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Growth of the ruminal bacteria Ruminococcus flavefaciens FD1, Selenomonas ruminantium HD4, and Butyrivibrio fibrisolvens 49 was limited by ester-linked feruloyl and p-coumaroyl groups. The limitation of growth on phenolic acid-carbohydrate complexes varied with individual bacteria and appeared to be influenced by ability to hydrolyze carbohydrate linkages.

Anaerobic fungi inhabit the rumen and actively degrade plant cell walls. Rumen fungi produce high levels of cellulases and hemicellulases and are particularly proficient in producing xylanases. These enzymes are regulated by substrate (especially soluble sugars) available to the organisms. Fungi degrade unlignified (i.e., no histochemical reaction for phenolics) plant walls totally, indicating that enzymes are able to hydrolyze or solubilize the entire plant wall. These organisms are better able to colonize and degrade the lignin-containing tissues than are bacteria; phenolics are solubilized but not metabolized from the plant wall by fungi. Anaerobic fungi are unique among rumen microorganisms in that they penetrate the cuticle. Residues after incubation with fungi are physically weaker than those incubated with whole rumen fluid or with rumen bacteria, suggesting that fungi could alter the fibrous residue for easier mastication by the animal. Data indicate that cocultures of anaerobic fungi with methanogenic bacteria stimulate cellulose degradation; other data suggest that fungi are inhibited by certain rumen microorganisms. The interaction of rumen fungi with other organisms in relation to fiber degradation in the rumen requires additional study. Rumen fungi have the potential to degrade the more recalcitrant plant walls in forages, but this potential is not always reached in the rumen.

Ruminal bacteria from axenic cultures of Ruminococcus flavefaciens FD1, Butyrivibrio fibrisolvens 49, and bacterial types from the ruminal ecosystem that were fixed with 50 mM lysine (l-lysine hydrochloride) added to glutaraldehyde had better-preserved capsules and extracellular material than bacteria fixed without lysine.

The plant cell wall of forages is a complex entity of cellulose fibers found in a matrix of hemicellulose and pectins. Different microbial types including bacteria, protozoa, and fungi associate with plant cell walls during incubation with rumen fluid. Bacteria, the major degraders of the forage cell wall, often firmly attach to the forage cell wall before digestion. Encapsulated cocci and irregularly shaped or pleomorphic bacteria are the predominant types of bacteria that adhere to and degrade cell walls. Although certain bacterial types adhere to particular forages, no consistent association was found between digestibility and the type of adhering bacteria. Bacteria adhere to the more rigid forage cell walls and adhere, or are close to, the more easily degraded cell walls. Tissues delignified with potassium permanganate or treated with sodium hydroxide (to improve forage digestibility) show a loss of electron denseness. These tissues separate into individual cells, which at times appear as microfibrils in the cell walls. Research is needed on forages of higher fiber content to mitigate the barriers that limit the attack on forage cell walls by rumen microorganisms.

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.

Ruminal bacteria were subjected to a series of phenolic compounds in various concentrations to acquire fundamental information on the influence on growth and the potential limits to forage utilization by phenolic monomers. Ruminococcus albus 7, Ruminococcus flavefaciens FD-1, Butyrivibrio fibrisolvens 49, and Lachnospira multiparus D-32 were tested against 1, 5, and 10 mM concentrations of sinapic acid, syringaldehyde, syringic acid, ferulic acid, vanillin, vanillic acid, p-coumaric acid, p-hydroxybenzaldehyde, p-hydroxybenzoic acid, and hydrocinnamic acid. Responses were variable and dependent on the phenolic compound and microbial species. Compounds especially toxic (i.e., resulting in poor growth, effect on several species, dose-related response) were p-coumaric acid and p-hydroxybenzaldehyde, and adaptation to the toxins did not occur after three 24-h periods. Syringic, p-hydroxybenzoic, and hydrocinnamic acids stimulated growth of all four species and also stimulated filter paper degradation by R. flavefaciens. None of the stimulatory compounds supported microbial growth in the absence of carbohydrates. In vitro dry matter digestibility of cellulose (Solka-Floc) was not stimulated by any of the phenolic compounds (10 mM), but the cinnamic acids and benzoic aldehydes (10 mM) reduced (P less than 0.05) digestion by the mixed population in ruminal fluid. Growth of R. flavefaciens in the presence of p-hydroxybenzoic acid (10 mM) or p-coumaric acid (5 mM) resulted in recognizable alterations in cell ultrastructure. Both phenolics caused a reduction in cell size (P less than 0.05), and p-coumaric acid caused a reduction in capsular size (P less than 0.05) and produced occasional pleomorphic cells.

