct growth strategy. We find that M. elsdenii maximizes growth rate when lactate transiently accumulates, which contrasts M. hexanoica's invariably high-yield strategy. This trade-off, which is supported by the analysis of growth kinetics, metabolic flux, and bioreactors simulating the rumen microbiome, ultimately contributes to co-existence on lactate and may have driven niche differentiation. Lastly, we demonstrate how lactate utilization in the Megasphaera is threatened by toxins widespread in feed, which points to dietary interventions to support calf health.

Alfalfa (Medicago sativa) has a high nutritional value, but poor digestibility of the stems limits its value as an energy source in ruminant diets. Xylan and lignin negatively impact cell wall digestibility, whereas pectins have high digestibility in the rumen. In plants, UDP-xylose synthase (UXS) catalyzes the decarboxylation of UDP-glucuronic acid to form UDP-xylose in an irreversible step that is key for xylan synthesis. We functionally characterized two UXS genes in alfalfa (MsaUXS2 and MsaUXS4) and investigated their impact on ruminal digestibility. Both genes are more highly expressed in stems than leaves, and the enzymes have UDP-glucuronic acid decarboxylase activity in vitro. Silencing MsaUXS2/MsaUXS4 via RNAi altered plant growth and resulted in a 40% decrease in xylose, a 115% increase in arabinose, and a 60% increase in galacturonic acid in the polysaccharide matrix as well as a 20% decrease in lignin in the cell wall. Together, our data show a major role for MsaUXS2 and MsaUXS4 in xylan synthesis and secondary cell wall deposition in alfalfa. Additionally, silenced lines had on average 30% increased gas production at 24 h in in vitro rumen digestibility assays, demonstrating the potential of targeting UXS genes to increase stem digestibility.

The Gram-negative, strictly anaerobic bacterium Megasphaera elsdenii was first isolated from the rumen in 1953 and is common in the mammalian gastrointestinal tract. Its ability to use either lactate or glucose as its major energy sources for growth has been well documented, although it can also ferment amino acids into ammonia and branched-chain fatty acids, which are growth factors for other bacteria. The ruminal abundance of M. elsdenii usually increases in animals fed grain-based diets due to its ability to use lactate (the product of rapid ruminal sugar fermentation), especially at a low ruminal pH (<5.5). M. elsdenii has been proposed as a potential dietary probiotic to prevent ruminal acidosis in feedlot cattle and high-producing dairy cows. However, this bacterium has also been associated with milk fat depression (MFD) in dairy cows, although proving a causative role has remained elusive. This review summarizes the unique physiology of this intriguing bacterium and its functional role in the ruminal community as well as its role in the health and productivity of the host animal. In addition to its effects in the rumen, the ability of M. elsdenii to produce C2–C7 carboxylic acids—potential precursors for industrial fuel and chemical production—is examined.

We evaluated the influence of diets differing in non-fiber carbohydrates and rumen-degradable protein (RDP) levels on changes in the ruminal bacterial populations in lactating Holstein cows. In all, 12 ruminally cannulated cows were assigned to diets with high or low RDP levels. Within each RDP level, molasses was substituted for corn grain at a concentration of 0%, 5.25%, or 10.5% of diet dry matter in a replicated 3 × 3 Latin square design with 28-day periods. Liquid and solid rumen digesta fractions collected at the end of each period underwent 16S rRNA gene sequencing to identify operational taxonomic units and were analyzed for short-chain fatty acids. Protein degradability affected 6 bacterial genera, whereas carbohydrate alteration impacted 13 genera (p < 0.05). Of the 30 genera with the highest relative abundance, 26 differed by digesta fraction (p < 0.05), with Bacteroidetes genera showing a greater abundance in solids and Firmicutes genera demonstrating a greater prevalence in liquids. Regarding relative abundances, with increasing molasses, Succiniclasticum decreased in liquid (p < 0.05), and CF231, YRC22, Clostridium, Desulfovibrio, BF311, and Oscillospira increased in solids (p < 0.05). In contrast, at higher RDP levels, Succiniclasticum increased while YRC22 and Pseudobutyrivibrio decreased in solids (p < 0.05). Genera with abundances found to be correlated with fermentation products in the liquid included Shuttleworthia, Treponema, Lachnospira, and Schwartzia, which typically have lower relative abundances, showing strong positive correlations with molar proportions (mol%) of propionate, butyrate, and valerate (p < 0.05), and negative correlations with pH and acetate mol% (p < 0.05). Fibrobacter was positively correlated with lactate mol% (p < 0.05). Butyrate mol% exhibited a quadratic increase as molasses increased (p = 0.017), and lactate mol% rose with increased RDP levels (p = 0.042). No treatment effects were detected for pH propionate and valerate mol%; however, we observed a tendency (p = 0.075) for a quadratic effect of molasses treatment on the mol% of acetate. These findings substantiate the pivotal role of diet in shaping rumen microbiota and metabolism, elucidating a nuanced relationship between dietary components, bacterial community structure, and metabolic output. This offers a more detailed understanding of rumen function and the potential for high-precision dietary management in lactating cows.

