Rumen Cellulolytic Bacteria Research Articles

Dose-response effects of dietary pequi oil on fermentation characteristics and microbial population using a rumen simulation technique (Rusitec)

The effect of increasing the concentration of commercial pequi (Caryocar brasiliense) oil on fermentation characteristics and abundance of methanogens and fibrolityc bacteria was evaluated using the rumen simulation technique (Rusitec). In vitro incubation was performed over 15 days using a basal diet consisting of ryegrass, maize silage and concentrate in equal proportions. Treatments consisted of control diet (no pequi oil inclusion, 0 g/kg DM), pequi dose 1 (45 g/kg DM), and pequi dose 2 (91 g/kg DM). After a 7 day adaptation period, samples for fermentation parameters (total gas, methane, and VFA production) were taken on a daily basis. Quantitative real time PCR (q-PCR) was used to evaluate the abundance of the main rumen cellulolytic bacteria, as well as abundance of methanogens. Supplementation with pequi oil did not reduce overall methane production (P = 0.97), however a tendency (P = 0.06) to decrease proportion of methane in overall microbial gas was observed. Increasing addition of pequi oil was associated with a linear decrease (P < 0.01) in dry matter disappearance of maize silage. The abundance of total methanogens was unchanged by the addition of pequi oil, but numbers of those belonging to Methanomassiliicoccaceae decreased in liquid-associated microbes (LAM) samples (P < 0.01) and solid-associated microbes (SAM) samples (P = 0.09) respectively, while Methanobrevibacter spp. increased (P < 0.01) only in SAM samples. Fibrobacter succinogenes decreased (P < 0.01) in both LAM and SAM samples when substrates were supplemented with pequi oil. In conclusion, pequi oil was ineffective in mitigating methane emissions and had some adverse effects on digestibility and selected fibrolytic bacteria.

Co-inoculation of cellulolytic rumen bacteria with methanogenic sludge to enhance methanogenesis of rice straw

Ruminal microbiota co-inoculated with methanogenic sludge were applied in a 2 L anaerobic system. The experiment lasted 48 days in three stages with different substrate loading rates of 3 g d−1, 7 g d−1 and 14 g d−1. The fermentation performance and microbial characteristics were assessed. The mean methane yields of different stages increased 1.79-, 2.07- and 2.26-fold compared with control samples, while the methane content of biogas varied from 32 to 44%, significantly different (P < 0.05) from the control reactor. There was no corresponding accumulation of volatile fatty acids (VFAs) and pH values varied between 6.8 and 7.68 during the whole anaerobic digestion, indicating system stability. The cellulolytic activities improved significantly (P < 0.05) compared with control samples. Co-inoculation not only added methanogens but also increased the proportion of bacteria containing GH 5 genes even though the bacterial composition changed by PCR-DGGE phylogenetic analysis. The proportion of cellulolytic genera of Clostridium and Ruminococcus increased, whereas Bacteroides, Fibrobacter and Acetivibrio disappeared due to their inability to acclimatize to in vitro lignocellulosic conditions. The analysis of GH and 16S rRNA genes also provided a better description of cellulolytic function and phylogenetic profile. Mutual cooperation formed between Methanobrevibacter and Ruminococcus was achieved for high cellulolytic activity and methanogenic efficiency in co-inoculated system. Moreover, this hypothesis was further strengthened by observations on morphological characteristics of cellulolytic consortia.

Digestibility of Juncus acutus and its effects on ruminal cellulolytic bacteria

The objectives of this study were to estimate the digestibility of Juncus acutus and to investigate its effects on rumen cellulolytic bacteria to consider J. acutus as an alternative roughage source in ruminant nutrition. Juncus acutus samples were collected from three different stations in Kizilirmak Delta and their proximate analysis was carried out. Organic matter digestibility (OMD) and metabolisable energy (ME) values of J. acutus were estimated from gas measured by the in vitro gas production method. The effects of J. acutus on the abundance of rumen cellulolytic bacteria Fibrobacter succinogenes, Ruminococcus flavefaciens and Ruminococcus albus were determined by real-time PCR method. Crude nutrient values of J. acutus collected from three stations were analysed. Mean OMD %, MEOMD and MEGP values of J. acutus from three stations were ranged from 42.64 to 42.48%, 6.78 to 6.82 MJ/kg DM and 5.05 to 5.26 MJ/kg DM respectively. Ruminal F. succinogenes, R. flavefaciens and R. albus abundance calculated from threshold (CT) values in rumen fluids obtained from 0 to 96 h incubations showed an increases in following order F. succinogenes> R. flavefaciens> R. albus. The CP % of J. acutus was found higher from cereal straw and close to low-quality alfalfa hay and dry meadow grass. Juncus acutus increased the amount of rumen cellulolytic bacteria. In the light of obtained results, it can be concluded that J. acutus may be considered as a new alternative roughage source in ruminant nutrition.

