Non-cellulolytic Bacteria Research Articles

Direct conversion of cellulose to l-lactic acid by a novel thermophilic Caldicellulosiruptor strain

BackgroundConsolidated bioprocessing (CBP) of lignocellulosic biomass to l-lactic acid using thermophilic cellulolytic/hemicellulolytic bacteria provides a promising solution for efficient lignocellulose conversion without the need for additional cellulolytic/hemicellulolytic enzymes. Most studies on the mesophilic and thermophilic CBP of lignocellulose to lactic acid concentrate on cultivation of non-cellulolytic mesophilic and thermophilic bacteria at temperatures of 30–55 °C with external addition of cellulases/hemicellulases for saccharification of substrates.Resultsl-Lactic acid was generated by fermenting microcrystalline cellulose or lignocellulosic substrates with a novel thermophilic anaerobic bacterium Caldicellulosiruptor sp. DIB 104C without adding externally produced cellulolytic/hemicellulolytic enzymes. Selection of this novel bacterium strain for lactic acid production is described as well as the adaptive evolution towards increasing the l-lactic acid concentration from 6 to 70 g/l on microcrystalline cellulose. The evolved strains grown on microcrystalline cellulose show a maximum lactic acid production rate of 1.0 g/l*h and a lactic acid ratio in the total organic fermentation products of 96 wt%. The enantiomeric purity of the l-lactic acid generated is 99.4%. In addition, the lactic acid production by these strains on several other types of cellulose and lignocellulosic feedstocks is also reported.ConclusionsThe evolved strains originating from Caldicellulosiruptor sp. DIB 104C were capable of producing unexpectedly large amounts of l-lactic acid from microcrystalline cellulose in fermenters. These strains produce l-lactic acid also from lignocellulosic feedstocks and thus represent an ideal starting point for development of a highly integrated commercial l-lactic acid production process from such feedstocks.

Characterization of an Anaerobic, Thermophilic, Alkaliphilic, High Lignocellulosic Biomass-Degrading Bacterial Community, ISHI-3, Isolated from Biocompost

The generation of a complex microbial consortium is a promising approach for efficient biomass decomposition. An anaerobic thermophilic alkaliphilic microbial consortium with efficient degradation ability was screened from bovine manure compost using non-pretreated milling corn stover (CS) and rice straw (RS). A stable microbial consortium ISHI-3 with high degradation ability for CS and RS was isolated by the roll tube technique. ISHI-3 comprised Herbivorax saccincola and bacteria belonging to the classes Pelotomaculum, Tepidanaerobacter, and Tepidimicrobium, as determined by DGGE of the PCR-generated 16S rRNA genes. Furthermore, metagenomics analysis using a 16S rRNA library was carried out to determine the bacterial distribution during degradation of CS and RS. H. saccincola and bacteria belonging to Pelotomaculum were relatively abundant in the beginning to middle periods of culture with CS and RS whereas bacteria belonging to Tepidanaerobacter and Tepidimicrobium gradually increased in the population during the later stages. To understand the role of non-cellulolytic bacteria in the consortium, novel strains ET1 and GL4, which were most closely related to Tepidimicrobium ferriphilum and Tepidanaerobacter acetatoxydans, were isolated from ISHI-3. Based on their carbon source usage, morphology, and phylogenetic analysis, we propose that strains ET1 and GL4 should be classified as a novel genus or species. Bacteria ET1 and GL4 can utilize different organic compounds as carbon and energy sources such as organic acids, alcohols, sugars, and amino acids, showing a preference for organic acids and alcohols rather than sugars such as glucose and cellobiose. These results indicated that ET1 and GL4 help to accelerate efficient lignocellulose degradation of H. saccincola.

Systems analysis in Cellvibrio japonicus resolves predicted redundancy of β-glucosidases and determines essential physiological functions.

Degradation of polysaccharides forms an essential arc in the carbon cycle, provides a percentage of our daily caloric intake, and is a major driver in the renewable chemical industry. Microorganisms proficient at degrading insoluble polysaccharides possess large numbers of carbohydrate active enzymes (CAZymes), many of which have been categorized as functionally redundant. Here we present data that suggests that CAZymes that have overlapping enzymatic activities can have unique, non-overlapping biological functions in the cell. Our comprehensive study to understand cellodextrin utilization in the soil saprophyte Cellvibrio japonicus found that only one of four predicted β-glucosidases is required in a physiological context. Gene deletion analysis indicated that only the cel3B gene product is essential for efficient cellodextrin utilization in C. japonicus and is constitutively expressed at high levels. Interestingly, expression of individual β-glucosidases in Escherichia coli K-12 enabled this non-cellulolytic bacterium to be fully capable of using cellobiose as a sole carbon source. Furthermore, enzyme kinetic studies indicated that the Cel3A enzyme is significantly more active than the Cel3B enzyme on the oligosaccharides but not disaccharides. Our approach for parsing related CAZymes to determine actual physiological roles in the cell can be applied to other polysaccharide-degradation systems.