The carboxylate platform employs a diverse microbial consortium of anaerobes in which the methanogens are inhibited. Nearly all biomass components are digested to a mixture of C1-C8 monocarboxylic acids and their corresponding salts. The methane-arrested anaerobic digestion proceeds readily without needing to sterilize biomass or equipment. It accepts a wide range of feedstocks (e.g., agricultural residues, municipal solid waste, sewage sludge, animal manure, food waste, algae, and energy crops), and produces high product yields. This review highlights several important aspects of the platform, including its thermodynamic underpinnings, influences of inoculum source and operating conditions on product formation, and downstream chemical processes that convert the carboxylates to hydrocarbon fuels and oxygenated chemicals. This review further establishes the carboxylate platform as a viable and economical route to industrial biomass utilization.

As major structural components of plant cell walls, cellulose and hemicellulose are degraded and fermented by anaerobic microbes in the rumen to produce volatile fatty acids, the main nutrient source for the host. Cellulose degradation is carried out primarily by specialist bacteria, with additional contributions from protists and fungi, via a variety of mechanisms. Hemicelluloses are hydrolyzed by cellulolytic bacteria and by generalist, non-cellulolytic microbes, largely via extracellular enzymes. Cellulose hydrolysis follows first-order kinetics and its rate is limited by available substrate surface area. Nevertheless, its rate is at least an order of magnitude more rapid than in anaerobic digesters, due to near-obligatory adherence of microbial cells to the cellulose surface, and a lack of downstream inhibitory effects; in the host animal, fiber degradation rate is also enhanced by the unique process of rumination. Cellulolytic and hemicellulolytic microbes exhibit intense competition and amensalism, but they also display mutualistic interactions with microbes at other trophic levels. Collectively, the fiber-degrading community of the rumen displays functional redundancy, partial niche overlap, and convergence of catabolic pathways that all contribute to stability of the ruminal fermentation. The superior hydrolytic and fermentative capabilities of ruminal fiber degraders make them promising candidates for several fermentation technologies.

