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.     

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.     

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.     

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.     

Degradation of maize stem by two rumen fungal species, Piromyces communis and Caecomyces communis, in pure cultures or in association with cellulolytic bacteria.

Two species of rumen fungi, Piromyces (Piromonas) communis FL and Caecomyces (Sphaeromonas) communis FG10, were cultured alone or in association with the cellulolytic bacteria Ruminococcus flavefaciens or Fibrobacter succinogenes on maize stem. A kinetic study of the degradation of the substrate was then made. After 48 h of culture, all non-lignified tissues observed by scanning electron microscopy disappeared with P communis and degradation was as complete as that observed in the rumen. In contrast, C communis degraded little of the plant cell walls. The ability of P communis to more rapidly degrade maize stem was probably due to the presence of filamentous rhizoids. The extent of dry matter loss after 8 days of incubation was practically the same in all the monocultures and in the 4 cocultures. However, the rate of degradation was faster in the bacterial than in the fungal monocultures and the co-cultures. No metabolic interaction was observed.     

Effects of glycerol on the growth,adhesion, and cellulolytic activity of rumen cellulolytic bacteria and anaerobic fungi

The effect of glycerol on the growth, adhesion, and cellulolytic activity of two rumen cellulolytic bacterial species,Ruminococcus flavefaciens andFibrobacter succinogenes subsp.succinogenes, and of an anaerobic fungal species,Neocallimastix frontalis, was studied. At low concentrations (0.1–1%), glycerol had no effect on the growth, adhesion, and cellulolytic activity of the two bacterial species. However, at a concentration of 5%, it greatly inhibited their growth and cellulolytic activity. Glycerol did not affect the adhesion of bacteria to cellulose. The growth and cellulolytic activity ofN. frontalis were inhibited by glycerol, increasingly so at higher concentrations. At a concentration of 5%, glycerol totally inhibited the cellulolytic activity of the fungus. Thus, glycerol can be added to animal feed at low concentrations.     

Degradation and utilization of cellulose and straw by three different anaerobic fungi from the ovine rumen

Three different ruminal fungi, a Neocallimastix sp. (strain LM-1), a Piromonas sp. (strain SM-1), and a Sphaeromonas sp. (strain NM-1), were grown anaerobically in liquid media which contained a suspension of either 1% (wt/vol) purified cellulose or finely milled wheat straw as the source of fermentable carbon. Fungal biomass was estimated by using cell wall chitin or cellular protein in cellulose cultures and chitin in straw cultures. Both strains LM-1 and SM-1 degraded cellulose with a concomitant increase in fungal biomass. Maximum growth of both fungi occurred after incubation for 4 days, and the final yield of protein was the same for both fungi. Cellulose degradation continued after growth ceased. Strain NM-1 failed to grow in the cellulose medium. All three anaerobic fungi grew in the straw-containing medium, and loss of dry weight from the cultures indicated degradation of straw to various degrees (LM-1 greater than SM-1 greater than NM-1). The total fiber component and the cellulose component of the straw were degraded in similar proportions, but the lignin component remained undegraded by any of the fungi. Maximum growth yield on straw occurred after 4 days for strain LM-1 and after 5 days for strains SM-1 and NM-1. The calculated yield of cellular protein for strain LM-1 was twice that of both strains SM-1 and NM-1. The cellular protein yield of strain SM-1 was the same in both cellulose and straw cultures. In contrast to cellulose, straw degradation ceased after the end of the growth phase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Degradation and utilization of cellulose and straw by three different anaerobic fungi from the ovine rumen.

