The effect of rumen chitinolytic bacteria on cellulolytic anaerobic fungi

J. KOPEČNÝ, B. HODROVÁ AND C. S. STEWART. 1996. The polycentric anaerobic fungus Orpinomyces joyonii A4 was cultivated on microcrystalline cellulose alone and in association with the rumen chitinolytic bacterium Clostridium sp. strain ChK5, which shows strong phenotypic similarity to Clostridium tertium . The presence of strain ChK5 significantly depressed the solubilization of microcrystalline cellulose, the production of short-chain fatty acids (SCFA) and the release of endoglucanase by the fungus. Co-culture of the monocentric anaerobic fungus Neocallimastix frontalis strain RE1, Neocallimastix sp. strain G-1 and Caecomyces sp. strain SC2 with strain ChK5 also resulted in depressed fungal cellulolysis. Cell-free supernatant fluids from strain ChK5 inhibited the release of reducing sugars from carboxymethylcellulose by cell-free supernatant fluids from O. joyonii strain A4. Strain 007 of the cellulolytic anaerobe Ruminococcus flavefaciens was also shown to produce small amounts of soluble products upon incubation with colloidal chitin. Mixtures of culture supernates from this bacterium and from O. joyonii strain A4 showed cellulase activity that was less than that of the component cultures. It is suggested that the ability of some rumen bacteria to hydrolyse or transform chitin may be an important factor in the interactions between bacteria and fungi in the rumen.     

Degradation and fermentation of cellulose by the rumen anaerobic fungi in axenic cultures or in association with cellulolytic bacteria

Three rumen anaerobic fungi—Neocallinastix frontalis MCH3,Piromyces (Piromonas) communis FL, andCaecomyces (Sphaeromonas) communis FG10—were cultured on cellulose filter paper alone or in association with one of two rumen cellulolytic bacteria,Ruminococcus flavefaciens 007 andFibrobacter succinogenes S85. Cocultures ofN. frontalis orP. communis andR. flavefaciens were markedly less effective than the fungal monocultures in degrading cellulose but more effective than the bacterial monocultures.R. flavefaciens had an antagonistic effect against both of the fungal species. In contrast, no interaction was observed between the two fungal species andF. succinogenes. Cellulose was more effectively degraded by the cocultureC. communis-R. flavefaciens than by the corresponding fungal and bacterial monocultures. The effectiveness of degradation of the cocultureC. communis-F. succinogenes was comparable to that of the bacterial strains but greater than that of the fungi; no interaction was observed between these two microorganisms.     

Effects of an organophosphorus insecticide on the growth and cellulolytic activity of fungi

The organophosphorus insecticide Selecron [O-(4-bromo-2-chlorophenyl) O-ethyl S-n-propyl-phosphorotioate] at 10 and 50 ppm significantly decreased respiration, mycelial protein, extracellular protein and mycelial dry weight of Aspergillus fumigatus, A. terreus and Myceliophthora thermophila when grown at 45°C. C_x and C₁ cellulases of tested fungi were significantly decreased. However, C₁ cellulase of A. fumigatus was slightly increased.     

Effects of some ionophore antibiotics and polyoxins on the growth of anaerobic rumen fungi

The growth of mixed rumen fungi in vitro was suppressed by both ionophore antibiotics (salinomycin, monensin and portmicin) and polyoxins (polyoxin B and D: inhibitors of chitin synthesis). The fungistatic effect of the ionophores on a Piromonas spp. was more pronounced than on a Neocallimastix spp. The polyoxins, however, were more potent fungistatically against the Neocallimastix spp. than the Piromonas spp. Higher concentrations of the polyoxins were required to elicit the same effect as that observed with the ionophores. Salinomycin administration decreased fungal count in the rumen of sheep, but fungal count increased after the cessation of the feeding of the antibiotic. Polyoxin D also suppressed the growth of fungi in vivo, but the effect was short-lived. Nevertheless, both bacterial and protozoal counts tended to increase during and after the administration of polyoxin D. Total volatile fatty acid concentrations in the rumen tended to increase during the period of polyoxin D administration. This increasing tendency was maintained for 10 d after the cessation of antibiotic administration. Offering polyoxin D to sheep increased production of propionate ( P < 0·05), while decreasing that of acetate. The results indicate that the rumen fungi are sensitive to chitin synthesis inhibitors as well as ionophores, and are essential members of microbes in the rumen ecosystem.     