An error has been noted in values for DMI in Table 3 (page 270). The corrected table is shown below, with corrected values shown in bold. The authors regret the error. Table 3Production and ruminal chemistry data for cows in the different milk fat response groups1Response groups: cows with milk fat percentage affected by starch fermentability (S-responsive), monensin (M-responsive; Rumensin 80, Elanco Animal Health, Greenfield, IN), or starch fermentability and monensin (SM-responsive), and cows not affected by either starch fermentability or monensin (nonresponsive).MeasurementS-responsiveM-responsiveSM-responsiveNonresponsiveCow 1664Cow 2464Cow 1272Cow 2088Cow 1884Cow 2082Cow 1692Cow 2470Production data,2Production data from last 5 d (10 milkings) of each 28-d period, averaged across all 4 treatment periods. kg/d DMI25.024.226.622.025.224.126.624.2 Milk yield48.035.938.432.734.922.142.636.8 3.5% FCM40.234.442.832.738.427.642.237.6 Protein1.251.161.230.971.180.861.221.06 Lactose2.181.892.341.982.241.482.251.82Ruminal chemistry3Ruminal chemistry from samples collected 6 h postfeeding on last 3 d of each 28-d period, averaged across all 4 treatment periods. pH5.725.945.876.096.056.125.885.96 Total VFA, mM159156149135136145151129 Acetate, mol %57.658.758.862.764.364.363.063.7 Propionate, mol %22.923.723.217.519.217.720.219.6 Butyrate, mol %13.212.111.614.312.413.312.612.3 Acetate:propionate ratio2.722.562.693.643.413.683.173.30Total number of ARISA peaks,4ARISA = automated ribosomal intergenic spacer analysis; AL = amplicon length. Includes total number of unique AL identified in full set of 24 samples (3 daily samples × 4 experimental periods × 2 phases) for each cow. AL1761751881731761821711681 Response groups: cows with milk fat percentage affected by starch fermentability (S-responsive), monensin (M-responsive; Rumensin 80, Elanco Animal Health, Greenfield, IN), or starch fermentability and monensin (SM-responsive), and cows not affected by either starch fermentability or monensin (nonresponsive).2 Production data from last 5 d (10 milkings) of each 28-d period, averaged across all 4 treatment periods.3 Ruminal chemistry from samples collected 6 h postfeeding on last 3 d of each 28-d period, averaged across all 4 treatment periods.4 ARISA = automated ribosomal intergenic spacer analysis; AL = amplicon length. Includes total number of unique AL identified in full set of 24 samples (3 daily samples × 4 experimental periods × 2 phases) for each cow. Open table in a new tab Shifts in bacterial community composition in the rumen of lactating dairy cows under milk fat-depressing conditionsJournal of Dairy ScienceVol. 93Issue 1PreviewEighteen 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). Full-Text PDF Open Archive

Rumen fluid (RF) as inocula is useful for evaluating biomass digestibility and has potential for producing volatile fatty acids (VFA) via the carboxylate platform. However, RF is not readily available, necessitating evaluation of potential preservation methods. Glycerol (50% v/v) and DMSO (5% v/v) were used to preserve rumen inocula for 3 months at -80 degrees C. Effects of cryo-preservation on digestibility, VFA production and community composition with beta-diversity distance metrics were compared to fresh RF using apple, citrus and grape pomace as substrates. For all substrates, DMSO cryo-preserved rumen digestibility parameters, VFA yield and product distribution were more significantly comparable to fresh RF (P > 0.05) than was glycerol cryo-preserved RF. Similarly, beta-diversity coefficient (unweighted unifrac) between DMSO cryo-preserved RF and fresh RF was 0.250 while the coefficient was 0.359 for the glycerol cryo-preserved RF compared to fresh RF. This showed that a DMSO cryo-preserved RF is less affected by preservation effects and is a more promising alternative to fresh RF.

Characterisation of pomace from citrus fruits, grapes and apples has shown to be rich in compounds that contain untapped energetic value with potential to serve as feedstock for biochemical and biofuel production. This study aimed to investigate the potential of recovering the energy in citrus (CtP), grape (GP) and apple (AP) pomace by extraruminal fermentations to produce VFA with utility to serve as precursors for hydrocarbon fuels. Chemical composition, neutral detergent fiber digestibility (seven incubation time points) and volatile fatty acid yield (after 96 h extraruminal fermentation) were analysed and compared among the three-pomace substrates. There were no significant differences (P > 0.05) in the apparent digestibility of CtP (77.3%) and AP (75.2%) while that of GP (55.2%) differed significantly from both (P < 0.05). VFA yields corresponded to the digestibility in that GP (97.1 mM concentration, with a fractional yield 0.38 out of a possible 0.75) yielded the lowest amount of VFA compared to AP (126.4 mM concentration, with a fractional yield of 0.48 out of a possible 0.75) and CtP (136.4 mM concentration, with a fractional yield of 0.52 out of a possible 0.75). Analysis of the alkyl chain lengths of the VFA showed that both CtP (12.16 mmol alkyl g−1) and AP (11.31 mmol alkyl g−1) can serve as substrates for VFA electrochemical conversion to hydrocarbons. The results reported here have industrial implications as the produced VFA and the amount of alkyl chain lengths are the direct determinants of the mixture of hydrocarbons that can be produced.