Three different ruminal fungi, a Neocallimastix sp. (strain LM-1), a Piromonas sp. (strain SM-1), and a Sphaeromonas sp. (strain NM-1), were grown anaerobically in liquid media which contained a suspension of either 1% (wt/vol) purified cellulose or finely milled wheat straw as the source of fermentable carbon. Fungal biomass was estimated by using cell wall chitin or cellular protein in cellulose cultures and chitin in straw cultures. Both strains LM-1 and SM-1 degraded cellulose with a concomitant increase in fungal biomass. Maximum growth of both fungi occurred after incubation for 4 days, and the final yield of protein was the same for both fungi. Cellulose degradation continued after growth ceased. Strain NM-1 failed to grow in the cellulose medium. All three anaerobic fungi grew in the straw-containing medium, and loss of dry weight from the cultures indicated degradation of straw to various degrees (LM-1 greater than SM-1 greater than NM-1). The total fiber component and the cellulose component of the straw were degraded in similar proportions, but the lignin component remained undegraded by any of the fungi. Maximum growth yield on straw occurred after 4 days for strain LM-1 and after 5 days for strains SM-1 and NM-1. The calculated yield of cellular protein for strain LM-1 was twice that of both strains SM-1 and NM-1. The cellular protein yield of strain SM-1 was the same in both cellulose and straw cultures. In contrast to cellulose, straw degradation ceased after the end of the growth phase.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Influence of hydrogen-consuming bacteria on cellulose degradation by anaerobic fungi

The presence of methanogens Methanobacterium arboriphilus, Methanobacterium bryantii, or Methanobrevibacter smithii increased the level of cellulose fermentation by 5 to 10% in cultures of several genera of anaerobic fungi. When Neocallimastix sp. strain L2 was grown in coculture with methanogens the rate of cellulose fermentation also increased relative to that for pure cultures of the fungus. Methanogens caused a shift in the fermentation products to more acetate and less lactate, succinate, and ethanol. Formate transfer in cocultures of anaerobic fungi and M. smithii did not result in further stimulation of cellulolysis above the level caused by H2 transfer. When Selenomonas ruminatium was used as a H2-consuming organism in coculture with Neocallimastix sp. strain L2, both the rate and level of cellulolysis increased. The observed influence of the presence of methanogens is interpreted to indicate a shift of electrons from the formation of electron sink carbon products to H2 via reduced pyridine nucleotides, favoring the production of additional acetate and probably ATP. It is not known how S. ruminantium exerts its influence. It might result from a lowered production of electron sink products by the fungus, from consumption of electron sink products or H2 by S. ruminantium, or from competition for free sugars which in pure culture could exert an inhibiting effect on cellulolysis.     

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。     

Influence of hydrogen-consuming bacteria on cellulose degradation by anaerobic fungi.

The presence of methanogens Methanobacterium arboriphilus, Methanobacterium bryantii, or Methanobrevibacter smithii increased the level of cellulose fermentation by 5 to 10% in cultures of several genera of anaerobic fungi. When Neocallimastix sp. strain L2 was grown in coculture with methanogens the rate of cellulose fermentation also increased relative to that for pure cultures of the fungus. Methanogens caused a shift in the fermentation products to more acetate and less lactate, succinate, and ethanol. Formate transfer in cocultures of anaerobic fungi and M. smithii did not result in further stimulation of cellulolysis above the level caused by H2 transfer. When Selenomonas ruminatium was used as a H2-consuming organism in coculture with Neocallimastix sp. strain L2, both the rate and level of cellulolysis increased. The observed influence of the presence of methanogens is interpreted to indicate a shift of electrons from the formation of electron sink carbon products to H2 via reduced pyridine nucleotides, favoring the production of additional acetate and probably ATP. It is not known how S. ruminantium exerts its influence. It might result from a lowered production of electron sink products by the fungus, from consumption of electron sink products or H2 by S. ruminantium, or from competition for free sugars which in pure culture could exert an inhibiting effect on cellulolysis.     

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.     

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.     

