Diauxic growth of Fibrobacter succinogenes S85 on cellobiose and lactose.

Fibrobacter succinogenes rapidly colonizes the preruminant calf rumen and becomes a dominant cellulolytic bacterium in the rumen after weaning. Although F. succinogenes actively degrades cellulose in the rumen, it seems that there is no or little of its substrate, cellulose, in the rumen of preweaned calves. We thus evaluated the ability of F. succinogenes to utilize lactose, a main sugar of milk, with or without the presence of cellobiose. We grew F. succinogenes S85 on media containing 2.5% lactose combined with 0%-0.2% cellobiose or a medium with 0.2% cellobiose but without lactose. The generation times on the 0.2% cellobiose medium and the 2.5% lactose medium were 1.9 and 16.2 h, respectively. The bacterium showed rapid growth on cellobiose and diauxic growth on the lactose media containing 0.05%-0.2% cellobiose. Moreover, the production of ß-galactosidase was low in the presence of 0.1%-0.2% cellobiose. Since the ß-galactosidase contained a signal peptide and a Por secretion system C-terminal sorting domain, we speculate that the ß-galactosidase would be secreted from the bacterial cells by the Por secretion system. Our data indicate the possibility that F. succinogenes could colonize preruminant calf rumen, consuming the lactose present in cow milk.

Modulatory effects of condensed tannin fractions of different molecular weights from a Leucaena leucocephala hybrid on the bovine rumen bacterial community in vitro.

BACKGROUND: Condensed tannin (CT) fractions of different molecular weights (MWs) may affect rumen microbial metabolism by altering bacterial diversity. In this study the effects of unfractionated CTs (F0) and five CT fractions (F1-F5) of different MWs (F1, 1265.8 Da; F2, 1028.6 Da; F3, 652.2 Da; F4, 562.2 Da; F5, 469.6 Da) from Leucaena leucocephala hybrid-Rendang (LLR) on the structure and diversity of the rumen bacterial community were investigated in vitro. RESULTS: Real-time polymerase chain reaction assay showed that the total bacterial population was not significantly (P > 0.05) different among the dietary treatments. Inclusion of higher-MW CT fractions F1 and F2 significantly (P < 0.05) increased the Fibrobacter succinogenes population compared with F0 and CT fractions F3-F5. Although inclusion of F0 and CT fractions (F1-F5) significantly (P < 0.05) decreased the Ruminococcus flavefaciens population, there was no effect on the Ruminococcus albus population when compared with the control (without CTs). High-throughput sequencing of the V3 region of 16S rRNA showed that the relative abundance of genera Prevotella and unclassified Clostridiales was significantly (P < 0.05) decreased, corresponding with increasing MW of CT fractions, whereas cellulolytic bacteria of the genus Fibrobacter were significantly (P < 0.05) increased. Inclusion of higher-MW CT fractions F1 and/or F2 decreased the relative abundance of minor genera such as Ruminococcus, Streptococcus, Clostridium XIVa and Anaeroplasma but increased the relative abundance of Acinetobacter, Treponema, Selenomonas, Succiniclasticum and unclassified Spirochaetales compared with the control and lower-MW CT fractions. CONCLUSION: This study indicates that CT fractions of different MWs may play an important role in altering the structure and diversity of the rumen bacterial community in vitro, and the impact was more pronounced for CT fractions with higher MW. © 2016 Society of Chemical Industry.

Influence of Substrates on the Surface Characteristics and Membrane Proteome of Fibrobacter succinogenes S85.

