Proteolytic activity of a rumen anaerobic fungus

Abstract A strain of the anaerobic phycomycetous fungus Neocallimastix frontalis isolated from the rumen of a sheep had a high proteolytic activity which became predominantly extracellular during growth. Proteolytic activity appeared to be due to a metalloprotease, as it was inhibited by 1,10-phenanthroline, EDTA and other chelators but not by phenylmethylsulphonyl fluoride (PMSF). Inhibition by EDTA was fully reversed by the addition of Zn²⁺, Ca²⁺ or Co²⁺, whereas addition of metal ions in the presence of 1,10-phenanthroline restored only a little activity. p -Chloromercuribenzoate (PCMB) was also inhibitory in dialysed supernatant fluid. N-α-p-Tosyl- l -lysine chloromethylketone (TLCK) inhibited proteolysis, suggesting that the protease(s) has a trypsin-like specificity, but benzoylarginine p -nitroanilide was not hydrolysed. Protease activity has a broad pH profile with a maximum at pH 7.5. Gel fractionation indicated that most of the activity was in a high- M _r form.     

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.     

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 effect of the presence of glucose on the fermentation of mannose by the anaerobic fungus Neocallimastix frontalis strain RE1

Abstract The disappearance of mannose and the formation of formate, acetate, lactate, ethanol and succinate by Neocallimastix frontalis strain RE1 occurred slowly when mannose was the only substrate present. When an equal quantity of glucose was present, the fermentation of mannose increased. Incubations with ¹³C-labelled mannose and glucose confirmed that the presence of both substrates resulted in increased product formation from mannose and reduced product formation from glucose. The relative proportions of products formed from the two substrates varied, possibly in part due to differences in the rates of growth of the fungus. The strains of N. frontalis able to utilize mannose may have a competitive advantage in the rumen ecosystem.     

Purification and characterization of an extracellular β-xylosidase from the rumen anaerobic fungus Neocallimastix frontalis

The purification of beta-xylosidase (beta-D-xyloside xylohydrolase, EC 3.2.1.37) from Neocallimastix frontalis was performed by ammonium sulphate precipitation, ion exchange chromatography, gel filtration and preparative isoelectric focusing. The enzyme had a molecular mass of 180,000 Da, an isoelectric point at pH 4.35 and catalysed the hydrolysis of p-nitrophenyl-beta-D-xylopyranoside optimally at pH 6.5 and 35 degrees C with a Km of 0.33 mg ml-1. The enzymatic activity was strongly increased by the presence of Ca2+, Mn2+, Zn2+, Co2+ or Mg2+ and completely inhibited by Hg2+ and p-chloromercuribenzoate. The purified protein also had a low level of xylanase activity.     

Effect of cocultivation of ruminal chytrid fungi with Methanobrevibacter smithii on lucerne stem degradation and extracellular fungal enzyme activities

Pentoses and hexoses were removed simultaneously from lucerne ( Medicago sativa ) stem by Piromonas communis and Neocallimastix frontalis growing axenically or in coculture with Methanobrevibacter smithii . In monocultures, more arabinose (68–71%) than glucose (52–53%) or xylose (25–30%) was removed. In cocultures, xylose solubilization from cell walls increased for P. communis whereas glucose solubilization increased for both fungi. The rates of sugar removal were monosaccharide-dependent. For both fungi, the specific activities of extracellular hydrolytic enzymes were markedly elevated in methanogenic cocultures.     

Effect of anaerobic fungi on in vitro feed digestion by mixed rumen microflora of buffalo

Five strains of anaerobic fungi isolated from the faeces of wild (hog deer, Cervus porcinus; blackbuck, Antelope cervicapra; spotted deer, Axis axis; nilgai, Baselophus tragocamelus) and rumen liquor of domestic (sheep, Ovies aries) ruminants showing high fibrolytic enzyme producing ability were added to mixed rumen microflora of buffalo to study their effect on the digestibility of lignocellulosic feed (wheat straw and wheat bran in the ratio of 80:20), enzyme production and fermentation end products in in vitro conditions. Among the 5 isolates studied, FNG5 (isolated from nilgai) showed the highest stimulating effect on apparent digestibility (35.31 +/- 1.61% vs. 28.61 +/- 1.55%; P < 0.05), true digestibility (43.64 +/- 1.73% vs. 35.37 +/- 1.65%; P < 0.01), neutral detergent fiber digestibility (29.30 +/- 2.58% vs. 18.47 +/- 2.12; P < 0.01) of feed 24 h after inoculation compared to the control group. The production of carboxymethyl cellulase, xylanase, acetyl esterase and beta-glucosidase was significantly (P < 0.05) higher in the FNG5 inoculated incubation medium. There was no improvement in the digestibility and enzyme production on the addition of the other 4 isolates. Total volatile fatty acid levels as well as the concentration of acetate, propionate, isobutyrate and valerate were significantly higher in the FNG5 added group as compared to the control group. The fungal isolate FNG5 from nilgai, a wild ruminant, was found to be superior to the other isolates tested and appears to have a potential to be used as a feed additive for improving fiber degradation in domestic ruminants.     