Contributing factors in the improvement of cellulosic H2 production in Clostridium thermocellum/Thermoanaerobacterium co-cultures

Lignocellulosic biohydrogen is a promising renewable energy source that could be a potential alternative to the unsustainable fossil fuel-based energy. Biohydrogen production could be performed by Clostridium thermocellum that is the fastest known cellulose-degrading bacterium. Previous investigations have shown that the co-culture of C. thermocellum JN4 and a non-cellulolytic bacterium Thermoanaerobacterium thermosaccharolyticum GD17 produces more hydrogen than the C. thermocellum JN4 mono-culture, but the mechanism of this improvement is unknown. In this work, we carried out genomic and evolutionary analysis of hydrogenase-coding genes in C. thermocellum and T. thermosaccharolyticum, identifying one Ech-type [NiFe] hydrogenase complex in each species, and, respectively, five and four monomeric or multimeric [FeFe] hydrogenases in the two species. Further transcriptional analysis showed hydrogenase-coding genes in C. thermocellum are regulated by carbon sources, while hydrogenase-coding genes in T. thermosaccharolyticum are not. However, comparison between transcriptional abundance of hydrogenase-coding genes in mono- and co-cultures showed the co-culturing condition leads to transcriptional changes of hydrogenase-coding genes in T. thermosaccharolyticum but not C. thermocellum. Further metabolic analysis showed T. thermosaccharolyticum produces H2 at a rate 4-12-fold higher than C. thermocellum. These findings lead to the suggestion that the improvement of H2 production in the co-culture over mono-culture should be attributed to changes in T. thermosaccharolyticum but not C. thermocellum. Further suggestions can be made that C. thermocellum and T. thermosaccharolyticum perform highly specialized tasks in the co-culture, and optimization of the co-culture for more lignocellulosic biohydrogen production should be focused on the improvement of the non-cellulolytic bacterium.

Isolation and characterization of Achromobacter sp. CX2 from symbiotic Cytophagales, a non-cellulolytic bacterium showing synergism with cellulolytic microbes by producing β-glucosidase

A Gram-negative, obligately aerobic, non-cellulolytic bacterium was isolated from the cellulolytic association of Cytophagales. It exhibits biochemical properties that are consistent with its classification in the genus Achromobacter. Phylogenetic analysis together with the phenotypic characteristics suggest that the isolate could be a novel species of the genus Achromobacter and designated as CX2 (= CGMCC 1.12675 = CICC 23807). The strain CX2 is the symbiotic microbe of Cytophagales and produces β-glucosidase. The results showed that the non-cellulolytic Achromobacter sp. CX2 has synergistic activity with cellulolytic microbes by producing β-glucosidase. To our knowledge, this is the first report on the synergetic effect of the combination of non-cellulolytic and cellulolytic microbes, which is significant to help understand the cellulolytic mechanism of cellulose-digesting Cytophagales.

Enhancing the Cellulose-Degrading Activity of Cellulolytic Bacteria CTL-6 (Clostridium thermocellum) by Co-Culture with Non-cellulolytic Bacteria W2-10 (Geobacillus sp.)

The effect of a non-cellulolytic bacterium W2-10 (Geobacillus sp.) on the cellulose-degrading activity of a cellulolytic bacterium CTL-6 (Clostridium thermocellum) was determined using cellulose materials (paper and straw) in peptone cellulose solution (PCS) medium under aerobic conditions. The results indicated that in the co-culture, addition of W2-10 resulted in a balanced medium pH, and may provide the required anaerobic environment for CTL-6. Overall, addition of W2-10 was beneficial to CTL-6 growth in the adverse environment of the PCS medium. In co-culture with W2-10, the CTL-6 cellulose degradation efficiency of filter paper and alkaline-treated wheat straw significantly increased up to 72.45 and 37.79 %, respectively. The CMCase activity and biomass of CTL-6 also increased from 0.23 U ml(-1) and 45.1 μg ml(-1) (DNA content) up to 0.47 U ml(-1) and 112.2 μg ml(-1), respectively. In addition, co-culture resulted in accumulation of acetate and propionate up to 4.26 and 2.76 mg ml(-1). This was a respective increase of 2.58 and 4.45 times, in comparison to the monoculture with CTL-6.