Although Fibrobacter succinogenes S85 is one of the most proficient cellulose degrading bacteria among all mesophilic organisms in the rumen of herbivores, the molecular mechanism behind cellulose degradation by this bacterium is not fully elucidated. Previous studies have indicated that cell surface proteins might play a role in adhesion to and subsequent degradation of cellulose in this bacterium. It has also been suggested that cellulose degradation machinery on the surface may be selectively expressed in response to the presence of cellulose. Based on the genome sequence, several models of cellulose degradation have been suggested. The aim of this study is to evaluate the role of the cell envelope proteins in adhesion to cellulose and to gain a better understanding of the subsequent cellulose degradation mechanism in this bacterium. Comparative analysis of the surface (exposed outer membrane) chemistry of the cells grown in glucose, acid-swollen cellulose and microcrystalline cellulose using physico-chemical characterisation techniques such as electrophoretic mobility analysis, microbial adhesion to hydrocarbons assay and Fourier transform infra-red spectroscopy, suggest that adhesion to cellulose is a consequence of an increase in protein display and a concomitant reduction in the cell surface polysaccharides in the presence of cellulose. In order to gain further understanding of the molecular mechanism of cellulose degradation in this bacterium, the cell envelope-associated proteins were enriched using affinity purification and identified by tandem mass spectrometry. In total, 185 cell envelope-associated proteins were confidently identified. Of these, 25 proteins are predicted to be involved in cellulose adhesion and degradation, and 43 proteins are involved in solute transport and energy generation. Our results supports the model that cellulose degradation in F. succinogenes occurs at the outer membrane with active transport of cellodextrins across for further metabolism of cellodextrins to glucose in the periplasmic space and inner cytoplasmic membrane.

Monitoring of gene expression in Fibrobacter succinogenes S85 under the co-culture with non-fibrolytic ruminal bacteria.

Fibrobacter succinogenes is one of the most pivotal fibrolytic bacterial species in the rumen. In a previous study, we confirmed enhancement of fiber digestion in a co-culture of F. succinogenes S85 with non-fibrolytic ruminal strains R-25 and/or Selenomonas ruminantium S137. In the present study, mRNA expression level of selected functional genes in the genome of F. succinogenes S85 was monitored by real-time RT-PCR. Growth profile of F. succinogenes S85 was similar in both the monoculture and co-cultures with non-fibrolytics. However, expression of 16S rRNA gene of F. succinogenes S85 in the co-culture was higher (P < 0.01) than that of the monoculture. This finding suggests that metabolic activity of F. succinogenes S85 was enhanced by coexistence with strains R-25 and/or S. ruminantium S137. The mRNA expression of fumarate reductase and glycoside hydrolase genes was up-regulated (P < 0.01) when F. succinogenes S85 was co-cultured with non-fibrolytics. These results indicate the enhancement of succinate production and fiber hydrolysis by F. succinogenes S85 in co-cultures of S. ruminantium and R-25 strains.

The effect of fibre source on the numbers of some fibre-degrading bacteria of Arabian camel's (Camelus dromedarius) foregut origin.

The total bacterial community of Fibrobacter succinogenes and Ruminococcus flavefaciens in fibre-enriched culture of the foregut contents of 12 adult feral camels (Camelus dromedaries) fed on native vegetation in Australia was investigated using quantitative PCR. Foregut contents were collected postmortem, pooled and filtered before divided into two fractions. One fraction was used for extraction of DNA, while the other fraction was inoculated straight away into BM 10 contained filter paper (FP), cotton thread (CT) or neutral detergent fibre (NDF) as the sole carbohydrate sources in Hungate tubes. The tubes were incubated anaerobically at 39 °C for 1 week. After a near complete degradation of the FP and CT and extensive turbidity in the NDF, media subculturing was carried out into fresh media tubes. This was repeated twice before genomic DNA was extracted and used for quantification of bacteria. Using an absolute quantification method, the numbers of cells in 1 ml of each sample ranged from 4.07 × 10(6) to 2.73 × 10(9) for total bacteria, 1.34 × 10(3) to 2.17 × 10(5) for F. succinogenes and 5.78 × 10(1) to 3.53 × 10(4) for R. flavefaciens. The mean cell number of F. succinogenes was highest in the FP enrichment medium at approximately 107-fold, whereas for the R. flavefaciens targeted primer, the NDF enrichment media had the highest mean cell number at approximately 4-fold when compared to the rumen content. The data presented here provide evidence of fibre type preference by the two main fibre-degrading bacteria and would help us understand the interaction between fibre type and fibre-degrading microorganisms, which has ramification on camel nutrition at different seasons and environments.

