Degradation and Utilization of Hemicellulose from Intact Forages by Pure Cultures of Rumen Bacteria

Several pure strains of rumen bacteria have previously been shown to degrade isolated hemicelluloses from a form insoluble in 80% acidified ethanol to a soluble form, regardless of the eventual ability of the organism to utilize the end products as energy sources. This study was undertaken to determine whether similar hemicellulose degradation or utilization, or both, occurs from intact forages. Fermentations by pure cultures were run to completion by using three maturity stages of alfalfa and two maturity stages of bromegrass as individual substrates. Organisms capable of utilizing xylan or isolated hemicelluloses could degrade and utilize intact forage hemicellulose, with the exception of two strains of Bacteroides ruminicola which were unable to degrade or utilize hemicellulose from grass hays. Intact forage hemicelluloses were extensively degraded by three cellulolytic strains that were unable to use the end products; in general, these strains degraded a considerably greater amount of hemicelluloses than the hemicellulolytic organisms. Hemicellulose degradation or utilization, or both, varied markedly with the different species and strains of bacteria, as well as with the type and maturity stage of the forage. Definite synergism was observed when a degrading nonutilizer was combined with either one of two hemicellulolytic strains on the bromegrass substrates. One hemicellulolytic strain, which could not degrade or utilize any of the intact bromegrass hemicellulose alone, almost completely utilized the end products solubilized by the nonutilizer. Similar synergism, although of lesser magnitude, was observed when alfalfa was used as a substrate.     

Mechanism of Isolated Hemicellulose and Xylan Degradation by Cellulolytic Rumen Bacteria

Although certain strains of cellulolytic rumen bacteria cannot utilize isolated hemicelluloses or xylan as a source of energy, all strains examined can degrade or solubilize these materials from an 80% ethyl alcohol insoluble to a soluble form. Centrifugation and washing of the cellobiose-grown bacterial cells did not affect the rate or extent of utilization or degradation or both. When the level of a nonutilizing culture inoculum (either normal or washed) was doubled, a corresponding increase in the initial rate of degradation was observed. With a nitrogen-free medium, utilization of xylan was almost completely inhibited for a utilizing strain, whereas degradation by either type of organism was not markedly affected. Cellobiose medium cell-free culture filtrates from a nonutilizing strain were able to degrade or solubilize xylan. The percentage of degradation increased with the volume of cell-free filtrate, and all activity was lost when the filtrate was boiled. No utilization (loss in total pentose) was observed with cell-free filtrates from utilizing or nonutilizing strains. The release of free hexose from insoluble cellulose by culture filtrates from a nonutilizing strain was very limited. On the other hand, carboxymethylcellulose (CMC-70L) and cellulodextrins were degraded to an 80% ethyl alcohol soluble form by filtrates from both types of organisms. Similar enzyme activity was obtained in cell-free culture filtrates from four additional strains of cellulolytic rumen bacteria (one xylan utilizer and three nonutilizers). When the assays were carried out aerobically, CMC-70L solubilization was reduced to a much greater extent than xylan or cellulodextrin solubilization. The enzyme or enzymes responsible for the degradation of hemicellulose by cellololytic rumen bacteria unable to utilize the hemicellulose as an energy source appear to be constitutive in nature, and this activity may be a nonspecific action of a beta-1, 4-glucosidase or -cellulase.     