Effects of live Saccharomyces cerevisiae cells on zoospore germination,growth, and cellulolytic activity of the rumen anaerobic fungus,Neocallimastix frontalis MCH3

The effects of a live yeast strain of Saccharomyces cerevisiae have been investigated on zoospore germination, metabolism, and cellulolytic activity of the anaerobic rumen fungus Neocallimastix frontalis MCH3. The addition of yeast cells to a vitamin-deficient medium stimulated the germination of fungal zoospores, increased cellulose degradation and hydrogen, formate, lactate, and acetate production. Responses depended on the concentration of yeast cells added and on their viability. Yeast supplementation provided vitamins such as thiamine, which is essential for fungal growth and activity. These results demonstrate that yeasts could enhance plant cell wall colonization by N. frontalis. With certain diets, yeasts could therefore be a good tool to optimize the microbial degradation of lignocellulosic materials, but more research is needed to understand their mechanisms of action, so that they can be used with maximum efficiency as feed supplements.     

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 of cellulolytic anaerobic bacteria from the bovine rumen: Comparison of three methods

Three methods—the most probable number technique, a cellulose agar overlay on basal carbohydrate plates, and carboxymethylcellulose in basal carbohydrate plates—were compared for ease of preparation, interpretation of results, and agreement in estimation of size of the cellulolytic bacterial population in digesta samples from the rumen. The most probable number method yielded consistent detection of cellulose hydrolysis in liquid medium but required at least a 10-day incubation, and its mean was associated with wide 95% confidence intervals. The cellulose overlay method was the least consistent, and zones of hydrolysis often were difficult to see. The carboxymethylcellulose method was the easiest method for preparation and required only a 2-day incubation. The three methods estimated the same population size (all within one-half log unit of each other), but the carboxymethylcellulose method had the lowest coefficient of variation.     

Detection of cellulolytic activity of bacteria and fungi growing on agar surfaces

Summary Cellulolytic activity of four fungal species growing on solid medium containing acid-swollen cellulose could be detected much more easily if fungal growth was partly inhibited by the detergent Triton X-100. The dye, aniline blue-black, did not affect growth but increased the sensitivity of detection of cellulolytic activity of both fungi and bacteria. Separating fungi from cellulose fibres by a layer of agar or by filters showed that cell-fibre contact is not necessary for cellulose degradation. Such degradation is clearer when contact is prevented.     

Quantitative analysis of cellulose degradation and growth of cellulolytic bacteria in the rumen

Ruminant animals digest cellulose via a symbiotic relationship with ruminal microorganisms. Because feedstuffs only remain in the rumen for a short time, the rate of cellulose digestion must be very rapid. This speed is facilitated by rumination, a process that returns food to the mouth to be rechewed. By decreasing particle size, the cellulose surface area can be increased by up to 10⁶-fold. The amount of cellulose digested is then a function of two competing rates, namely the digestion rate ( K _d) and the rate of passage of solids from the rumen ( K _p). Estimation of bacterial growth on cellulose is complicated by several factors: (1) energy must be expended for maintenance and growth of the cells, (2) only adherent cells are capable of degrading cellulose and (3) adherent cells can provide nonadherent cells with cellodextrins. Additionally, when ruminants are fed large amounts of cereal grain along with fiber, ruminal pH can decrease to a point where cellulolytic bacteria no longer grow. A dynamic model based on stella ^® software is presented. This model evaluates all of the major aspects of ruminal cellulose degradation: (1) ingestion, digestion and passage of feed particles, (2) maintenance and growth of cellulolytic bacteria and (3) pH effects.     

Dinitrogen fixation by obligate and facultative anaerobic bacteria in association with cellulolytic fungi

The anaerobic bacteriumClostridium butyricum is the major contributor to nitrogen gains by a cellulolytic/nitrogen-fixing population isolated from straw. Growth of the anaerobe is supported by the products of fungal cellulases. The facultative anaerobeEnterobacter cloacae does not make a significant direct contribution to nitrogen fixation but in association withC. butyricum allows the anaerobe to grow under aerobic conditions. The major function ofE. cloacae is though to be provision of oxygen-depleted microsites.     

In vitro study of the proteolytic activity of rumen anaerobic fungi

Abstract To better define the antigenic structure of the outer cell membranes of Legionellae, a panel of 6 monoclonal antibodies was raised against partially purified outer membranes of Legionella pneumophila serogroup 1, Corby strain. This study describes the purification and characterization of one of these monoclonal antibodies reacting with a 135-kDa protein, which was shown to be common to all 14 serogroups of Legionella pneumophila . It shows no cross-reactivity with 20 other Legionella species, or 9 other Gram-negative species tested by SDS-PAGE and Western blotting procedures. The epitope would appear to be predominantly surface exposed and, from preliminary detergent extraction studies, not peptidoglycan-associated.     

The effect of ammonia treatment on the solubilization of straw and the growth of cellulolytic rumen bacteria

Pre-treatment of straw with anhydrous ammonia increased its susceptibility to solubilization by the predominant cellulolytic bacteria from the rumen, Bacteroides succinogenes, Ruminococcus albus and R. flavefaciens. Ammonia treatment also increased the production of microbial protein and fermentation products by all three species. Scanning electron microscope observations of straw during digestion suggested that the attack of straw by these bacteria was accompanied by the formation of substantial numbers of adherent microcolonies.     