Electron microscopic study of the methylcellulose-mediated detachment of cellulolytic rumen bacteria from cellulose fibers

The presence of methylcellulose prevents the attachment of cellulolytic rumen bacteria to cellulose fibers. The addition of methylcellulose to pure cultures of these organisms in which the cells are already adherent to cellulose causes their detachment from this insoluble substrate and the inhibition of their growth. Methylcellulose is not used as a carbon source by these organisms and has no effect on their growth when glucose and cellobiose are the carbon sources. Attached cells of Bacteroides succinogenes orient themselves in the plane of the individual cellulose fibers and their methylcellulose-induced detachment, which is complete (almost 100%), leaves grooves where the cellulose has been digested. Attached cells of Ruminococcus albus colonize the cellulose in a looser and less regular pattern and their almost complete methylcellulose-induced detachment leaves less regular pits in the cellulose surface. On the other hand, attached cells of Ruminococcus flavefaciens colonize the cellulose surface in a random orientation by means of a discernible exopolysaccharide network, and their less complete methylcellulose-induced detachment leaves no residual impressions on the cellulose surface. These data support the suggestion that bacterial attachment is necessary for the digestion of highly ordered crystalline cellulose, and that cellulolytic species differ in the nature of their attachment to this insoluble substrate and in the nature of their enzymatic attack. Methylcellulose is an effective agent for detaching major rumen cellulolytic bacteria from their cellulosic substrate.     

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.     

Biohydrogenation of linoleic acid by rumen fungi compared with rumen bacteria

AIMS: To investigate biohydrogenation of linoleic acid by rumen fungi compared with rumen bacteria, and to identify the fungus with the fastest biohydrogenation rate. METHODS AND RESULTS: Biohydrogenation of linoleic acid by mixed rumen fungi and mixed rumen bacteria were compared in vitro. With mixed rumen bacteria, all biohydrogenation reactions were finished within 100 min of incubation and the end product of biohydrogenation was stearic acid. With mixed rumen fungi, biohydrogenation proceeded more slowly over a 24-h period. Conjugated linoleic acid (CLA; cis-9, trans-11 C18 : 2) was an intermediate product, and vaccenic acid (VA; trans-11 C18 : 1) was the end product of biohydrogenation. Fourteen pure fungal isolates were tested for biohydrogenation rate. DNA sequencing showed that the isolate with the fastest rate belonged to the Orpinomyces genus. CONCLUSIONS: It is concluded that rumen fungi have the ability to biohydrogenate linoleic acid, but biohydrogenation is slower in rumen fungi than in rumen bacteria. The end product of fungal biohydrogenation is VA, as for group A rumen bacteria. Orpinomyces is the most active biohydrogenating fungus. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to demonstrate that rumen fungi can biohydrogenate fatty acids. Fungi could influence CLA content of ruminant products.     

Fermentation of cellulose and production of cellulolytic and xylanolytic enzymes by anaerobic fungi from ruminant and non-ruminant herbivores

Four anaerobic fungi were grown on filter paper cellulose and monitored over a 7–8 days period for substrate utilisation, fermentation products, and secretion of cellulolytic and xylanolytic enzymes. Two of the fungi (N1 and N2) were Neocallimastix species isolated from a ruminant (sheep) and the other two fungi were Piromyces species (E2 and R1) isolated from an Indian Elephant and an Indian Rhinoceros, respectively. The tested anaerobic fungi degraded the filter paper cellulose almost completely and estimated cellulose digestion rates were 0.25, 0.13, 0.21 and 0.18 g · 1^-1 · h^-1 for strains E2, N1, N2, R1, respectively. All strains secreted cellulolytic and xylanolytic enzymes, including endoglucanase, exoglucanase, -glucosidase and xylanase. Strain E2 secreted the highest levels of enzymes in a relatively short time. The product formation on avicel by enzymes secreted by the four fungi was studied. Both in the presence and absence of glucurono-1,5--lactone, a specific inhibitor of -glucosidase, mainly glucose was formed but no cellobiose. Therefore the exoglucanase secreted by the four fungi is probably a glucohydrolase.