Pectin-fermenting Bacteria Isolated from the Bovine Rumen

Thirty-two strains of pectin-fermenting rumen bacteria were isolated from bovine rumen contents in a rumen fluid medium which contained pectin as the only added energy source. Based on differences in morphology and the Gram stain, 10 of these strains were selected for characterization. Two strains were identified as Lachnospira multiparus, four strains were identified as Butyrivbrio fibrisolvens, and three strains were identified as Bacteroides ruminicola. Characteristics of the remaining strain did not correspond with any previously described species. It was a gram-positive anaerobic coccus, 1.0 to 1.2 mum in diameter, and occurred primarily as single cells or diplococci. The strain fermented pectin rapidly but showed little or no growth on any other energy sources tested. The only detectable end products were acetic acid and gas, a portion of which was identified as hydrogen. Although the physiological characteristics of this organism differ markedly from other described species, it has been placed in the genus Peptostreptococcus on the basis of morphology, Gram stain, relations to oxygen, and the occurrence of cell division in only one plane. End products of fermentation are somewhat similar to those of the cellulolytic ruminococci. Eight previously characterized strains of cellulolytic bacteria isolated in nonselective media were unable to ferment pectin, whereas ten strains of hemicellulolytic rumen bacteria, eight of which were isolated with a xylan medium, showed considerable variation in this characteristic.     

Evaluation by electron microscopy and anaerobic culture of types of rumen bacteria associated with digestion of forage cell walls.

Different morphological types of rumen bacteria which degraded cell walls of forage grasses with various in vitro digestibilities were evaluated with electron microscopy. The majority of these bacteria (i.e., about 70% or more) consisted of two distinct types: (i) encapsulated cocci and (ii) irregularly shaped bacteria, resembling major fiber digesters found in the rumen. Each type was capable of degrading structurally intact cell walls. Differences (P less than or equal to 0.02) in the percent ratio of encapsulated cocci to irregularly shaped bacteria were observed between Bermuda grass and fescue; the ratio of encapsulated cocci to irregularly shaped bacteria between Bermuda grass and orchard grass was similar and variations were high. The proportion of irregularly shaped bacteria usually increased with increased time of digestion. Differences (P greater than 0.1) were not found in the percentage ratio of encapsulated cocci to irregularly shaped bacteria attached to specific tissue types in either Bermuda grass or fescue. However, encapsulated cocci tended to be more prevalent on sclerenchyma than other tissues in Bermuda grass, but less prevalent on sclerenchyma than other tissues in fescue. Transmission electron microscopy of tissue digestion of rapidly degraded orchard grass blades revealed that mesophyll, parenchyma bundle sheath, and parts of the epidermal cell wall apparently were degraded without direct attachment of bacteria although bacteria were near the cell walls undergoing digestion. Anaerobic growth studies showed that the total culturable bacteria developing on medium 10 and media containing carbohydrates similar to those in forage cell walls (i.e., pectin, xylan, and cellobiose) were 80% higher from rumen bacterial populations adapted in vitro to cell walls of orchard grass compared to those from Bermuda grass; the number of colonies from the orchard grass-adapted population was significantly (P less than or equal to 0.05) greater on the medium containing xylan. Filter paper tests showed that the cellulolytic activity of populations adapted to fescue was greater than that of orchard grass or Bermuda grass.     

Digestion of polysaccharide constituents of tropical pasture herbage in the bovine rumen. IV. The hydrolysis of hemicelluloses from spear grass by cell-free enzyme systems from rumen fluid

Hemicelluloses have been isolated from spear grass (Heteropogon contortus), before and after digestion in the rumen, and separated into linear and branched fractions. Similar fractions have also been obtained from a pasture sample and from the faeces fibre of a steer fed on the same pasture. The rates of hydrolysis of all of these hemicellulose fractions have been determined in the presence of extracellular enzymes from rumen fluid and of enzymes liberated by disruption of rumen microorganisms. The oligosaccharide products of such enzymic degradations have been partially identified. The results confirm that the incomplete digestion of hemicelluloses in the rumen is due to physical protection (e.g. by lignin), rather than to structural differences between different components of the hemicelluloses. There is no difference between rates of digestion of branched and linear hemicelluloses, and previous results which indicated such differences were probably caused by presence of a readily digested glucan in linear hemicellulose fractions.     

Interactions between anaerobic cellulolytic bacteria and fungi in the presence of Methanobrevibacter smithii

A mixed inoculum of cellulolytic rumen bacteria depressed straw degradation by a mixed culture of cellulolytic fungi grown in the presence of Methanobrevibacter smithii. The inhibitory effect appeared to be caused by Ruminococcus albus strain JI and R. flavefaciens strain 007. Ruminococcus albus strain J1 also depressed straw degradation by the fungi, but R. albus strain SY3 and three strains of Bacteroides (Fibrobacter) succinogenes tested showed little or no inhibitory activity. It seems that some ruminococci show competitive or antagonistic activity towards certain rumen fungi.     