Information on the interaction between mixed populations in the rumen and plant phenolics is required to fully elucidate the limitations of phenolic compounds on forage digestibility. The objective of this study was to examine the degradation of Italian ryegrass (Lolium multiflorum L.) hay incubated with mixed ruminal populations in consecutive batch culture (CBC) with or without phenolic acids or phenolic compounds extracted from plant cell walls. Each CBC consisted of a series of 10 cultures (3 replicates per culture) inoculated (10%, vol/vol) in sequence at 48-h intervals with microbial suspension from the previous set of cultures. All cultures were grown on a semidefined medium containing Italian ryegrass hay, and each CBC was initiated with an inoculum from the rumen. Rumenlike fermentation characteristics were maintained in control CBCs by repeated inoculum transfer. Treatment CBCs were transferred as described above, but cultures 5, 6, and 7 were incubated in the presence of trans-p-coumaric, cis-p-coumaric, or trans-ferulic acid or phenolics extracted from the cell walls of maize stem or barley straw. Mean apparent dry matter disappearance in control CBC cultures was 495 mg per g of hay, whereas the presence of phenolics reduced the initial dry matter disappearance by 6.3 to 25.6%. trans-p-Coumaric acid and, to a lesser extent, the phenolics from cell walls of maize stem were the most inhibitory compounds for dry matter disappearance and for the production of volatile fatty acids; trans-p-coumaric acid altered the molar ratio of acetate/propionate/butyrate. The CBC further showed variations in the ability of the rumen microbial population to adapt to phenolic compounds.

Five anaerobic fungal isolates from the bovine rumen were grown on Coastal Bermuda grass (CBG) leaf blades and monitored over a 9-day period for substrate utilization, fermentation products, cellulase, and xylanase activities. Two of the fungal isolates showed monocentric growth patterns; one (isolate MC-1) had monoflagellated zoospores and morphologically resembled members of the genus Piromyces; the other (isolate MC-2) had multiflagellated zoospores and resembled members of the genus Neocallimastix. Three other isolates (PC-1, PC-2, and PC-3) exhibited polycentric growth and have not yet been described in the literature; these isolates were characterized by differences in morphology. All of the isolates degraded CBG to approximately the same extent (70% [dry weight]) in 9 days. Fermentation product accumulation was concurrent with substrate utilization. The major fermentation products for all isolates were formate, acetate, D-(-)-lactate, L-(+)-lactate, ethanol, carbon dioxide, and hydrogen. Succinate was produced by all cultures, with the exception of MC-1. Fermentation balances revealed different profiles for each isolate. As a group, monocentric isolates produced a greater ratio of oxidized to reduced products when grown on glucose or CBG than did the polycentric isolates, which produced a nearly equal ratio of these products. All isolates exhibited cellulolytic and xylanolytic activities, including endoglucanase, exoglucanase, beta-glucosidase, xylanase, and beta-xylosidase activities. Increasing enzyme activity correlated with the accumulation of fermentation products and substrate utilization. The optimum pH for the enzymatic activity of polycentric isolates was within a more narrow range (pH 6.4 to 7.0) than that of the monocentric isolates (pH 5.5 to 7.5). Activity toward cellulosic substrates was not detected until after the disappearance of reducing sugars. Xylanase activity was found to be five to seven times that of carboxymethyl cellulase activity for all cultures grown on CBG.