Effects of Different Juncus acutus: Maize Silage Ratios on Digestibility and Rumen Cellulolytic Bacteria

The objectives of this study were to estimate the digestibility of different ratios of Juncus acutus and maize silage and to investigate the effects of them on rumen bacteria. Three different ratios of Juncus acutus and maize silage 100:0 (A), 50:50 (B) and 0:100 (C) were prepared and their gas productions were determined at 0, 3, 6, 12, 24, 48, 72 and 96 h incubation times by ANKOM RF gas production system. OMD%, MEOMD, MEGP, and b values of A, B, C were 42.06, 51.06 and 60.21%; 6.72, 8.16 and 9.63 MJ/kg DM; 5.15, 6.28 and 7.55 MJ/kg DM; 20.85, 35.24 and 48.11 mL respectively. There were significant variations between the chemical composition, gas production, OMD%, MEGP and MEOMD values of A, B and C (P Ruminococcus flavefaciens>Ruminococcus albus values at all incubation times. In conclusion, mixing of Juncus acutus with maize silage in 50:50 ratio increased the amount of rumen cellulolytic bacteria and 22% of both OMD and ME of Juncus acutus. Supplementation of maize silage to Juncus acutus in ruminant diet may improve the utilization of Juncus acutus through providing of nitrogen and fermentable carbohydrates to rumen bacteria.

Improved culturability of cellulolytic rumen bacteria and phylogenetic diversity of culturable cellulolytic and xylanolytic bacteria newly isolated from the bovine rumen

The phylotypes of rumen bacteria have increased by the accumulation of 16S rRNA gene sequences, and they show a complex microbial community structure in the rumen. However, most of the biochemical properties of rumen bacteria defined by phylotypes are still unknown. We attempted to improve the culturability of cellulolytic bacteria from the rumen using an agar medium (CA) and a gellan gum medium (CG) containing azo-carboxymethylcellulose as a carbon source. We isolated 129 strains from these media, and the numbers of isolates that showed filter paperase, carboxymethylcellulase and xylanase activity were 51, 117 and 105, respectively. The isolates were classified into six phyla by 16S rRNA gene sequences. In accordance with other studies, fibre-adherent rumen bacteria from the phylum Firmicutes were the most abundant cultured isolates obtained (82.2%). Isolates that were unclassified (< 97% similarity) totalled 19.4%, indicating that the media used in this study was successfully able to improve the culturability of rumen cellulolytic bacteria. Moreover, as the Chao1 richness of CG was higher than that of CA, we estimated that, compared with CA, CG supports the growth of a wide variety of rumen bacteria. These results demonstrate that culturable species of ruminal cellulolytic bacteria can be increased using improved culture media.

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.

English

Phanerochaete chrysosporium was widely used to delignify agricultural waste product and improve biodegradation of the substrate as animal feed. The experiment was carried out to increase the use of palm oil fronds as a substitute material for napier grass through biodegradation process with P. chysosporium. A completely randomized design with four treatments and four replications was used. The treatments were ration containing 60% napier grass (R1), ration containing 40% napier grass and 20% fermented palm oil frond (R2), ration containing 20% napier grass and 40% fermented palm oil frond (R3), ration containing 60% fermented palm oil frond (R4). Fourty percent concentrate was included in all treatment rations. Parameters measured were in vitro digestibilities of dry matter, organic matter, crude fiber, NDF, ADF, NH3, TVFA, and ruminal cellulolitic bacteria. Results showed that increasing level of fermented palm oil frond in the ration reduced (P&lt;0.05) digestibilities of dry matter, organic matter, crude fiber, NDF, ADF, N-NH3, TVFA concentration and number of ruminal cellulolytic bacteria. It is concluded that fermentation of palm oil frondwith P. chysosporium decrease lignin content by 47.79%, but increasing the fermented palm oil frond in the ration reduces nutrient digestibilities, N-NH3 and TVFA concentrations and rumen cellulolytic bacteria counts. Fermented palm oil frond up to 40% could be used as a substitute for forages in ruminant rations.