Semi-quantitative analysis of Ruminococcus flavefaciens, Fibrobacter succinogenes and Streptococcus bovis in the equine large intestine using real-time polymerase chain reaction

There is a need to further our understanding of the role that the equine hindgut ecosystem plays in digestive processes and diseases. The aim of the present study was to utilise the real-time PCR technique to determine the abundance of candidate cellulolytic (Ruminococcus flavefaciens; Fibrobacter succinogenes) and non-cellulolytic (Streptococcus bovis) bacteria in lumen contents from the caecum, ventral and dorsal colon, and rectum of healthy horses (n 14). Total DNA was extracted from frozen and lyophilised lumen contents, and PCR primers and Taqman probes were designed based on 16S rDNA sequences for specific detection of candidate bacterial species. Overall, in frozen and lyophilised digesta, there were significantly (P F. succinogenes > S. bovis (P < 0.05), while in lyophilised digesta R. flavefaciens was present in significantly (P < 0.05) greater amounts than F. succinogenes and S. bovis. R. flavefaciens and F. succinogenes were abundant at significantly (P < 0.05) greater levels in lyophilised digesta v. frozen digesta, with no difference in S. bovis levels. These data indicate that for these bacteria at least, faeces are a suitable model for studying the bacterial ecosystem within the equine colon. The present study also indicates that the preservation method of digesta affects levels of bacteria detected.

Effective cellulose degradation by a mixed-culture system composed of a cellulolytic Clostridium and aerobic non-cellulolytic bacteria

A stable cellulose-degrading microflora enriched from composting materials has been analyzed in our laboratory. Cellulose-degrading efficiency of an anaerobic cellulolytic isolate, Clostridium straminisolvens CSK1, was remarkably lower than that of the original microflora. We successfully constructed bacterial communities with effective cellulose degradation by mixing C. straminisolvens CSK1 with aerobic non-cellulolytic bacteria isolated from the original microflora. Comparison of the cellulose degradation processes of the pure culture of C. straminisolvens CSK1 and the mixed-culture indicated that non-cellulolytic bacteria essentially contribute to cellulose degradation by supplying anaerobic environment, consuming metabolites, which otherwise deteriorate the cellulolytic activity, and by neutralizing pH.

Effects of Non-ionic Surfactants on Enzyme Distributions of Rumen Contents, Anaerobic Growth of Rumen Microbes, Rumen Fermentation Characteristics and Performances of Lactating Cows

A series of experiments was carried out to determine the possibility for the non-ionic surfactant (NIS) as a feed additive for ruminant animals. The effect of the NIS on c the enzyme distribution in the rumen fluids of Hereford bulls, 2 the growth of pure culture of rumen bacteria and e rumen anaerobic fungi, f the ruminal fermentation characteristics of Korean native cattle (Hanwoo), and g the performances of Holstein dairy cows were investigated. When NIS was added to rumen fluid at the level of 0.05 and 0.1% (v/v), the total and specific activities of cell-free enzymes were significantly (p<0.01) increased, but those of cell-bound enzymes were slightly decreased, but not statistically significant. The growth rates of ruminal noncellulolytic species (Ruminobacter amylophilus, Megasphaera elsdenii, Prevotella ruminicola and Selenomonas ruminantium) were significantly (p<0.01) increased by the addition of NIS at both concentrations tested. However, the growth rate of ruminal cellulolytic bacteria (Fibrobacter succinogenes, Ruminococcus albus, Ruminococcus flavefaciens and Butyrivibrio fibrisolvens) were slightly increased or not affected by the NIS. In general, NIS appears to effect Gram-negative bacteria more than Gram-positive bacteria; and non-cellulolytic bacteria more than cellulolytic bacteria. The growth rates of ruminal monocentric fungi (Neocallimastix patriciarum and Piromyces communis) and polycentric fungi (Orpinomyces joyonii and Anaeromyces mucronatus) were also significantly (p<0.01) increased by the addition of NIS at all concentrations tested. When NIS was administrated to the rumen of Hanwoo, Total VFA and ammonia-N concentrations, the microbial cell growth rate, CMCase and xylanase activities in the rumen increased with statistical difference (p<0.01), but NIS administration did not affect at the time of 0 and 9 h post-feeding. Addition of NIS to TMR resulted in increased TMR intake and increased milk production by Holstein cows and decreased body condition scores. The NEFA and corticoid concentrations in the blood were lowered by the addition of NIS. These results indicated that the addition of NIS may greatly stimulate the release of some kinds of enzymes from microbial cells, and stimulate the growth rates of a range of anaerobic ruminal microorganisms, and also stimulate the rumen fermentation characteristics and animal performances. Our data indicates potential uses of the NIS as a feed additive for ruminant animals. (Asian-Aust. J. Anim. Sci. 2003. Vol. 16, No. 1 : 104-115)