Pretreatment of biomass feedstocks is considered necessary to increase their conversion to biofuels and other bioproducts. However, even the best chemical and physical pretreatments have disadvantages that make the development of alternative strategies to improve biomass fermentability a worthy pursuit. An interesting natural process is that of rumination, by which ruminant animals regurgitate their food for re-chewing to reduce particle size, increase surface area, and accelerate its biodegradation by the animal's mutualistic microbial community. Detailed examination of the process reveals that it is a unique, unusually effective, and energy-efficient type of physical treatment of fibrous biomass. Effectiveness and energy efficiency are gained by several unappreciated aspects of the process, including: full wetting of the biomass prior to its exposure to re-chewing; a hypsodont dentition pattern and jaw movement designed to maximize shearing and delamination (rather than cutting) of the biomass; and an effective feed sorting mechanism that results in a preferential processing of the larger particles that are in need of further grinding, rather than smaller particles whose additional grinding would provide little benefit. These nuances of the rumination process suggest designs for accomplishing similar particle size reduction and surface area increases within bioreactors during cellulosic biomass fermentation ("co -treatment"). While the rumination process has evolved within the context of allowing the ruminant animal to exploit widely available but highly fibrous feeds, it has the potential for broad applicability and improved efficiency for industrial conversion of cellulosic biomass by both pure and mixed cultures.

Abstract The rumen microbial community is the agent that allows cattle and other ruminants to process complex plant polymers into digestible fatty acids. Traditional methods to sample rumen microbes often involve labor-intensive stomach tubing, or invasive surgeries to access the rumen lumen via cannula ports, thereby limiting the number of animals that could be sampled in a specific study. In this study, we tested the viability of using buccal swabs as a proxy of the rumen microbial contents in a timecourse experiment on eight cannulated cows. Rumen contents and buccal swabs were collected at six equally spaced timepoints, with the first timepoint being 2 hours prior to feeding. Simpson diversity and Shannon evenness estimates of the microbial counts of each sample revealed that the first timepoint had the lowest diversity and highest evenness (Tukey HSD < 0.05) out of all other timepoints. Principal component analysis confirmed that the buccal swab samples from the first timepoint were the most similar to paired rumen samples taken at the same times. Using a Random Forest Classifier analysis, we estimated the Gini importance scores for individual microbial taxa as a proxy of their uniqueness to the rumen or oral environments of the cows. We identified 18 oral-only microbial taxa that are contaminants and could be removed from future comparisons using this method. Finally, we attempted to estimate the exact relative abundance of rumen microbial taxa from buccal swab samples using paired rumen-swab data in a Random Forest Regression model. The model was found to have moderate (~38%) accuracy in cross-validation studies. Our data suggests that buccal swabs can serve as fast and suitable proxies for rumen microbial contents of dairy cattle, but that additional factors must be measured to improve direct regression of results to those of the rumen.

We describe a method that adds long-read sequencing to a mix of technologies used to assemble a highly complex cattle rumen microbial community, and provide a comparison to short read-based methods. Long-read alignments and Hi-C linkage between contigs support the identification of 188 novel virus-host associations and the determination of phage life cycle states in the rumen microbial community. The long-read assembly also identifies 94 antimicrobial resistance genes, compared to only seven alleles in the short-read assembly. We demonstrate novel techniques that work synergistically to improve characterization of biological features in a highly complex rumen microbial community.