Enumeration and presumptive identification of some functional groups of bacteria in the rumen of dairy cows fed grass silage-based diets

Abstract Samples of rumen ingesta from two rumen-fistulated dairy cows fed grass silage-based diets were examined for numbers and types of bacteria that developed colonies on rumen fluid-agar media designated to support the growth of (a) a wide range of species, (b) cellulolytic bacteria, (c) lactate-fermenting bacteria, (d) non-fermentative bacteria. The most numerous species was Bacteroides ruminicola followed by Butyrivibrio fibrisolvens . The most abundant cellulolytic species were Eubacterium cellulosolvens and Ruminococcus flavefaciens. Megasphaera elsdenii and Selenomonas ruminantium were important lactate fermenters but an unidentified bacterium that grew poorly on maintenance medium was by far the most numerous among bacteria isolated from lactate-containing medium. One strain remained sufficiently viable to show that it fermented lactate to propionate and acetate.     

Degradation of isolated grass mesophyll, epidermis and fibre cell walls in the rumen and by cellulolytic rumen bacteria in axenic culture

The degradation of cell walls of mesophyll, epidermis and fibre cells isolated from leaves of perennial and Italian ryegrass within the sheep rumen or by selected strains of rumen bacteria in vitro , was followed by estimation of dry matter loss, or loss of neutral sugar residues. Primary cell walls (mesophyll and epidermis) were fully degraded within 12 h in the rumen, while the more heavily lignified fibre cell walls showed only a 40% loss of dry matter over the same period. Neutral sugar residues were lost at a common rate from walls of all three cell types. Incubation of cell walls with cellulolytic bacteria showed that the extent to which cell walls were attacked was constantly ordered (epidermis > mesophyll > fibre). The rate of degradation of cell walls was less in axenic culture than within the rumen. Greatest weight losses were produced by Ruminococcus albus , followed by Bacteroides succinogenes , with Ruminococcus flavefaciens effecting the least change, regardless of the nature of the cell wall provided as a substrate. Xylose was more readily lost from primary cell walls than glucose during the early stages of attack, but both were lost at a common rate from fibre cell walls. Dry matter losses produced by the hemicellulolytic strain, Bacteroides ruminocola , were limited even after extended incubation. Electron microscopy indicated that R. albus was less commonly attached to cell walls than were the other cellulolytic strains, although evidence of capsular material was present. Bacteroides succinogenes was seen with an extensive capsule which enveloped clusters of cells, forming micro-colonies in association with the plant cell wall. Vesicle-like structures, commonly associated with the cellulolytic bacteria R. albus and B. succinogenes , were found on comparatively few occasions in this study.     

Rumen bacterial degradation of forage cell walls investigated by electron microscopy

The association of rumen bacteria with specific leaf tissues of the forage grass Kentucky-31 tall fescue (Festuca arundinacea Schreb.) during in vitro degradation was investigated by transmission and scanning electron microscopy. Examination of degraded leaf cross-sections revealed differential rates of tissue degradation in that the cell walls of the mesophyll and pholem were degraded prior to those of the outer bundle sheath and epidermis. Rumen bacteria appeared to degrade the mesophyll, in some cases, and phloem without prior attachment to the plant cell walls. The degradation of bundle sheath and epidermal cell walls appeared to be preceded by attachment of bacteria to the plant cell wall. Ultrastructural features apparently involved in the adhesion of large cocci to plant cells were observed by transmission and scanning electron microscopy. The physical association between plant and rumen bacterial cells during degradation apparently varies with tissue types. Bacterial attachment, by extracellular features in some microorganisms, is required prior to degradation of the more resistant tissues.     