Comparison of morphological and enzyme characteristics of anaerobic fungi isolated from Cervus dama

Seven anaerobic fungal isolates from Cervus dama (domesticated and free living) were grown on carboxymethyl cellulose (CMC) and avicel, and monitored over a five day period for substrate utilization and cellulase activities. All fungal isolates showed monocentric growth patterns; four of them had polyflagellated zoospores and morphologically resembled members of the genus Neocallimastix; the other three had monoflagellated zoospores and resembled members of the genus Piromyces. All of the isolates degraded CMC and avicel, and exhibited cellulolytic activities (carboxymethyl cellulase-(CMC-ase) and avicelase).     

The effects of co-cultivation with the acetogen Acetitomaculum ruminis on the fermentative metabolism of the rumen fungi Neocallimastix patriciarum and Neocallimastix sp. strain L2

Abstract The effects of co-cultivation with the hydrogen-utilizing acetogenic bacterium Acetitomaculum ruminis on the fermentative activities of the rumen fungi Neocallimastix patriciarum or Neocallimastix sp. L2 were investigated. In both co-cultures acetate production increased, making it the predominant fermentation product, as the accumulation of lactate, formate, ethanol, H₂ and (in the case of Neocallimastix sp. L2) succinate all decreased. The effects of co-cultivation with Methanobrevibacter smithii were more pronounced. Decreased activities of lactate dehydrogenase, alcohol dehydrogenase and (in the case of Neocallimastix sp. L2) fumarate reductase accompanied the shift in fermentation product formation. The rate of glucose utilization and the fungal biomass yield were also increased in the co-culture.     

Enhanced resistance of anaerobic rumen fungi to the ionophores monensin and lasalocid in the presence of methanogenic bacteria

The presence of Methanobrevibacter smithii altered the susceptibility of the anaerobic fungi Neocallimastix frontalis and Piromonas communis to the carboxylic ionophores monensin and lasalocid. The ionophores depressed growth (measured by chitin accretion), the uptake of glucose and the production of H2, formate and acetate by the fungi growing axenically in semi-solid medium. In the presence of M. smithii, the sensitivity of the fungi to monensin and lasalocid was decreased. For example, the uptake of glucose by N. frontalis strain RE1 in the culture was reduced to 50% of the control value by monensin at 0.5 microgram/ml. In the presence of M. smithii strain PS, approximately three times as much monensin was needed to bring about the same effect. In similar tests, the sensitivity of strain RE1 to lasalocid was decreased about nine-fold in the presence of M. smithii. The effect was not observed if the methanogens were killed by autoclaving before inoculation. It is suggested that the enhanced resistance to ionophores in the presence of M. smithii is a consequence of changes in the energy metabolism of the fungi growing in co-culture.     

Enhanced resistance of anaerobic rumen fungi to the ionophores monensin and lasalocid in the presence of methanogenic bacteria

The presence of Methanobrevibacter smithii altered the susceptibility of the anaerobic fungi Neocallimastix frontalis and Piromonas communis to the carboxylic ionophores monensin and lasalocid. The ionophores depressed growth (measured by chitin accretion), the uptake of glucose and the production of H2, formate and acetate by the fungi growing axenically in semi-solid medium. In the presence of M. smithii , the sensitivity of the fungi to monensin and lasalocid was decreased. For example, the uptake of glucose by N. frontalis strain RE1 in the culture was reduced to 50% of the control value by monensin at 0.5 mUg/ml. In the presence of M. smithii strain PS, approximately three tunes as much monensin was needed to bring about the same effect. In similar tests, the sensitivity of strain RE1 to lasalocid was decreased about nine-fold in the presence of M. smithii. The effect was not observed if the methanogens were killed by autoclaving before inoculation. It is suggested that the enhanced resistance to ionophores in the presence of M. smithii is a consequence of changes in the energy metabolism of the fungi growing in co-culture.     