The rumen is a specialized foregut in ruminant animals that is the site of an active, anaerobic microbial fermentation of feedstuffs to produce volatile fatty acids and microbial cell protein that serve as primary nutrient sources for the host. Sequence analysis of DNA from ruminal contents has revealed that the microbial community contains hundreds to thousands of species of bacteria, protozoa, fungi, and archaea, the vast majority of which have eluded isolation and characterization. The community provides numerous examples of interspecific interactions (competition, amensalism, mutualism, commensalism and predation) that maintain a balanced fermentative activity. From birth the ruminal community changes from one characteristic of nonruminants to one more compatible with the developing rumen organ. The mature community displays substantial functional redundancy among its members, along with resilience when subjected to perturbation. Although diet is the major driver of ruminal microbial composition, there is substantial individuality of the community for each host. Progress is being made in identifying important species and assemblages that contribute to rumen function, and to specific physiological outputs (e.g., feed efficiency and milk composition) that are important in ruminant agriculture.

Biomass derived from low-value, high-volume invasive plant species is an attractive, alternative feedstock to produce biofuels and biochemicals. This study aimed to use the carboxylate platform to valorize the invasive leguminous shrub, Prosopis juliflora (Mesquite), by utilizing in vitro rumen fermentations without chemical pretreatment to produce volatile fatty acids. The three fractions of the mesquite: leaves (ProL), stems (ProS) and branches (ProB) were compared regarding chemical composition, neutral detergent fiber (NDF) digestibility at 7 time points and VFA production after 72 h with sugarcane bagasse (SCB) as a reference. NDF digestibility was significantly (P < 0.05) higher in ProL (35.8%) than ProS (30.4%) and ProB (20.9%) compared to SCB (21.9%). VFA concentrations from 20 g biomass L-1 showed significant differences with 8.07, 6.71 and 6.51 g L-1 for ProL, ProS and ProB respectively, while SCB yielded 4.02 g L-1. These concentrations were comparable with other platforms that employ chemically pretreated biomass for VFA production.

In vitro ruminal fermentations resemble in vivo fermentations with respect to substrate consumption and distribution of fermentation products in short term (1-5 d) incubations. However, little is known regarding changes in in vitro fermentations over prolonged incubation or multiple transfers. Gas production, pH, fermentation products, and bacterial community composition were examined in duplicate in vitro fermentations of switchgrass plus distillers grains that were transferred at 3-4 d intervals over 900 d. Additionally, duplicate fermentations inoculated from 160 d-old enrichments into the same medium but supplemented with ethanol, and transferred at 3-4 d over a 730 d period were characterized. SWG and SWG + E fermentation showed marked differences in community composition, pH, total product concentrations and ratios, relative to each other and to the original inoculum. The results have implications for the use of ruminal inocula for industrial production of short- and medium-chain fatty acids via the carboxylate platform.

A major goal in dairy research is to improve milk production efficiency (MPE). With the advent of next-generation sequencing, efforts are underway to improve MPE by manipulating the rumen microbiota. MPE correlates to ruminal bacterial community composition (BCC), but the adult rumen microbiota is highly stable and returns to a baseline BCC after heavy perturbation. We seek to influence rumen BCC by early intervention in pre-weaning calves. Two cannulated Holstein donors of disparate MPE were selected. Three cohorts of 6 bull calves were established and dosed by gavage with a rumen inoculum sourced from the high-efficiency donor (HE), the low-efficiency donor (LE), or an autoclaved 50:50 mix as a microbe-free control (C). Dosing occurred at birth, then biweekly through 6 weeks. Feces were collected at each dosing. Daily preweaning intake of calf starter, which correlates to downstream feed efficiency, was greatest in HE calves and lowest in C calves (P < 0.05), though preweaning average daily gain did not differ between cohorts (P = 0.210). Calves were sacrificed at 8 weeks to access rumen contents and rumen wall sections were collected to assess papillation. Fecal and rumen samples were subjected to 16S rRNA amplicon sequencing. BCC differed by cohort in fecal and rumen samples (P < 0.05), with HE calf samples most similar to adult rumen samples and C calves least similar. Additionally, HE calves tended to have elongated papillae (P =0.062), the development of which is dependent on byproducts of microbial metabolism in the rumen and which points to differences in potential absorptive capacity of the epithelium. These data demonstrate that the rumen BCC can be influenced by early intervention. Future work includes expansion of this dosing protocol to a cohort of 60 female calves, following rumen BCC development and the impact on MPE in through the first lactation.

