Interactions among cellulolytic bacteria from an anaerobic digester

High cellulolytic activity of particular strains did not cause dominance of one, or a few, species of fiber-digesting bacteria in a cattlewaste anaerobic digester. The population contained a large number of species and varieties with different cellulolytic and fiber-digesting activities. Although mixed cultures of some of these bacteria showed no intereffects, with others, cellulolysis was less or in some cases greater than that shown by individual components of the cultures. The interactions were probably related to effects on growth of the bacteria rather than on activities of components of the cellulase enzyme complex, and culture filtrates of two of the more numerous cellulolytic species ofClostridium affected growth of other cellulolytic bacteria. The inhibitory factor(s) appeared to be of bacteriocin type, but the stimulatory factor(s) was unknown. It was suggested that these interactions are localized or short-lived in the digester, and so the population remains in a dynamic steady state.Some inhibitions of growth of rumen cellulolytic bacteria were caused by the digester bacteria, but it was suggested that factors other than these inhibitions are responsible for the absence of rumen bacteria from anaerobic digesters.     

Citrate metabolism in anaerobic bacteria

Abstract The regulation of anaerobic citrate metabolism is very diverse among different groups of bacteria. In organisms like Streptococcus lactis and Clostridium sporosphaeroides which lack citrate synthase, the activity of its antagonistic enzyme, citrate lyase, need not be regulated. Many anaerobes like Rhodocyclus gelatinosus and Clostridium sphenoides are able to synthesize their own l -glutamate and contain citrate synthase. In these bacteria the activity of citrate metabolizing enzymes which are involved in a cascade system are under strict control. In Rc. gelatinosus activation/inactivation of citrate lyase is controlled by acetylation/deacetylation which is catalyzed by its corresponding regulatory enzymes, citrate lyase ligase and citrate lyase deacetylase. In C. sphenoides inactivation of citrate lyase is accomplished by deacetylation as well as by changing in the enzyme conformation. Activation of citrate lyase is catalyzed by citrate lyase ligase whose activity in addition is modulated by phosphorylation/dephosphorylation. Further, electron transport process also seems to play a role in the inactivation of citrate metabolizing enzymes in enteric bacteria.     

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.     

高效厌氧纤维素降解细菌的分离及酶特性研究

采用透明圈初筛和滤纸降解率复筛的方法从内蒙古绵羊瘤胃内容物中分离到高效厌氧纤维素降解细菌4株.通过形态学、生理生化反应、生态特性和遗传型的鉴定,所分离的4株菌WHQ、LYQ、LBG-1和NDF-3分别归为溶纤维丁酸弧菌(Butyrivibrio fibrisollvens)、黄色瘤胃球菌(Ruminococcus flavefaciens)、产琥珀酸丝状杆菌(Fibrobacter succinogenes)和解多糖梭菌(Clostridium polysaccharolyticum).测定了4株菌对滤纸的降解率,WHQ、LYQ、LBG-1和NDF-3的2周滤纸降解率分别为25.1%、14.3%、21.0%和20.6%.本研究同时对4株菌的滤纸酶活力、羧甲基纤维素酶活力和β-葡萄糖苷酶活力进行了测定.     

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.     

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.     

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.     

Characterization of 13 newly isolated strains of anaerobic, cellulolytic, thermophilic bacteria

Characteristics of 13 newly isolated thermophilic, anaerobic, and cellulolytic strains were compared with previously described strains of Clostridium thermocellum: ATCC 27405 and JW20 (ATCC 31549). Colony morphology, antibiotic sensitivity, fermentation end-products, and cellulose degradation were documented. All 13 strains were sensitive to erythromycin (5 μg/ml) and chloramphenicol (25 μg/ml), and all strains but one were sensitive to kanamycin (20 μg/ml). Polymerase chain reaction (PCR) amplification using primers based on gene sequences from C. thermocellum ATCC 27405 was successful for all 13 strains in the case of the hydrogenase gene and 11 strains in the case of phosphotransacetylase/acetate kinase genes. Ten strains amplified a product of the expected size with primers developed to be specific for C. thermocellum 16SrRNA primers. Two of the 13 strains did not amplify any product with the PCR primers designed for the phosphotransacetylase/acetate kinase and 16SrRNA primers. A MboI-like GATC- recognizing restriction activity was present in all of the five strains examined. The results of this study have several positive implications with respect to future development of a transformation system for cellulolytic thermophiles. Journal of Industrial Microbiology & Biotechnology (2001) 27, 275–280. Received 12 September 2000/ Accepted in revised form 20 November 2000     