Treatment of fibrous residue from in vitro digestion trials with chitinase was evaluated gravimetrically and microscopically to determine the associated fungal biomass. The percent dry weight removed by chitinase with time paralleled changes in the number of sporangia associated with leaves. The fungal biomass contributed about 12% dry matter to the residue of leaves incubated with ruminal fluid plus streptomycin and penicillin.

Sheep fed the forage Digitaria pentzii fertilized with sulfur were compared with those fed unfertilized forage for the rumen microbial population involved with fiber degradation. No differences were detected in the bacterial population as determined by anaerobic cultures on a habitat-simulating medium, xylan, or pectin, by 35S labeling techniques for microbial protein, or by transmission electron microscopic studies of bacterium-fiber interactions. Rumen volume and water flow from the rumen were not different for sheep fed each of the forages. Rumen fungi were prevalent in sheep fed sulfur-fertilized D. pentzii as shown by sporangia adhering to forage fiber and by colonies developing from zoospores in roll tubes with cellobiose plus streptomycin and penicillin. Fungi were absent or in extremely small numbers in sheep fed unfertilized forage. Nylon bag digestibility studies showed that the fungi preferentially colonized the lignified cells of blade sclerenchyma by 6 h and caused extensive degradation by 24 h. In the absence of bacteria in in vitro studies, extensive hyphal development occurred; other lignified tissues in blades (i.e., mestome sheath and xylem) were attacked, resulting in a residue with partially degraded and weakened cell walls. Nonlignified tissues were also degraded. Breaking force tests of leaf blades incubated in vitro with penicillin and streptomycin and rumen fluid from sheep fed sulfur-fertilized forage or within nylon bags in such sheep showed a residue at least twice as fragile as that from sheep fed unfertilized forage. In vitro tests for dry matter loss showed that rumen fungi, in the absence of actively growing bacteria, could remove about 62% of the forage material. The response of rumen fungi in sheep fed sulfur-fertilized D. pentzii afforded a useful in vivo test to study the role of these microbes in fiber degradation. Our data establish that rumen fungi can be significant degraders of fiber and further establish a unique role for them in attacking and weakening lignocellulosic tissues. The more fragile residues resulting from attack by fungi could explain the greater intake consistently observed by sheep eating sulfur-fertilized compared with unfertilized D. pentzii forage.

The degradation of forage cell walls by rumen bacteria was investigated with critical-point drying/scanning electron microscopy and ruthenium red staining/transmission electron microscopy. Differences were observed in the manner of attachment of different morphological types of rumen bacteria to plant cell walls during degradation. Cocci, constituting about 22% of the attached bacteria, appeared to be attached to degraded plant walls via capsule-like substances averaging 58 nm in width (range, 21 to 84 nm). Many bacilli appeared to adhere to forage substrates without distinct capsule-like material, although unattached bacteria with capsules were observed occasionally. Certain bacili appeared to be attached to degraded tissue via small amounts of extracellular material, but others apparently had no extracellular material. Bacilli with a distinct morphology due to an irregularly folded, electron-dense outer layer or layers (about 15 nm thick) and without fibrous extracellular material consituted about 37% of the attached bacteria and were observed to adhere so closely to degraded plant walls that the bacterial shape conformed to the shape of the degraded zone. In the rumen ecosystem, bacteria appeared to adhere to plant substrates during degradation by capsule-like material and by small amounts of extracellular material, as well as by the other means not observable by electron microscopy.