Growth Factor Requirements of Ruminococcus flavefaciens Isolated from the Rumen of Cattle Fed Purified Diets

Eight strains of cellulolytic cocci were isolated from a 10^-8 dilution of rumen ingesta and were presumptively identified as Ruminococcus flavefaciens. Four strains were isolated from a steer fed a purified diet which contained isolated soy protein, and four strains were isolated from a steer fed a purified diet which contained urea. Certain growth factor requirements of these bacteria were determined. All strains grew with clarified rumen fluid added to the medium. However, fatty acids could substitute for rumen fluid in four strains. Two strains isolated from each steer either required or their growth was stimulated by isobutyric and/or isovaleric and/or 2-methyl-butyric acid. These results indicate that, even when a diet was fed which contained no branched-chain amino acids, the carbon skeleton precursors of branched-chain fatty acids, the cattle were still able to maintain a large population of cellulolytic bacteria that require fatty acids for growth. Therefore, the fatty acids appear to be provided by other bacteria, by protozoa, or by the host animal.     

Expression of a modified Neocallimastix patriciarum xylanase in Butyrivibrio fibrisolvens digests more fibre but cannot effectively compete with highly fibrolytic bacteria in the rumen

AIMS: This study investigated the competitive abilities of two Neocallimastix patriciarum-derived xylanases constructs in Butyrivibrio fibrisolvens H17c (xynA and pUMSX) and their ability to compete in vivo. METHODS AND RESULTS: The digestibility of neutral detergent fibre (NDF) increased during co-culture of xynA or pUMSX and weakly cellulolytic, but not with highly cellulolytic, ruminococci. Competition studies among xynA, pUMSX and cellulolytic consortia demonstrated that xynA was the fittest. XynA did not persist at high levels in the rumen and was undetectable after 22 days. CONCLUSION: The construction of recombinant xylanolytic B. fibrisolvens does improve the digestibility of fibre above that of the native, but digestibility is still less than that of the most potent fibre digesters such as ruminococci. SIGNIFICANCE AND IMPACT OF THE STUDY: Fibre digestion may be improved by genetic manipulation of ruminal bacteria but ecological parameters, such as persistence in vivo and the niche of the organism, must be taken into account.     

The production of plant cell wall polysaccharide-degrading enzymes by hemicellulolytic rumen bacterial isolates grown on a range of carbohydrate substrates

Several cultures of bacteria, isolated from the rumen, that were able to utilize plant cell wall structural polysaccharides were grown on a range of carbohydrate substrates and the activities of the principal polysaccharide-degrading enzymes determined. The esterase activity was also monitored. The extent of hemicellulose degradation and utilization by the isolates was comparable with that of the hemicellulolytic type strains. Enzyme activities in all of the cultures examined were affected by the carbon source in the growth medium. Many responses were strain specific, although growth on glucose (or cellobiose and maltose to a lesser extent) resulted in reduced activities in most of the organisms examined, whilst polysaccharidic substrates resulted in higher levels of the appropriate polysaccharidase. However, enzyme activity was detectable in some isolates after culture on mono- or disaccharides in the absence of the principal or related polysaccharide substrate.     

Hemicellulose-degrading bacteria and yeasts from the termite gut

Termites play a major role in the recycling of photosynthetically fixed carbon. With the aid of their symbiotic intestinal flora, they are able to degrade extensively wood constituents such as cellulose and hemicellulose. Nevertheless, the microbial species involved in the degradation of hemicelluloses are poorly defined. The purpose of this paper was to examine the microflora involved in hemicellulose degradation. Different aerobic and facultatively anaerobic bacteria and yeasts were isolated using xylan, arabinogalactan and carboxymethylcellulose as substrates. Gram-positive isolates belonged to the genera Bacillus, Paenibacillus, Streptomyces or the actinobacteria group, while the Gramnegative strains were assigned to the genera Pseudomonas, Acinetobacter, Ochrobactrum , and to genera belonging to the family Enterobacteriaceae. The spectrum and activity of xylan- and arabinogalactan-hydrolysing glycosidases of these new isolates, together with additional bacterial strains originally obtained from enrichments with aromatic compounds were determined.     