The Fibrobacteres: an Important Phylum of Cellulose-Degrading Bacteria

The phylum Fibrobacteres currently comprises one formal genus, Fibrobacter, and two cultured species, Fibrobacter succinogenes and Fibrobacter intestinalis, that are recognised as major bacterial degraders of lignocellulosic material in the herbivore gut. Historically, members of the genus Fibrobacter were thought to only occupy mammalian intestinal tracts. However, recent 16S rRNA gene-targeted molecular approaches have demonstrated that novel centres of variation within the genus Fibrobacter are present in landfill sites and freshwater lakes, and their relative abundance suggests a potential role for fibrobacters in cellulose degradation beyond the herbivore gut. Furthermore, a novel subphylum within the Fibrobacteres has been detected in the gut of wood-feeding termites, and proteomic analyses have confirmed their involvement in cellulose hydrolysis. The genome sequence of F. succinogenes rumen strain S85 has recently suggested that within this group of organisms a "third" way of attacking the most abundant form of organic carbon in the biosphere, cellulose, has evolved. This observation not only has evolutionary significance, but the superior efficiency of anaerobic cellulose hydrolysis by Fibrobacter spp., in comparison to other cellulolytic rumen bacteria that typically utilise membrane-bound enzyme complexes (cellulosomes), may be explained by this novel cellulase system. There are few bacterial phyla with potential functional importance for which there is such a paucity of phenotypic and functional data. In this review, we highlight current knowledge of the Fibrobacteres phylum, its taxonomy, phylogeny, ecology and potential as a source of novel glycosyl hydrolases of biotechnological importance.

Overproduction, purification, crystallization and preliminary X-ray characterization of a novel carbohydrate-binding module of endoglucanase Cel5A fromEubacterium cellulosolvens

The anaerobic cellulolytic rumen bacterium Eubacterium cellulosolvens produces a large array of cellulases and hemicellulases that are responsible for the hydrolysis of plant cell-wall polysaccharides. One of these enzymes, endoglucanase Cel5A, comprises two tandemly repeated novel carbohydrate-binding modules (CBMs) and two catalytic domains belonging to glycoside hydrolase family 5 joined by flexible linker sequences. The novel CBM located at the N-terminus of the endoglucanase has been crystallized. The crystals belonged to the hexagonal space group P6(1)22 and contained a single molecule in the asymmetric unit. The structure of the L-selenomethionine derivative has been solved by a MAD experiment on crystals that diffracted to 1.75 Å resolution.

Green House Gas Control and Agricultural Biomass for Sustainable Animal Agriculture in Developing Countries

Important green house gases (GHG) attributed to animal agriculture are methane (CH 4 ) and nitrous oxide (N 2 O), though carbon dioxide (CO 2 ) contributes almost half of total greenhouse effect. Rumen CH 4 production in an enteric fermentation can be accounted as the biggest anthropogenic source. Some of prebiotics and probiotics have been innovated to mitigate rumen CH 4 emission. The possible use of agricultural biomass consisted of non-edible parts of crop plants such as cellulose and hemi cellulose and animal wastes was proposed as a renewable energy and nitrogen sources. The ammonia stripping from digested slurry of animal manure in biogas plant applied three options of nitrogen recycling to mitigate nitrous oxide emission. In the first option of the ammonia stripping, the effect of ammonolysis on feed value of cellulose biomass was evaluated on digestibility, energy metabolism and protein utilization. Saccharification of the NH 3 treated cellulose biomass was confirmed in strictly anaerobic incubation with rumen cellulolytic bacteria, Ruminoccous flavefaciens , to produce bio-ethanol as the second option of ammonia stripping. In an attempt of NH 3 fuel cell, the reformed hydrogen from the NH 3 stripped from 20 liter of digested slurry in thermophilic biogas plant could generate 0.12 W electricity with proton exchange membrane fuel cell (PEM) as the third option. Key words: GHG, rumen, methane, probiotics, ammonia stripping, biomass

Detection and identification of rumen bacteria constituting a fibrolytic consortium dominated by Fibrobacter succinogenes