Magnesium requirement of some of the principal rumen cellulolytic bacteria.

Information available on the role of Mg for growth and cellulose degradation by rumen bacteria is both limited and inconsistent. In this study, the Mg requirements for two strains each of the cellulolytic rumen species Fibrobacter succinogenes (A3c and S85), Ruminococcus albus (7 and 8) and Ruminococcus flavefaciens (B34b and C94) were investigated. Maximum growth, rate of growth and lag time were all measured using a complete factorial design, 2(3)×6; factors were: strains (2), within species (3) and Mg concentrations (6). R. flavefaciens was the only species that did not grow when Mg was singly deleted from the media, and both strains exhibited a linear growth response to increasing Mg concentrations (P<0.001). The requirement for R. flavefaciens B34b was estimated as 0.54 mM; whereas the requirement for R. flavefaciens C94 was >0.82 as there was no plateau in growth. Although not an absolute requirement for growth, strains of the two other species of cellulolytic bacteria all responded to increasing Mg concentrations. For F. succinogenes S85, R. albus 7 and R. albus 8, their requirement estimated from maximum growth was 0.56, 0.52 and 0.51, respectively. A requirement for F. succinogenes A3c could not be calculated because there was no solution for contrasts. Whether R. flavefaciens had a Mg requirement for cellulose degradation was determined in NH3-free cellulose media, using a 2×4 factorial design, 2 strains and 4 treatments. Both strains of R. flavefaciens were found to have an absolute Mg requirement for cellulose degradation. Based on reported concentrations of Mg in the rumen, 1.0 to 10.1 mM, it seems unlikely that an in vivo deficiency of this element would occur.

Eremophila glabra reduces methane production and methanogen populations when fermented in a Rusitec.

Eremophila glabra Juss. (Scrophulariaceae), a native Australian shrub, has been demonstrated to have low methanogenic potential in a batch in vitro fermentation system. The present study aimed to test longer-term effects of E. glabra on rumen fermentation characteristics, particularly methane production and the methanogen population, when included as a component of a fermentation substrate in an in vitro continuous culture system (Rusitec). E. glabra was included at 150, 250, 400 g/kg DM (EG15, EG25, and EG40) with an oaten chaff and lupin-based substrate (control). Overall, the experiment lasted 33 days, with 12 days of acclimatization, followed by two periods during which fermentation characteristics (total gas, methane and VFA productions, dry matter disappearance, pH) were measured. The number of copies of genes specifically associated with total bacteria and cellulolytic bacteria (16S rRNA gene) and total ruminal methanogenic archaeal organisms (the methyl coenzyme M reductase A gene (mcrA)) was also measured during this time using quantitative real-time PCR. Total gas production, methane and volatile fatty acid concentrations were significantly reduced with addition of E. glabra. At the end of the experiment, the overall methane reduction was 32% and 45% for EG15 and EG25 respectively, compared to the control, and the reduction was in a dose-dependent manner. Total bacterial numbers did not change, but the total methanogen population decreased by up to 42.1% (EG40) when compared to the control substrate. The Fibrobacter succinogenes population was reduced at all levels of E. glabra, while Ruminococcus albus was reduced only by EG40. Our results indicate that replacing a portion of a fibrous substrate with E. glabra maintained a significant reduction in methane production and methanogen populations over three weeks in vitro, with some minor inhibition on overall fermentation at the lower inclusion levels.

Effects of monolaurin on ruminal methanogens and selected bacterial species from cattle, as determined with the rumen simulation technique.