Cellulolytic cocci isolated from the cecum of guinea pigs (Cavia porcellus).

Five strains of anaerobic, gram-variable cellulolytic cocci, belonging to the genus Ruminococcus, were isolated from the cecum of a guinea pig. They differed from most previously described strains of cellulolytic ruminococci as follows. (i) Lactate was the major fermentation product; lesser amounts of formate and ethanol and a trace of succinate were also produced, along with an uptake of acetate. (ii) No growth occurred at 30 degrees C; however, good growth was observed at 38 and 45 degrees C, (iii) Glucose, cellobiose, cellulose, xylose, arabinose, xylan, sucrose, and lactose were fermented by all strains. Rumen fluid was required for growth in a complete medium containing all nutrients previously found to be required by species in this genus. Limited growth occurred when rumen fluid was replaced by yeast extract, and maximum, but delayed, growth occurred when a water extract of alfalfa was added to the complete medium. No qualitative differences were found in the cell wall amino acids and sugar composition of these strains as compared to Ruminococcus flavefaciens and Ruminococcus albus; however, cell walls of the guinea pig strains appeared to contain a higher proportion of glucose.     

Hemicellulose-degrading enzymes in rumen ciliate protozoa

Hemicellulose-degrading enzymes were detected in cell-free extracts of protozoa representing ten genera of rumen entodiniomorphid and holotrich ciliates. The enzyme preparations released monosaccharides, disaccharides, and oligomers fromLolium perenne hemicellulose B and oat spelt xylan; the activity was present both in cells isolated directly from rumen contents and in those cultured in vitro. The specific activities were higher in the cellulolytic entodiniomorphid genera (Polyplastron, Diploplastron, Eremoplastron, Epidinium, Ophryoscolex, Eudiplodinium) than in the holotrich ciliates (Dasytrichia ruminantium, Isotricha intestinalis/I. prostoma) and the entodinia examined (Entodinium bursa, E. simplex, E. caudatum). The rate of hemicellulose-B degradation to alcohol-soluble products was approximately 5–10 times higher than the rate of reducing sugar accumulation; this indicates an initial depolymerization to intermediate oligosaccharide fragments. Examination of the hemicellulose degradation products by thin-layer and gas-liquid chromatography confirmed oligosaccharide formation, revealed markedly different rates of arabinose and xylose release, and indicated that the mode of polysaccharide degradation was similar in the protozoal preparations examined.     

Degradation and utilization of forage hemicellulose by rumen bacteria, singly in coculture or added sequentially

M. FONDEVILA AND B.A. DEHORITY 1994. Procedures for sequential addition experiments were developed to study the mechanisms involved in the synergistic and inhibitory interactions observed in forage hemicellulose digestion by rumen bacterial cocultures. One organism was allowed to ferment a forage substrate, the culture tube was sterilized and then inoculated with a second organism. No differences were found in the extent of degradation or utilization between fermentations sterilized by oxidation or heat, and based on ease of handling, heat was used in all subsequent experiments. Studies were conducted with Fibrobacter succinogenes A3c, Ruminococcus flavefaciens B34b and Prevotella ruminicola H2b, singly and in all possible combinations. Results from the sequential addition studies substantiated earlier suggestions that the increase observed in hemicellulose utilization results from initial solubilization of the hemicellulose from the forage by the non-utilizer and subsequent utilization of this solubilized polysaccharide by the utilizing, but non-degrading organism.     

利用基本培养基初步筛选牛瘤胃中纤维素降解菌

目的初步筛选牛瘤胃中纤维素降解菌。方法分别采用基本培养基（牛肉膏蛋白胨培养基、马丁培养基）,利用好氧、兼性和厌氧3种不同的培养方法进行初选,初步分离牛瘤胃中的细菌与真菌,再通过复选培养基（加入微晶纤维素）,筛选降解纤维素的菌种。结果筛选分离出降解纤维素的1株厌氧细菌和1株厌氧真菌。结论此实验方法简单易行,能够有效地从牛瘤胃中筛选出生长良好的纤维素降解菌。     