A fibrolytic consortium, dominated by the rumen cellulolytic bacterium Fibrobacter succinogenes, was artificially constructed on hay stems to detect and identify rumen bacteria that can potentially interact with F. succinogenes. Consortium-bacterial members were determined by DGGE and sequencing analysis targeted bacterial 16S rDNA. An artificial consortium was formed in a 2-step incubation of hay stems; the first step with group 1, 2 or 3 F. succinogenes strains, the second step with rumen fluid. After consortium formation, morphologically different bacteria were observed in association with F. succinogenes. DGGE exhibited more than 30 bands, the pattern of which depended on the F. succinogenes group. Sequencing suggested that Butyrivibrio fibrisolvens, Pseudobutyrivibrio ruminis, Clostridium sp., F. succinogenes group 2, Prevotella ruminicola and unclassified Bacteroides were prominent in the group 1 consortium and that Treponema bryantii, B. fibrisolvens, Acinetobacter sp, and Wolinella succinogenes were prominent in the group 2 consortium. However, in the group 3 consortium, F. succinogenes-like bacteria were microscopically undetectable, whereas cellulolytic Ruminococcus albus and F. succinogenes group 1 were prominent, suggesting that the group 3 cannot be a core member of this consortium. This study is the first attempt to identify bacterial members of a fibrolytic consortium dominated by a specific bacterium.

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 0.42mM and for S85, >0.64mM. 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.

Diversity and Strain Specificity of Plant Cell Wall Degrading Enzymes Revealed by the Draft Genome of Ruminococcus flavefaciens FD-1

Background Ruminococcus flavefaciens is a predominant cellulolytic rumen bacterium, which forms a multi-enzyme cellulosome complex that could play an integral role in the ability of this bacterium to degrade plant cell wall polysaccharides. Identifying the major enzyme types involved in plant cell wall degradation is essential for gaining a better understanding of the cellulolytic capabilities of this organism as well as highlighting potential enzymes for application in improvement of livestock nutrition and for conversion of cellulosic biomass to liquid fuels.Methodology/Principal FindingsThe R. flavefaciens FD-1 genome was sequenced to 29x-coverage, based on pulsed-field gel electrophoresis estimates (4.4 Mb), and assembled into 119 contigs providing 4,576,399 bp of unique sequence. As much as 87.1% of the genome encodes ORFs, tRNA, rRNAs, or repeats. The GC content was calculated at 45%. A total of 4,339 ORFs was detected with an average gene length of 918 bp. The cellulosome model for R. flavefaciens was further refined by sequence analysis, with at least 225 dockerin-containing ORFs, including previously characterized cohesin-containing scaffoldin molecules. These dockerin-containing ORFs encode a variety of catalytic modules including glycoside hydrolases (GHs), polysaccharide lyases, and carbohydrate esterases. Additionally, 56 ORFs encode proteins that contain carbohydrate-binding modules (CBMs). Functional microarray analysis of the genome revealed that 56 of the cellulosome-associated ORFs were up-regulated, 14 were down-regulated, 135 were unaffected, when R. flavefaciens FD-1 was grown on cellulose versus cellobiose. Three multi-modular xylanases (ORF01222, ORF03896, and ORF01315) exhibited the highest levels of up-regulation.Conclusions/SignificanceThe genomic evidence indicates that R. flavefaciens FD-1 has the largest known number of fiber-degrading enzymes likely to be arranged in a cellulosome architecture. Functional analysis of the genome has revealed that the growth substrate drives expression of enzymes predicted to be involved in carbohydrate metabolism as well as expression and assembly of key cellulosomal enzyme components.

Influence of different periods of exposure to hot environment on rumen function and diet digestibility in sheep

Effects of different periods of exposure to hot environments on rumen function, diet digestibility and digesta passage rate were studied in four adult not-pregnant Sardinian ewes housed in a climatic chamber. The ewes were kept in individual metabolic cages. The trial lasted 83 days; 17 days were spent under thermal comfort conditions (TC) [temperature-humidity index (THI) = 65.0 +/- 2.0], followed by 49 days under elevated THI (ETHI: THI = 82.0 +/- 2.5) and 17 days under thermal comfort (TC; THI = 65.0 +/- 1.0). Five digestibility and passage rate trials were carried out during the 83 days. Trials 1 and 5 were carried out under TC; trials 2, 3 and 4 were carried out under ETHI. Values of rectal temperatures (39.7 +/- 0.3 degrees C) and respiratory rate (118.4 +/- 31.8 breaths/min) indicated that sheep under ETHI were heat-stressed. Heat stress caused an increase (P < 0.01) in water intake, and reductions (P < 0.05) in dry matter intake, rumen pH, rumen cellulolytic and amylolytic bacteria count, rumen osmolarity, organic matter, dry matter, neutral detergent fibre, acid detergent fibre and non-structural carbohydrates digestibility coefficients, and a reduction of digesta passage rates. Under ETHI, diet digestibility and passage rate of digesta were reduced in a time-dependent fashion. Variation of diet digestibility under ETHI was not related to passage rate of digesta and feed intake. Reduction of cellulolytic and amylolytic bacteria and the adaptive response to hot environment seem to be related to alteration of digestibility observed in ewes chronically exposed to hot environment.