Efficacy of coumarin on hepatic key enzymes of glucose metabolism in chemical induced type 2 diabetic rats

The study was undertaken to evaluate the antidiabetic effect of coumarin on carbohydrate metabolic key enzymes in control and streptozotocin (STZ)-nicotinamide (NA)-induced diabetic rats. On oral administration of coumarin at a dose of 100 mg/kg body weight per day to diabetic rats for 45 days; resulted in a significant reduction in the levels of plasma glucose, glycosylated hemoglobin (HbA₁c) and increase in the levels of insulin and hemoglobin. Administration of coumarin caused a significant increase in the levels of glycolytic enzyme (hexokinase) and hepatic shunt enzyme (glucose-6-phophate dehydrogenase) whereas significant decrease in the levels of gluconeogenic enzymes (glucose-6-phosphatase and fructose-1,6-bisphosphatase) in diabetic treated rats. Furthermore, protection against body weight loss of diabetic animals also observed. This study indicates that the administration of coumarin to diabetic rats resulted in alterations in the metabolism of glucose with subsequent reduction in plasma glucose levels.     

Effects of aromatic amino acids, phenylacetate and phenylpropionate on fermentation of xylan by the rumen anaerobic fungi, Neocallimastix frontalis and Piromyces communis

AIMS: Anaerobic fungi are important members of the fibrolytic community of the rumen. The aim of this study was to study their requirement for aromatic amino acids (AA) and related phenyl acids (phenylpropionic and phenylacetic acids) for optimal xylan fermentation. METHODS AND RESULTS: Neocallimastix frontalis RE1 and Piromyces communis P were grown in a defined medium containing oat spelts xylan as the sole energy source, plus one of the following N sources: ammonia; ammonia plus a complete mixture of 20 AA commonly found in protein; ammonia plus complete AA mixture minus aromatic AA; ammonia plus phenyl acids; ammonia plus complete AA mixture without aromatic AA plus phenyl acids. Both species grew in all the media, indicating no absolute requirement for AA. The complete AA mixture increased (P<0.05) acetate concentration by 18% and 15%, sugar utilization by 33% and 22% and microbial yield by about 22% and 15% in N. frontalis and P. communis, respectively, in comparison with the treatments that had ammonia as the only N source. Neither the supply of aromatic AA or phenol acids, nor their deletion from the complete AA mixture, affected the fermentation rate, products or yield of either species. CONCLUSIONS: AA were not essential for N. frontalis and P. communis, but their growth on xylan was stimulated. The effects could not be explained in terms of aromatic AA alone. SIGNIFICANCE AND IMPACT OF THE STUDY: Ruminant diets should contain sufficient protein to sustain optimal fibre digestion by ruminal fungi. Aromatic AA or phenyl acids alone cannot replace the complete AA mixture.     

The effect of lasalocid on glucose uptake, hydrogen production and the solubilization of straw by the anaerobic rumen fungus Neocallimastix frontalis

Three isolates of Neocallimastix frontalis grown in pure culture with glucose as substrate differed in their response to the presence of lasalocid in the growth medium. For two strains (RE1 and RK21) hydrogen production and glucose uptake were reduced to 50% or less of the control values in the presence of lasalocid at 0–25 μg/ ml. A third strain (PNK2) retained significant fermentative ability in the presence of at least four times this amount of lasalocid. Lasalocid was rather less inhibitory to the solubilization of straw by these fungi, PNK2 again providing the most resistant strain. It is concluded that the use of lasalocid in ruminant feeds is likely to have a strain selective effect on the rumen population of Neocallimastix.     

Degradation of lignified secondary cell walls of lucerne (Medicago sativa L.) by rumen fungi growing in methanogenic co-culture

Aims: To compare the abilities of the monocentric rumen fungi Neocallimastix frontalis, Piromyces communis and Caecomyces communis, growing in coculture with Methanobrevibacter smithii, to colonize and degrade lignified secondary cell walls of lucerne (alfalfa) hay. Methods and Results: The cell walls of xylem cylinders isolated from stems of lucerne contained mostly xylans, cellulose and lignin together with a small proportion of pectic polysaccharides. All of these major components were removed during incubation with the three fungi, and differing cell wall polysaccharides were degraded to different extents. The greatest dry weight loss was found with N. frontalis and least with C. communis, and scanning electron microscopy revealed that these extensively colonized different cell types. C. communis specifically colonized secondary xylem fibres and showed much less degradation than N. frontalis and P. communis. Conclusions: Neocallimastix frontalis and P. communis were efficient degraders of the cell walls of lucerne xylem cylinders. Degradation occurred of pectic polysaccharides, xylan and cellulose. Loss of lignin from the xylem cylinders probably resulted from the cleavage of xylan releasing xylan–lignin complexes. Significance and Impact of the Study: Unlike rumen bacteria, the rumen fungi N. frontalis, P. communis and C. communis are able to degrade lignified secondary walls in lucerne stems. These fungi could improve forage utilization by ruminants and may have potential in the degradation of lignocellulosic biomass in the production of biofuels.     