The effect of ammonia treatment on the solubilization of straw and the growth of cellulolytic rumen bacteria

Pre-treatment of straw with anhydrous ammonia increased its susceptibility to solubilization by the predominant cellulolytic bacteria from the rumen, Bacteroides succinogenes, Ruminococcus albus and R. flavefaciens. Ammonia treatment also increased the production of microbial protein and fermentation products by all three species. Scanning electron microscope observations of straw during digestion suggested that the attack of straw by these bacteria was accompanied by the formation of substantial numbers of adherent microcolonies.     

Fermentation of cellodextrins by cellulolytic and noncellulolytic rumen bacteria

Water-soluble cellodextrins were prepared from microcrystalline cellulose by using fuming hydrochloric acid and acetone precipitation. This cellodextrin preparation contained only trace amounts of glucose and cellobiose and was primarily composed of cellotetraose and cellopentaose. When various species of cellulolytic and noncellulolytic bacteria were cultured with cellodextrins, their growth rates and maximal optical densities were in most cases similar to those observed with cellobiose. Time course samplings and analyses of cellodextrins by high-pressure liquid chromatography indicated that longer-chain cellodextrins were hydrolyzed extracellularly to cellobiose and cellotriose. Cellodextrin utilization by noncellulolytic rumen bacteria and extracellular hydrolysis of cellodextrins increase the possibility that cross-feeding occurs in the rumen and help to explain the high numbers of noncellulolytic bacteria in ruminants fed fibrous diets.     

不同精粗比底物下瘤胃真菌和纤维降解细菌共培养发酵特性及菌群变化

采用体外厌氧共培养技术,研究了瘤胃真菌和纤维降解细菌在不同精粗比(A组为全粗料,B组3∶7,C组5∶5,D组7∶3,E组为全精料)底物下菌群变化及其共培养发酵特性。结果表明:与0h相比,发酵至24h时B组和C组的厌氧真菌数量有较大幅度的上升,A组和D组则有所下降,E组未检测到真菌生长;纤维降解细菌随精粗比的增加呈上升趋势。发酵至48h时,各组均未检测到真菌生长;从A组到C组细菌数量呈上升趋势,此后急剧下降。DGGE结果表明,A、B和C组(精粗比低于5∶5)的DGGE图谱相似,有11条共有条带,但是当精粗比上升到7:3时,条带数目显著下降。随精料比例的增加,整个发酵期共培养系统中pH值显著下降(P<0.05)。整个发酵期间,共培养系统发酵产生的VFA主要为乙酸,丙酸和丁酸的量较少,乙酸与丙酸比值从A组到C组呈下降趋势,此后呈上升趋势。随精料比例的上升,发酵48h时总挥发性脂肪酸浓度从A组到C组呈上升趋势,此后呈下降趋势。发酵48h的羧甲基纤维素酶活和木聚糖酶活均以A组最高,而α-淀粉酶活从A组到D组逐渐增大,而E组最低,仅为B、C、D组的1/4~1/3。     

Isolation and characterization of cellulolytic anaerobic fungi and associated mycoplasmas from the rumen of a steer fed a roughage diet

The isolation of 12 strains of cellulolytic fungi from the rumen of a roughage-fed steer is described. These represented three different genera, including one new genus and species (Orpinomyces bovis). The organisms were indistinguishable on the basis of fermentation products from cellulose, and their fermentation patterns were very similar to those of rumen fungi isolated in other countries. Mycoplasmas were found to be associated with 7 of the 12 isolates. The ecological role of the association of the mycoplasmas with rumen fungi is still unknown.     

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.     

Fermentation of woods by rumen anaerobic fungi

Abstract The potential of rumen anaerobic fungi for fermenting untreated woods has been assessed using two Neocallimastix species isolated from sheep. When a strain of N. frontalis was incubated for 11 days with wood from 12 hardwood (angiosperm) species, many woods were measurably fermented, with wood from Populus tremuloides (32%) and Fagus sylvatica (21%) being the most highly degraded. This N. frontalis solubilised celulose, hemicellulose and lignin in P. tremuloides wood. Lower degradation (17%) of P. tremuloides wood by a different species of Neocallimastix showed that the choice of fungus as well as the structure and chemistry of the wood influenced the amount of wood cell wall degraded by anaerobic fungi. The amount of degradation was not related to the length of fungal rhizoids.     

The fermentative characteristics of anaerobic rumen fungi

Substrate utilization and fermentation characteristics of rumen fungi of the genus Neocallimastix are described. Preliminary observations on the removal of monosaccharides from plant cell walls and the effect of fermentation products on growth of Neocallimastix sp. (isolate R1) are presented. The properties of rumen fungi are discussed in relation to their role in the rumen.