Proteomic identification of CBM37-containing cellulases produced by the rumen cellulolytic bacterium Ruminococcus albus 20 and their putative involvement in bacterial adhesion to cellulose

The objective of this study was to identify and characterize other proteins than fimbrial proteins potentially involved in R. albus 20 adhesion to cellulose using an adhesion-related antiserum preparation (i.e. anti-Adh serum). From protein fractions of R. albus 20 grown on cellulose, the serum recognized at least 10 cellulose-binding proteins (CBPs), among which homologs of glycoside hydrolases (family 5, 9 and 48) of R. albus 8 (i.e. Cel5G, Cel9B and Cel48A) were identified by a proteomic approach. In strain 20, Cel9B and Cel48A were identified as two major CBPs and as bacterial cell-associated proteins. The anti-Adh serum was also shown to target the C-terminal family 37 carbohydrate-binding module (CBM37) of Cel9B and Cel48A, indicating that this module, unique to R. albus, may play a significant role in bacterial adhesion to cellulose as suggested previously for R. albus 8. Overall, our results support the hypothesis of an adhesion mechanism involving the CBM37 of Cel9B and Cel48A. This adhesion mechanism may not be restricted to these two enzymes but may also involve other CBM37-containing proteins such as Cel5G and the other uncharacterised proteins recognized by the anti-Adh serum.

Isolation and identification of cellulose-binding proteins from sheep rumen contents.

To extend our understanding of the mechanisms of plant cell wall degradation in the rumen, cellulose-binding proteins (CBPs) from the contents of a sheep rumen were directly isolated and identified using a metaproteomics approach. The rumen CBPs were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and some CBPs revealed endoglucanase activities toward carboxymethyl cellulose. Using mass spectrometry analyses, four CBPs were identified and annotated as known proteins from the predominant rumen cellulolytic bacterium Fibrobacter succinogenes: tetratricopeptide repeat domain protein, OmpA family protein, fibro-slime domain protein, and cellulose-binding endoglucanase F (EGF). Another CBP was identified as the cellulosomal glycosyl hydrolase family 6 exoglucanase, Cel6A, of Piromyces equi. F. succinogenes cells expressing EGF were found to be major members of the bacterial community on the surface or at the inner surface of hay stems by immunohistochemical analyses using anti-EGF antibody. The finding that four of the five CBPs isolated and identified from sheep rumen contents were from F. succinogenes indicates that F. succinogenes is significantly involved in cellulose degradation in the rumen.

Effect of Roughage Sources on Cellulolytic Bacteria and Rumen Ecology of Beef Cattle

The effect of different tropical feed sources on rumen ecology, cellulolytic bacteria, feed intake and digestibility of beef cattle was investigated. Four fistulated, castrated male crossbred cattle were randomly allocated to a 4×4 Latin square design. The treatments were: T1) urea-treated (5%) rice straw (UTS); T2) cassava hay (CH); T3) fresh cassava foliage (FCF); T4) UTS:FCF (1:1 dry matter basis). Animals were fed concentrates at 0.3% of body weight on a DM basis and their respective diets on an ad libitum basis. The experimental period was 21 days. The results revealed that the use of UTS, CH, FCF and UTS:FCF as roughage sources could provide effective fiber and maintain an optimal range of ruminal pH and NH3-N. Total viable and cellulolytic bacterial populations were enhanced (p<0.05) with UTS as the roughage source. Animals fed FCF had a higher rumen propionate production (p<0.05) with a lower cellulolytic bacteria count. Moreover, three predominant cellulolytic bacteria species, namely Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens, were found in all treatment groups. Roughage intake and total DM intake were highest with UTS (2.2 and 2.5% BW, respectively) as the roughage source (p<0.05). Nutrient intake in terms of organic matter intake (OMI) was similar in UTS, CH and UTS:FCF treatments (8.0, 6.8 and 8.7 kg/d, respectively), while crude protein intake (CPI) was enhanced in CH, FCF and UTS:FCF as compared to the UTS treatment (p<0.05). Digestion coefficients of DM and organic matter (OM) were similar among treatments, while the CP digestion coefficients were similar in CH, FCF and UTS:FCF treatments, but were higher (p<0.05) in CH than in UTS. CP and ADF digestible intakes (kg/d) were highest (p<0.05) on the CH and UTS treatments, respectively. It was also observed that feeding FCF as a full-feed resulted in ataxia as well as frequent urination; therefore, FCF should only be fed fresh as part of the feed or be fed wilted. Hence, combined use of FCF and UTS as well as CH and FCF were recommended. (Key Words : Cassava Hay, Cassava Foliage, Urea-treated Rice Straw, Cellulolytic Bacteria Species, Roughage, Rumen)