Anaerobic fungi in ruminal fluid from cows eating Bermuda grass hay plus a grain and minerals supplement were evaluated for diversity in sporangial morphotypes and colony growth patterns and for the degradation of various lignocelluloses. In selective cultures containing streptomycin and penicillin, an active population of ruminal fungi colonized leaf blades and degraded fiber at rates and extents almost equal to that of the total ruminal population. Three major sporangial morphotypes were consistently observed on leaf blades: oval, globose, and fusiform. Fungal colonies representing three distinct growth types consistently developed in anaerobic roll tubes inoculated with strained ruminal fluid. Sporangial morphotypes could not be matched to colony types due to multiple sporangial forms within a colony. Under identical growth conditions, one type exhibited a monocentric growth pattern, while two types exhibited polycentric growth patterns previously unreported in ruminal fungi. Mixed ruminal fungi in selective cultures or in digesta taken directly from the rumen produced a massive clearing of the sclerenchyma. Quantitation of tissue areas in cross sections by light microscopic techniques showed that fungal incubations resulted in significant (P = 0.05) increases in sclerenchyma degradation compared to whole ruminal fluid incubations. The mestome cell wall was at times penetrated and partially degraded by fungi; the colonization was less frequent and to a lesser degree than with the sclerenchyma. Conversely, ruminal bacteria were not observed to degrade the mestome sheath. Phenolic monomers at 1 mM concentrations did not stimulate to a significant (P = 0.05) extent the dry weight loss or fungal colonization of leaf blades; at 10 mM concentrations cinnamic and benzoic acids were toxic to ruminal fungi.

Fungal colonies developing in anaerobic media from zoospores in rumen fluid from cows eating Cynodon dactylon or Medicago sativa included types showing monocentric and polycentric growth. High energy supplements added to diets of Sorghum bicolor silage increased fungal numbers in the rumen, but increases were also affected by the history and predisposition of the animal. Mixed fungal types in rumen fluid and pure cultures of isolates showing monocentric and polycentric growth degraded and weakened lignocellulosic tissues and penetrated the cuticle of C. dactylon leaf blades. By weakening or degrading recalcitrant structures in forages, rumen fungi may alter physical parameters of plants that influence utilization of fibre by ruminants.

Bermudagrass (Cynodon dactylon) leaf blades and whole cordgrass (Spartina alterniflora) fiber were evaluated for degradation of cell walls by microbial groups in ruminal fluid. The groups were selected by the addition of antibiotics to the inoculum as follows: (i) whole ruminal fluid (WRF), no antibiotics; (ii) cycloheximide (C) to inhibit fungi, thus showing potential bacterial activity; (iii) streptomycin and penicillin (S,P) to inhibit fiber-degrading bacteria, showing potential fungal activity; (iv) streptomycin, penicillin, and chloramphenicol (S,P,CAM) to inhibit all bacteria including methanogens; (v) streptomycin, penicillin, and cycloheximide (S,P,C) to inhibit all microbial activity as a control; and (vi) autoclaved ruminal fluid (ARF) to inhibit all biological activity as a second control. Scanning electron microscopy of tissue degradation indicated that tissues not giving a positive histological reaction for lignin were more readily degraded. Cordgrass was more highly lignified, with more tissues resisting degradation than in bermudagrass. Patterns of degradation due to treatment resulted in three distinct groups of data based on the extent of fiber or component losses: WRF and C greater than S,P and S,P,CAM greater than S,P,C and ARF. Therefore, bacterial activity was responsible for most of the fiber loss. Fiber degradation by anaerobic fungi was significantly less (P = 0.05). Cupric oxide oxidation of undigested and digested bermudagrass fiber indicated that phenolic constituents differed in their order of resistance to removal or solubilization. Vanillyl and syringyl components of lignin were the most resistant to decomposition, whereas ferulic acid was readily solubilized from fiber in the absence of microbial activity.(ABSTRACT TRUNCATED AT 250 WORDS)

The bovine rumen resembles many other ecosystems in that its component bacterial cells are universally surrounded and protected by extracellular structures. The most common form of these structures is a fibrous carbohydrate slime that extends away from the cell and may mediate the attachment of the bacterium to a surface. This attachment is relatively specific and it may occur at the surface of the rumen epithelium or on the cell walls of a specific tissue within the plant-derived food of the animal. The production of the extracellular slime is under nutritional control and slime may be overproduced when soluble carbohydrates are available in high concentration. This overproduction results in cell-cell adhesion among the rumen bacteria with the eventual formation of slime-enclosed microcolonies and, in extreme cases, the generation of sufficient viscosity to cause feedlot bloat.