Influence of Tween 80 on the Enzyme Distribution in Rumen Liquor and on the Growth of Rumen Bacteria and Fungi

Ha, J.K., Lee, S.S., Goto, M., Moon, Y.H. and Cheng, K.-J. 2002. Influence of Tween 80 on the enzyme distribution in rumen liquor and on the growth of rumen bacteria and fungi. J. Appl. Anim. Res., 21: 129–143. The effect of the surfactant Tween 80 on the enzyme distribution and growth of pure cultures of rumen anaerobic non-cellulolytic and cellulolytic bacteria and monoceiitric and polycentric fungi was investigated. When Tween 80 was added to rumen fluid at the level of 0.05 and 0.1 per cent (v/v), the total and specific activities of most of the cell-free enzymes were significantly (P<0.01) increased, but those of cell-bound enzymes did not follow a definite trend. The growth rates of rumen noncellulolytic bacteria (Ruminobacter amylophilus, Megasphaera elsdenii, Prevotella ruminicola and Selenomonas ruminantium) were significantly (P<0.01) increased by the addition of Tween 80 at both the concentrations tested. However, the growth rate of rumen cellulolytic bacteria (Fibrobacter succinogenes, Ruminococcus albus, Ruminococcus flavefaciens and Butyrivibrio fibrisolvens) were generally not affected by Tween 80. In general, Tween 80 appears to affect gram-negative bacteria more than gram-positive bacteria; and non-cellulolytic bacteria more than cellulolytic bacteria. The growth rates of ruminal monoceiitric fungi (Neocallimastixpatriciarum Piromyces communis) and polycentric fungi (Orpinomyces joyonii and Anaeromyces mucronatus) were also significantly (P<0.01) increased by the addition of 0.05 per cent Tween 80. These results indicate that the addition of 0.05 per cent Tween 80 may greatly stimulate the release of some enzymes from microbial cells and also stimulate the growth rates of a range of anaerobic ruminal microorganisms and thus can be used as a potential feed additive for ruminants.

Initial pH as a Determinant of Cellulose Digestion Rate by Mixed Ruminal Microorganisms In Vitro

In vitro fermentations of pure cellulose by mixed ruminal microorganisms were conducted under conditions in which pH declined within ranges similar to those observed in the rumen. At low cellulose concentrations (12.5g/L), the first-order rate constants (κ) of cellulose disappearance were successively lower at initial pH values of 6.86, 6.56, and 6.02, but in each case the value of κ was maintained over a pH range of 0.3 to 1.2 units, as the fermentation progressed. Plots of κ versus initial pH were linear, and κ displayed a relative decrease of approximately 7% per 0.1 unit decrease in pH. At high cellulose concentration (50g/L) and an initial pH of 6.8, cellulose digestion was initially zero-order, the absolute rate of digestion declined with pH, and digestion essentially ceased at pH 5.3 after only 30% of the added cellulose was digested. Further incubation resulted in a loss of bound N and P, suggesting that at low pH cells lysed or detached from the undigested fibers. Pure cultures of ruminal cellulolytic bacteria also were able to ferment cellulose to a minimum pH of 5.1 to 5.3, but the extent of fermentation was increased by coculture with noncellulolytic bacteria. A model is proposed in which the first-order rate constant of cellulose digestion is determined by the pH at which the fermentation is initiated, and end product ratios reflect greater activity of the noncellulolytic population as pH declines.

Competition among three predominant ruminal cellulolytic bacteria in the absence or presence of non-cellulolytic bacteria.

Competition among three species of ruminal cellulolytic bacteria - Fibrobacter succinogenes S85, Ruminococcus flavefaciens FD-1 and Ruminococcus albus 7 - was studied in the presence or absence of the non-cellulolytic ruminal bacteria Selenomonas ruminantium or Streptococcus bovis. Co-cultures were grown under either batch or continuous conditions and populations were estimated using species-specific oligonucleotide probes to 16S rRNA. The three cellulolytic species co-existed in cellobiose batch co-culture, but inclusion of either Sel. ruminantium or Str. bovis yielded nearly a monoculture of the non-cellulolytic competitor. In cellobiose chemostats, R. albus completely dominated the triculture, but R. flavefaciens became predominant over F. succinogenes and R. albus when Sel. ruminantium was co-inoculated into the chemostats. Similar effects on competition were observed in the presence of Str. bovis at a lower (0.021 h(-1)), but not at a higher (0.045 h(-1)) dilution rate. In cellulose batch co-cultures, R. albus was more abundant than both F. succinogenes and R. flavefaciens, regardless of the presence of the non-cellulolytic species. Co-existence among the three cellulolytic species was observed in almost all cellulose chemostats, but Sel. ruminantium altered the relative proportions of the cellulolytic species. R. albus and R. flavefaciens were found to produce inhibitors that suppressed growth of R. flavefaciens and F. succinogenes, respectively. These data indicate that interactions among cellulolytic bacteria, while complex, can be modified further by non-cellulolytic species.