Biochemical Characterization of Cellulose-Binding Proteins (CBPA and CBPB) from the Rumen Cellulolytic BacteriumEubacterium cellulosolvens5

The cellulose-binding proteins, CBPA and CBPB, of rumen cellulolytic bacterium Eubacterium cellulosolvens 5 were biochemically characterized, and their properties were compared. Recombinant CBPA and CBPB were a typical 1,4-beta-endoglucanase. Both proteins bound to insoluble polysaccharides such as Avicel cellulose, acid swollen cellulose, lichenan, chitin, and oat spelt xylan. On the other hand, only recombinant CBPB bound to agarose and starch.

Dockerin-like sequences in cellulases and xylanases from the rumen cellulolytic bacterium Ruminococcus flavefaciens.

Recent analysis of the endA cellulase gene from Ruminococcus flavefaciens 17 has revealed that it encodes a product of 759 amino acids that provides the first example of a multidomain cellulase from a Ruminococcus sp. Following the family 5 catalytic domain in the predicted EndA enzyme is a 282 amino acid domain of unknown function for which no close relationship was found to other protein sequences. However, the C-terminal sequences of EndA contain a 34 amino acid threonine-rich linker connected to an 81 amino acid region, both of which show strong similarities to sequences present in two xylanases from R. flavefaciens 17. A distant relationship is evident between regions of the 80 amino acid sequences of EndA, XynD and XynB and the duplicated 23 amino acid dockerin sequences found in cellulolytic Clostridium sp., suggesting that as in Clostridium sp. these sequences could mediate the binding of enzymatic polypeptides to another component in the cell surface enzyme complex of R. flavefaciens.

Efecto in vitro de extractos ricos en saponinas de Pithecellobium saman y Sapindus saponaria sobre el crecimiento de dos bacterias celulolíticas ruminales

Se evaluó el efecto de extractos ricos en saponinas (ERS) provenientes de frutos de árboles nativos sobre el crecimiento de dos especies de bacterias celulolíticas del rumen. Ruminococcus flavefaciens y Fibrobacter succinogenes se cultivaron en medios de cultivo que contenían 0, 3 y 5 mg·ml−1 de ERS de los frutos de Sapindus saponaria, Pithecellobium saman y saponinas purificadas de Quillaja saponaria. Se determinó la curva de crecimiento de las bacterias por cuantificación espectrofotométrica a 620 nm. La utilización de 3 y 5 mg·ml−1 de ERS de S. saponaria y 5 mg·ml−1 de ERS de P. saman redujeron (P &lt; 0,05) el crecimiento de R. flavefaciens; mientras que la adición de 3 mg·ml−1 de ERS de P. saman incrementó el crecimiento de la bacteria después de la hora 11 en comparación con el tratamiento control. Por otro lado, el crecimiento de F. succinogenes fue inhibido (P &lt; 0,05) completamente por la utilización de 3 y 5 mg·ml−1 de ERS de S. saponaria y 5 mg·ml−1 de P. saman. La adición de ERS de Q. saponaria sólo afectó el crecimiento de F. succinogenes cuando se adicionaron 5 mg·ml−1. Los resultados muestran que la adición de ERS provenientes de frutos de árboles tropi­cales afectan el crecimiento de dos de las más importantes bacterias celulolíticas del rumen, en especial, de la bacteria Gram-negativa F. succinogenes.