Before being able to implement effective ruminal methane mitigation strategies via feed supplementation, the assessment of side effects on ruminal fermentation and rumen microbial populations is indispensable. In this respect we investigated the effects of monolaurin, a methane-mitigating lipid, on methanogens and important carbohydrate-degrading bacteria present in ruminal fluid of dairy cattle in continuous culture employing the rumen simulation technique. In six experimental runs, each lasting for 10 days, four diets with different carbohydrate composition, based on hay, maize, wheat and a maize-wheat mixture, either remained non-supplemented or were supplemented with monolaurin and incubated in a ruminal-fluid buffer mixture. Incubation liquid samples from days 6 to 10 of incubation were analyzed with relative quantitative polymerase chain reaction (qPCR) of 16S rRNA genes to assess monolaurin-induced shifts in specific rumen microbial populations in relation to the corresponding non-supplemented diets. Monolaurin completely inhibited Fibrobacter succinogenes in all diets while the response of the other cellulolytic bacteria varied in dependence of the diet. Megasphaera elsdenii remained unaffected by monolaurin in the two diets containing maize, but was slightly stimulated by monolaurin with the wheat and largely with the hay diet. The supply of monolaurin suppressed Methanomicrobiales below the detection limit with all diets, whereas relative 16S rRNA gene copy numbers of Methanobacteriales increased by 7-fold with monolaurin in case of the hay diet. Total Archaea were decreased by up to over 90%, but this was significant only for the wheat containing diets. Thus, monolaurin exerted variable effects mediated by unknown mechanisms on important ruminal microbes involved in carbohydrate degradation, along with its suppression of methane formation. The applicability of monolaurin for methane mitigation in ruminants thus depends on the extent to which adverse effects on carbohydrate-degrading bacteria actually impair the supply of digested carbohydrates to the animal.

Influence of the composition of the cellulolytic flora on the development of hydrogenotrophic microorganisms, hydrogen utilization, and methane production in the rumens of gnotobiotically reared lambs.

We investigated the influence of the composition of the fibrolytic microbial community on the development and activities of hydrogen-utilizing microorganisms in the rumens of gnotobiotically reared lambs. Two groups of lambs were reared. The first group was inoculated with Fibrobacter succinogenes, a non-H(2)-producing species, as the main cellulolytic organism, and the second group was inoculated with Ruminococcus albus, Ruminococcus flavefaciens, and anaerobic fungi that produce hydrogen. The development of hydrogenotrophic bacterial communities, i.e., acetogens, fumarate and sulfate reducers, was monitored in the absence of methanogens and after inoculation of methanogens. Hydrogen production and utilization and methane production were measured in rumen content samples incubated in vitro in the presence of exogenous hydrogen (supplemented with fumarate or not supplemented with fumarate) or in the presence of ground alfalfa hay as a degradable substrate. Our results show that methane production was clearly reduced when the dominant fibrolytic species was a non-H(2)-producing species, such as Fibrobacter succinogenes, without significantly impairing fiber degradation and fermentations in the rumen. The addition of fumarate to the rumen contents stimulated H(2) utilization only by the ruminal microbiota inoculated with F. succinogenes, suggesting that these communities could play an important role in fumarate reduction in vivo.

Analysis of a continuous culture of Fibrobacter succinogenes S85 on a standardized glucose medium.

Continuous cultures of Fibrobacter succinogenes S85 were performed on a standardized fully synthetic culture medium with glucose as carbon source at a dilution rate (D = 0.02 h(-1)) in a 5-L bioreactor. The culture was stabilized during 20 days and demonstrated the ability of Fibrobacter succinogenes to grow in this synthetic medium. CO(2) partial pressure and redox potential probes were used to check the anaerobic state of the culture. The biomass yield was calculated 0.206 g (g glucose)(-1) and the production yield of succinate, the major end-product, was 0.63 mol (mol glucose)(-1). The consistency of the experimental data was checked by proton and mass (C, N) balances. The results were satisfactory (90-110% recovery) leading to derive a stoichiometric equation representative of the growth on glucose. The stoichiometric coefficients were calculated using data reconciliation and linear algebra methods enabling to obtain a complete modeling of all conversion yields possible.

Ionized calcium requirement of rumen cellulolytic bacteria.