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.     

共存于厌氧真菌分离培养液中瘤胃甲烷菌的检测及其多样性分析

对分离自山羊瘤胃的真菌分离培养液中甲烷菌进行16SrDNA扩增、DGGE分析、RFLP及测序分析,研究共存于真菌分离培养液中甲烷菌的种类及其多样性。DGGE结果显示:从厌氧真菌分离至第45代,甲烷菌多样性指数由1·32降至0·99,相似性最低为34·7%;第45代至62代,多样性指数由0·99升至1·15,相似性最低为89·2%。RFLP多态性分析69个克隆共得到5个操作分类单元,选择其中6个具有代表性的序列进行测序。序列及系统进化分析表明,属于其中3个操作分类单元的克隆最相似菌都是UnculturedarchaealsymbiontPA202,相似性均为95%,没有与这些克隆相似性较高的已培养甲烷菌;属于另外2个操作分类单元的克隆最相似菌都是Unculturedrumenmethanogen956,相似性均为97%,最相似已知菌为Methanobrevibactersp.NT7,相似性为97%。结果表明,真菌培养液中存在目前尚未分离培养的瘤胃甲烷菌。     

Dynamics of initial colonization of nonconserved perennial ryegrass by anaerobic fungi in the bovine rumen

Anaerobic fungi (Neocallimastigales) are active degraders of fibrous plant material in the rumen. However, only limited information is available relating to how quickly they colonize ingested feed particles. The aim of this study was to determine the dynamics of initial colonization of forage by anaerobic fungi in the rumen and the impact of different postsampling wash procedures used to remove loosely associated microorganisms. Neocallimastigales-specific molecular techniques were optimized to ensure maximal coverage before application to assess the population size (quantitative PCR) and composition (automated ribosomal intergenic spacer analysis) of the colonizing anaerobic fungi. Colonization of perennial ryegrass (PRG) was evident within 5 min, with no consistent effect of time or wash procedure on fungal population composition. Wash procedure had no effect on population size unlike time, which had a significant effect. Colonizing fungal population size continued to increase over the incubation period after an initial lag of c. 4 min. This dynamic differs from that reported previously for rumen bacteria, where substantial colonization of PRG occurred within 5 min. The observed delay in colonization of plant material by anaerobic fungi is suggested to be primarily mediated by the time taken for fungal zoospores to locate, attach and encyst on plant material.     

Structure of genes and an insertion element in the methane producing archaebacterium Methanobrevibacter smithii

Summary DNA fragments cloned from the methanogenic archaebacterium Methanobrevibacter smithii which complement mutations in the purE and proC genes of E. coli have been sequenced. Sequence analyses, transposon mutagenesis and expression in E. coli minicells indicate that purE and proC complementations result from the synthesis of M. smithii polypeptides with molecular weights of 36,697 and 27,836 respectively. The encoding genes appear to be located in operons. The M. smithii genome contains 69% A/T basepairs (bp) which is reflected in unusual codon usages and intergenic regions containing approximately 85% A/T bp. An insertion element, designated ISM1, was found within the cloned M. smithii DNA located adjacent to the proC complementing region. ISM1 is 1381 bp in length, has 29 bp terminal inverted repeat sequences and contains one major ORF encoded in 87% of the ISM1 sequence. ISM1 is mobile, present in approximately 10 copies per genome and integration duplicates 8 bp at the site of insertion. The duplicated sequences show homology with sequences within the 29bp terminal repeat sequence of ISM1. Comparison of our data with sequences from halophilic archaebacteria suggests that 5GAANTTTCA and 5TTTTAATATAAA may be consensus promoter sequences for archaebacteria. These sequences closely resemble the consensus sequences which precede Drosophila heatshock genes (Pelham 1982; Davidson et al. 1983). Methanogens appear to employ the eubacterial system of mRNA: 16SrRNA hybridization to ensure initiation of translation; the consensus ribosome binding sequence is 5AGGTGA.