Fermentation of barley straw by anaerobic rumen bacteria and fungi in axenic culture and in co-culture with methanogens

When incubated in axenic culture, strains of anaerobic rumen fungi were more active than cellulolytic bacteria in solubilizing barley straw stem fragments 5 to 10 mm in length. Pretreatment with ammonia had little effect on microbial attack. Of three species of methanogens tested, Methanobrevibacter smithii strain PS formed the most stable and reproducible co-cultures with the fungi and with Ruminococcus albus , and the presence of this organism enhanced the extent of degradation of straw, although this effect was less marked than that previously observed when pure cellulose was used as substrate.     

Bacteria associated with wood tissues of Esca-diseased grapevines: functional diversity and synergy with Fomitiporia mediterranea to degrade wood components

Fungi are considered to cause grapevine trunk diseases such as esca that result in wood degradation. For instance, the basidiomycete Fomitiporia mediterranea (Fmed) is overabundant in white rot, a key type of wood-necrosis associated with esca. However, many bacteria colonize the grapevine wood too, including the white rot. In this study, we hypothesized that bacteria colonizing grapevine wood interact, possibly synergistically, with Fmed and enhance the fungal ability to degrade wood. We isolated 237 bacterial strains from esca-affected grapevine wood. Most of them belonged to the families Xanthomonadaceae and Pseudomonadaceae. Some bacterial strains that degrade grapevine-wood components such as cellulose and hemicellulose did not inhibit Fmed growth in vitro. We proved that the fungal ability to degrade wood can be strongly influenced by bacteria inhabiting the wood. This was shown with a cellulolytic and xylanolytic strain of the Paenibacillus genus, which displays synergistic interaction with Fmed by enhancing the degradation of wood structures. Genome analysis of this Paenibacillus strain revealed several gene clusters such as those involved in the expression of carbohydrate-active enzymes, xylose utilization and vitamin metabolism. In addition, certain other genetic characteristics of the strain allow it to thrive as an endophyte in grapevine and influence the wood degradation by Fmed. This suggests that there might exist a synergistic interaction between the fungus Fmed and the bacterial strain mentioned above, enhancing grapevine wood degradation. Further step would be to point out its occurrence in mature grapevines to promote esca disease development.     

Synergism in Degradation and Utilization of Intact Forage Cellulose, Hemicellulose, and Pectin by Three Pure Cultures of Ruminal Bacteria

Pure cultures of ruminal bacteria characterized as using only a single forage polysaccharide (Fibrobacter succinogenes A3c, cellulolytic; Bacteroides ruminicola H2b, hemicellulolytic; Lachnospira multiparus D15d, pectinolytic) were inoculated separately and in all possible combinations into fermentation tubes containing orchard grass as the sole substrate. Fermentations were run to completion, and then cultures were analyzed for digestion of cellulose plus degradation and utilization of hemicellulose and pectin. Addition of the noncellulolytic organisms, in any combination, to the cellulolytic organism F. succinogenes had little effect on overall cellulose utilization. F. succinogenes degraded but could not utilize hemicellulose; however, when it was combined with B. ruminicola, total utilization of hemicellulose increased markedly over that by B. ruminicola alone. L. multiparus was inactive in hemicellulose digestion, alone or in any combination. Although unable to degrade and utilize purified pectin, B. ruminicola degraded and utilized considerable quantities of the forage pectin. In contrast, L. multiparus was very active against purified pectin, but had extremely limited ability to degrade and utilize pectin from the intact forage. Both degradation and utilization of forage pectin increased when F. succinogenes was combined with B. ruminicola. Sequential addition of two cultures, allowing one to complete its fermentation before adding the second, was used to study synergism between cultures on forage pectin digestion. In general, synergistic effects did not appear to be related to a particular sequence of utilization. The ability of F. succinogenes to degrade and B. ruminicola to degrade and utilize forage pectin contradicts both previous and present data obtained with purified pectin. Thus, isolation and characterization of ruminal bacteria on purified substrates may be misleading with regard to their role in the overall ruminal fermentation.