ABSTRACT 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 were correlated to growth and milk production of animals raised on calf starter grains and/or corn silage to determine if early-life diet has 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. SIGNIFICANCE The gut microbiota is essential to the survival of many organisms, including ruminants that rely on microorganisms for nutrient acquisition from dietary inputs toward 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, animal 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.

The Dietary Guidelines for Americans Advisory Committee suggested that a reduction of animal-source foods would enhance sustainability and healthfulness of US diets. This work quantified contributions of domestically consumable dairy products to human-edible nutrient supply and greenhouse gas (GHG) emissions of US agriculture. Data on US dairy production were obtained from the analysis conducted by White and Hall (2017; https://doi.org/10.1073/pnas.1707322114), which utilized data from USDA databases; the US Food and Drug Administration; the US Environmental Protection Agency; the United Nations Food and Agriculture Organization; and peer-reviewed, published sources. We disaggregated the reported animal metrics to specifically assess contributions of dairy products available for domestic consumption. All assessments terminated at fluid milk production and did not consider further processing. Census Bureau data describing the US population by age and sex groups were matched to nutrient requirements (USDA and World Health Organization) to identify weighted average requirements of the US population. Dairy products were estimated to meet the average energy, protein, and calcium requirements of 71.2, 169, and 254 million people, respectively. The contributions of consumable dairy products to available domestic supply of energy (11% of total consumable supply), protein (20%), lysine (28%), methionine (24%), calcium (73%), vitamin A (39%), vitamin D (54%), riboflavin (47%), vitamin B12 (47%), and choline (29%) were also quantified. Dairy products available for domestic consumption account for 1.4% of US anthropogenic emissions, 16% of agricultural emissions, and 32% of emissions from animal agriculture. Although this work provides an initial snapshot of how dairy products contribute to nutrient supply and GHG emissions, future work should explore economics, land and resource use efficiency, impact of processing, and targeted assessment of alternative nutrient sources (supplements, dietary fortification, etc.) to better quantify and understand the trade-offs.

BACKGROUND:Cellulose is the most abundant biological polymer on earth, making it an attractive substrate for the production of next-generation biofuels and commodity chemicals. However, the economics of cellulose utilization are currently unfavorable due to a lack of efficient methods for its hydrolysis. Fibrobacter succinogenes strain S85, originally isolated from the bovine rumen, is among the most actively cellulolytic mesophilic bacteria known, producing succinate as its major fermentation product. In this study, we examined the transcriptome of F. succinogenes S85 grown in continuous culture at several dilution rates on cellulose, cellobiose, or glucose to gain a system-level understanding of cellulose degradation by this bacterium.RESULTS:Several patterns of gene expression were observed for the major cellulases produced by F. succinogenes S85. A large proportion of cellulase genes were constitutively expressed, including the gene encoding for Cel51A, the major cellulose-binding endoglucanase produced by this bacterium. Moreover, other cellulase genes displayed elevated expression during growth on cellulose relative to growth on soluble sugars. Growth rate had a strong effect on global gene expression, particularly with regard to genes predicted to encode carbohydrate-binding modules and glycoside hydrolases implicated in hemicellulose degradation. Expression of hemicellulase genes was tightly regulated, with these genes displaying elevated expression only during slow growth on soluble sugars. Clear differences in gene expression were also observed between adherent and planktonic populations within continuous cultures growing on cellulose.CONCLUSIONS:This work emphasizes the complexity of the fiber-degrading system utilized by F. succinogenes S85, and reinforces the complementary role of hemicellulases for accessing cellulose by these bacteria. We report for the first time evidence of global differences in gene expression between adherent and planktonic populations of an anaerobic bacterium growing on cellulose at steady state during continuous cultivation. Finally, our results also highlight the importance of controlling for growth rate in investigations of gene expression.