Ionized calcium (Ca(+2)) appears to be required by the 3 predominant species of rumen cellulolytic bacteria, Fibrobacter succinogenes, Ruminococcus flavefaciens, and Ruminococcus albus. The present study evaluated the role of ionized calcium in growth and cellulose digestion. Maximum growth or rate and extent of digestion and lag time were the criteria used to evaluate Ca(+2) requirements. All cultures except F. succinogenes A3c grew when repeatedly transferred in a medium without added Ca(+2). As Ca(+2) concentration increased in cellobiose medium, the rate of growth increased and lag time decreased for F. succinogenes A3c, whereas F. succinogenes S85 exhibited increases in both maximum growth and rate of growth. No responses in any of the criteria were observed for the ruminococci in cellobiose medium. Both strains of F. succinogenes had an absolute requirement for Ca(+2) with cellulose as the only substrate. For strain A3c the requirement was 0.36 to 0.42 mM and for S85, >0.64 mM. Increases in extent of cellulose degradation occurred with all strains of ruminococci as Ca(+2) concentration increased; however, degradation in Ca(+2)-free medium was similar to that of F. succinogenes with Ca(+2). Although the ruminococci presumably have cellulosomes that require Ca(+2) in their structure, such was not evident in our studies. The function of Ca(+2) in cellulose degradation by F. succinogenes is unknown, but may be related to the secretion or activation of their cellulolytic enzymes. Based on reported concentrations of Ca(+2) in the rumen, it seems unlikely that an in vivo deficiency would occur for these bacteria.

Application of a data reconciliation method to the stoichiometric analysis of Fibrobacter succinogenes growth.

Fibrobacter succinogenes S85, a strictly anaerobic Gram-negative bacterium, was grown in continuous culture in a bioreactor at different dilution rates (0.02 to 0.092 h(-1)) on a fully synthetic culture medium with glucose as carbon source. Glucose and ammonium sulfate consumption, as well as biomass, succinate, acetate, formate, and carbohydrate production were regularly measured. The relevant biomass elemental compositions were established for each dilution rate. Robustness of the experimental information was checked by C and N mass balances estimation, which were satisfactory. A detailed overall stoichiometry analysis of the process, including all substrates and products of the culture, was proposed. Online and off-line parameters measured during the culture brought a large number of data which were weighted by their respective variance associated to the measured value. The material balance resulted in an overdetermined linear system of equations made of weighted relationships including experimental data, elemental balances (C, H, O, N, S, Na), and an additional constraint. The mass balances involved in stoichiometric equations were solved using data reconciliation and linear algebra methods to take into account error measurements. This methodology allowed to establish the overall stoichiometric equation for each dilution rate studied.

Outer membrane proteins of Fibrobacter succinogenes with potential roles in adhesion to cellulose and in cellulose digestion.

Comparative analysis of binding of intact glucose-grown Fibrobacter succinogenes strain S85 cells and adhesion-defective mutants AD1 and AD4 to crystalline and acid-swollen (amorphous) cellulose showed that strain S85 bound efficiently to both forms of cellulose while mutant Ad1 bound to acid-swollen cellulose, but not to crystalline cellulose, and mutant Ad4 did not bind to either. One- and two-dimensional electrophoresis (2-DE) of outer membrane cellulose binding proteins and of outer membranes, respectively, of strain S85 and adhesion-defective mutant strains in conjunction with mass spectrometry analysis of tryptic peptides was used to identify proteins with roles in adhesion to and digestion of cellulose. Examination of the binding to cellulose of detergent-solubilized outer membrane proteins from S85 and mutant strains revealed six proteins in S85 that bound to crystalline cellulose that were absent from the mutants and five proteins in Ad1 that bound to acid-swollen cellulose that were absent from Ad4. Twenty-five proteins from the outer membrane fraction of cellulose-grown F. succinogenes were identified by 2-DE, and 16 of these were up-regulated by growth on cellulose compared to results with growth on glucose. A protein identified as a Cl-stimulated cellobiosidase was repressed in S85 cells growing on glucose and further repressed in the mutants, while a cellulose-binding protein identified as pilin was unchanged in S85 grown on glucose but was not produced by the mutants. The candidate differential cellulose binding proteins of S85 and the mutants and the proteins induced by growth of S85 on cellulose provide the basis for dissecting essential components of the cellulase system of F. succinogenes.