One carbon metabolism in methanogenic bacteria

Two strains of Methanosarcina (M. Barkeri strain MS, isolated from sewage sludge, and strain UBS, isolated from lake sediments) were found to have similar cellular properties and to have DNA base compositions of 44 mol percent guanosine plus cytosine. Strain MS was selected for further studies of its one-carbon metabolism. M. barkeri grew autotrophically via H₂ oxidation/CO₂ reduction. The optimum temperature for growth and methanogenesis was 37°C. H₂ oxidation proceeded via an F₄₂₀-dependent NADP⁺-linked hydrogenase. A maximum specific activity of hydrogenase in cell-free extracts, using methyl viologen as electron acceptor, was 6.0 mol min · mg protein at 37°C and the optimum pH (9.0). M. barkeri also fermented methanol andmethylamine as sole energy sources for growth. Cell yields during growth on H₂/CO₂ and on methanol were 6.4 and 7.2 mg cell dry weight per mmol CH₄ formed, respectively. During mixotrophic growth on H₂/CO₂ plus methanol, most methane was derived from methanol rather than from CO₂. Similar activities of hydrogenase were observed in cell-free extracts from H₂/CO₂-grown and methanol-grown cells. Methanol oxidation apparently proceeded via carrierbound intermediates, as no methylotrophy-type of methanol dehydrogenase activity was observed in cell-free extracts. During growth on methanol/CO₂, up to 48% of the cell carbon was derived from methanol indicating that equivalent amounts of cell carbon were derived from CO₂ and from an organic intermediate more reduced than CO₂. Cell-free extracts lacked activity for key cell carbon synthesis enzymes of the Calvin cycle, serine path, or hexulose path.Abbreviations CAPS cycloaminopropane sulfonic acid - CH₃-SCoM methyl coenzyme M - DCPIP 2,6-dichlorophenolindophenol - DEAE diethylaminoethyl - dimethyl POPOP 1,4-bis-2-(4-mothyl-5-phenyloxazolyl)-benzene - DNA deoxyribonucleic acid - dpm dismtegrations per min - DTT dithiothreitol - EDTA ethylenediamine tetraacetic acid - F₄₂₀ factor 420 - G+C guanosine plus cytosine - NAD⁺ nicotinamide adenine dinucleotide - NADP⁺ nicotinamide adenine dinucleotide phosphate - PBBW phosphate buffered basal Weimer - PMS phenazine methosulfate - PPO 2,5-diphenyloxazole - rRNA ribosomal ribonucleic acid - RuBP ribulose-1,5-bisphosphate - Tris tris-hydroxymethyl-aminomethane - max maximum specific growth rate     

Carbon monoxide-dependent energy metabolism in anaerobic bacteria and archaea

Despite its toxicity for the majority of living matter on our planet, numerous microorganisms, both aerobic and anaerobic, can use carbon monoxide (CO) as a source of carbon and/or energy for growth. The capacity to employ carboxidotrophic energy metabolism anaerobically is found in phylogenetically diverse members of the Bacteria and the Archaea. The oxidation of CO is coupled to numerous respiratory processes, such as desulfurication, hydrogenogenesis, acetogenesis, and methanogenesis. Although as diverse as the organisms capable of it, any CO-dependent energy metabolism known depends on the presence of carbon monoxide dehydrogenase. This review summarizes recent insights into the CO-dependent physiology of anaerobic microorganisms with a focus on methanogenic archaea. Carboxidotrophic growth of Methanosarcina acetivorans, thought to strictly rely on the process of methanogenesis, also involves formation of methylated thiols, formate, and even acetogenesis, and, thus, exemplifies how the beneficial redox properties of CO can be exploited in unexpected ways by anaerobic microorganisms.     

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.     

Genomics of cellulolytic bacteria

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Aerobic and facultatively anaerobic cellulolytic bacteria from the gut of the termite Zootermopsis angusticollis

AIMS: To demonstrate the occurrence of cellulolytic bacteria in the termite Zootermopsis angusticollis. METHODS AND RESULTS: Applying aerobic cultivation conditions we isolated 119 cellulolytic strains from the gut of Z. angusticollis, which were assigned to 23 groups of aerobic, facultatively anaerobic or microaerophilic cellulolytic bacteria. 16S rDNA restriction fragment pattern and partial 16S rDNA sequence analysis, as well as numerical taxonomy, were used for the assignment of the isolates. The Gram-positive bacteria of the actinomycetes branch could be assigned to the order Actinomycetales including the genera Cellulomonas/Oerskovia, Microbacterium and Kocuria. The Gram-positive bacteria from the order Bacillales belonged to the genera Bacillus, Brevibacillus and Paenibacillus. Isolates related to the genera Afipia, Agrobacterium/Rhizobium, Brucella/Ochrobactrum, Pseudomonas and Sphingomonas/Zymomonas from the alpha-proteobacteria and Spirosoma-like from the "Flexibacteriaceae" represented the Gram-negative bacteria. CONCLUSIONS: A cell titre of up to 10(7) cellulolytic bacteria per ml, determined for some isolates, indicated that they may play a role in cellulose digestion in the termite gut in addition to the cellulolytic flagellates and termite's own cellulases. SIGNIFICANCE AND IMPACT OF THE STUDY: The impact of bacteria on cellulose degradation in the termite gut has always been a matter of debate. In the present survey we investigated the aerobic and facultatively anaerobic cellulolytic bacteria in the termite gut.     