Degradation of wheat straw by Fibrobacter succinogenes S85: a liquid- and solid-state nuclear magnetic resonance study.

Wheat straw degradation by Fibrobacter succinogenes was monitored by nuclear magnetic resonance (NMR) spectroscopy and chemolytic methods to investigate the activity of an entire fibrolytic system on an intact complex substrate. In situ solid-state NMR with 13C cross-polarization magic angle spinning was used to monitor the modification of the composition and structure of lignocellulosic fibers (of 13C-enriched wheat straw) during the growth of bacteria on this substrate. There was no preferential degradation either of amorphous regions of cellulose versus crystalline regions or of cellulose versus hemicelluloses in wheat straw. This suggests either a simultaneous degradation of the amorphous and crystalline parts of cellulose and of cellulose and hemicelluloses by the enzymes or degradation at the surface at a molecular scale that cannot be detected by NMR. Liquid-state two-dimensional NMR experiments and chemolytic methods were used to analyze in detail the various sugars released into the culture medium. An integration of NMR signals enabled the quantification of oligosaccharides produced from wheat straw at various times of culture and showed the sequential activities of some of the fibrolytic enzymes of F. succinogenes S85 on wheat straw. In particular, acetylxylan esterase appeared to be more active than arabinofuranosidase, which was more active than alpha-glucuronidase. Finally, cellodextrins did not accumulate to a great extent in the culture medium.

Use of community genome arrays (CGAs) to assess the effects of Acacia angustissima on rumen ecology.

This research developed a community genome array (CGA) to assess the effects of Acacia angustissima on rumen microbiology. A. angustissima produces non-protein amino acids as well as tannins, which may be toxic to animals, and CGA was used to assess the effects of this plant on the ecology of the rumen. CGAs were developed using a 7.5 cmx2.5 cm nylon membrane format that included up to 96 bacterial genomes. It was possible to separately hybridize large numbers of membranes at once using this mini-membrane format. Pair-wise cross-hybridization experiments were conducted to determine the degree of cross-hybridization between strains; cross-hybridization occurred between strains of the same species, but little cross-reactivity was observed among different species. CGAs were successfully used to survey the microbial communities of animals consuming an A. angustissima containing diet but quantification was not precise. To properly quantify and validate the CGA, Fibrobacter and Ruminococcus populations were independently assessed using 16S rDNA probes to extracted rRNA. The CGA detected an increase in these populations as acacia increased in the diet, which was confirmed by rRNA analysis. There was a great deal of variation among strains of the same species in how they responded to A. angustissima. However, in general Selenomonas strains tended to be resistant to the tannins in the acacia while Butyrivibrio fibrisolvens was sensitive. On the other hand some species, like streptococci, varied. Streptococcus bovis-like strains were sensitive to an increase in acacia in the diet while Streptococcus gallolyticus-like strains were resistant. Strep. gallolyticus has independently been shown to be resistant to tannins. It is concluded that there is significant variation in tannin resistance between strains of the same species. This implies that there are specific molecular mechanisms at play that are independent of the phylogenetic position of the organism.

The effect of cellobiose, glucose, and cellulose on the survival of Fibrobacter succinogenes A3C cultures grown under ammonia limitation.

The ruminal, cellulolytic bacterium, Fibrobacter succinogenes A3C, grew rapidly on cellulose, cellobiose, or glucose, but it could not withstand long periods of energy source starvation. If ammonia was limiting and either cellobiose or glucose was in excess, the viability declined even faster. The carbohydrate-excess, ammonia-limited cultures did not spill energy, but they accumulated large amounts of cellular polysaccharide. Cultures that were carbohydrate-limited had approximately 4 nmol ATP mg cell protein(-1), but ATP could not be detected in cultures that had an excess of soluble carbohydrates. However, if F. succinogenes A3C was provided with excess cellulose and ammonia was limiting, ATP did not decline, and the cultures digested the cellulose soon after additional nitrogen sources were added. From these results, it appears that excess soluble carbohydrates can promote the death of F. succinogenes, but cellulose does not.