Diversity of cellulolytic bacteria in landfill

Nine of 37 cellulolytic bacterial isolates obtained from landfill waste could be easily differentiated on the basis of gross morphological characteristics. Four isolates were selected for further characterization and on the basis of initial results appear to be previously unidentified cellulolytic species of bacteria. An aerotolerant anaerobic, cellulolytic Clostridium and three obligately anaerobic cellulolytic Eubacterium isolates are described. The Clostridium has an unusually high pH optimum for growth of 7.7. The optimum temperature for growth is 50°C. The pH growth optimum of each of the Eubacterium isolates is around pH 7.0 while temperature optima are 37° 45° and 50°C for LFI, LF4 and LF5 respectively. Most isolates had growth optima in the thermophilic range. The ease with which apparently previously unidentified species could be isolated is a reflection of the unique and highly variable, heterogeneous environment within landfill waste.     

Multiple mechanisms controlling carbon metabolism in bacteria

Catabolite repression is a universal phenomenon, found in virtually all living organisms. These organisms range from the simplest bacteria to higher fungi, plants, and animals. A mechanism involving cyclic AMP and its receptor protein (CRP) in Escherichia coli was established years ago, and this mechanism has been assumed by many to serve as the prototype for catabolite repression in all organisms. However, recent studies have shown that this mechanism is restricted to enteric bacteria and their close relatives. Cyclic AMP-independent mechanisms of catabolite repression occur in other bacteria, yeast, plants, and even E. coli. In fact, single-celled organisms such as E. coli, Bacillus subtilis, and Saccharomyces cerevisiae exhibit multiple mechanisms of catabolite repression, and most of these are cyclic AMP-independent. The mechanistic features of the best of such characterized processes are briefly reviewed, and references are provided that will allow the reader to delve more deeply into these subjects.     

Genetics of carbon metabolism in methylotrophic bacteria

Abstract The application of genetic techniques to the methylotrophic bacteria has greatly enhanced studies of these important organisms. Two methylotrophic systems have been studied in some detail, the serine cycle for formaldehyde assimilation and the methanol oxidation system. In both cases, genes have been cloned and mapped in Methylobacterium species (facultative serine cycle methanol-utilizers). In addition, methanol oxidation genes have been studied in an autotrophic methanol-utilizer ( Paracoccus denitrificans ) and three methanotrophs ( Methylosporovibrio methanica, Methylomonas albus and Methylomonas sp. A4). Although much remains to be learned in these systems, it is becoming clear that the order of C₁ genes has been conserved to some extent in methylotrophic bacteria, and that many C₁ genes are loosely clustered on the chromosome. Operons appear to be rare, but some examples have been observed. The extension of genetic approaches to both the obligate and facultative methylotrophs holds much promise for the future in understanding and manipulating the activities of these bacteria.     

A novel mode of lactate metabolism in strictly anaerobic bacteria

Lactate is a common substrate for major groups of strictly anaerobic bacteria, but the biochemistry and bioenergetics of lactate oxidation is obscure. The high redox potential of the pyruvate/lactate pair of E₀′ = ?190 mV excludes direct NAD⁺ reduction (E₀′ = ?320 mV). To identify the hitherto unknown electron acceptor, we have purified the lactate dehydrogenase (LDH) from the strictly anaerobic, acetogenic bacterium Acetobacterium woodii. The LDH forms a stable complex with an electron‐transferring flavoprotein (Etf) that exhibited NAD⁺ reduction only when reduced ferredoxin (Fd^2?) was present. Biochemical analyses revealed that the LDH/Etf complex of A. woodii uses flavin‐based electron confurcation to drive endergonic lactate oxidation with NAD⁺ as oxidant at the expense of simultaneous exergonic electron flow from reduced ferredoxin (E₀′ ≈ –500 mV) to NAD⁺ according to: lactate + Fd^2? + 2 NAD⁺ → pyruvate + Fd + 2 NADH. The reduced Fd^2? is regenerated from NADH by a sequence of events that involves conversion of chemical (ATP) to electrochemical and finally redox energy (Fd^2? from NADH) via reversed electron transport catalysed by the Rnf complex. Inspection of genomes revealed that this metabolic scenario for lactate oxidation may also apply to many other anaerobes.