Effect of steroidal saponin from Yucca schidigera extract on ruminal microbes.

The effects of steroidal saponins (SAP) isolated from Yucca schidigera extract on ruminal bacteria and fungi were investigated in pure culture studies. Prevotella bryantii, Ruminobacter amylophilus, Selenomonas ruminantium and Streptococcus bovis were cultured through ten 24-h transfers in ruminal fluid medium containing 0 or 25 microg SAP ml-1 (measured as smilagenin equivalents). The four strains, each non-exposed or pre-exposed to SAP, were then inoculated into medium containing 0 or 250 microgram smilagenin equivalents ml-1 and 24-h growth curves were determined. The cellulolytic ruminal bacteria Ruminococcus flavefaciens, Fibrobacter succinogenes and Rc. albus were cultured for 72 h on Whatman no. 1 filter paper in medium containing 0, 9, 90 or 180 microgram SAP ml-1 for the determination of filter paper digestion and endoglucanase activity. The ruminal bacteria differed in their responses to SAP. Steroidal saponins in the medium reduced the growth of Strep. bovis (P < 0.01 at 2, 3, 4, 5, 6 and 8 h), P. bryantii (P < 0.05 at 4, 5, 6, 8, 10 and 24 h) and Rb. amylophilus (P < 0.05 at 14 and 24 h), but the growth of S. ruminantium was enhanced (P < 0.05) at 10, 14 and 24 h. The growth curves of all four non-cellulolytic species were similar (P > 0.05) between pre-exposed and non-exposed cultures and the concentrations of total SAP and soluble (deglycosylated) SAP in the liquid fraction were unchanged (P > 0.05) over time. Steroidal saponins inhibited the digestion of filter paper by all three cellulolytic bacteria, but F. succinogenes was less (P < 0.05) sensitive to SAP and more (P < 0. 05) effective at deglycosylating SAP than were Rc. flavefaciens or Rc. albus. Transmission electron microscopy revealed that SAP altered the cell walls of the SAP-inhibited non-cellulolytic bacteria. The ruminal fungi, Neocallimastix frontalis and Piromyces rhizinflata, were cultured on filter paper in medium containing 0, 0. 45, 2.25 or 4.5 microgram SAP ml-1. Filter paper digestion by both fungi was completely inhibited by 2.25 microgram SAP ml-1. Steroidal saponins from Y. schidigera inhibit cellulolytic ruminal bacteria and fungi, but their effects on amylolytic bacteria are species dependent and similar to the effects of ionophores. As such, SAP may be useful in nutritional applications targeting starch-digesting ruminal micro-organisms.

The role of ruminal carboxymethylcellulases in the degradation of β-glucans from cereal grain

The non-cellulolytic ruminal bacterium, Prevotella bryantii, grew rapidly on water soluble β-glucans, but a mutant deficient in carboxymethylcellulase (CMCase) activity could not. Native activity gels prepared with CMC and β-glucan indicated that the CMCase and β-glucanase activities migrated at similar rates. When a plasmid carrying the P. bryantii CMCase was transferred to Escherichia coli, the clone had CMCase and β-glucanase activities. P. bryantii grew on mixed β-1,3-1,4 glucans, but not on β-1,3 glucan, and similar results were obtained with the cellulolytic ruminal bacteria (Fibrobacter succinogenes S85, Ruminococcus flavefaciens FD1 and Ruminococcus albus B199). Mixed ruminal bacteria from cattle fed hay had twice as much CMCase activity as bacteria from cattle fed 90% cereal grain (P<0.05), and the CMCase and β-glucanase activities were highly correlated (r2=0.93) for the 22 samples tested. The CMCase and β-glucanase activities of mixed ruminal bacteria migrated slowly through polyacrylamide gels, but the migration distances were approximately the same. When β-glucan-utilizing ruminal bacteria were isolated from cattle fed hay or 90% cereal grain, 70 and 38% of the strains, respectively, had CMCase activity. A similar trend was observed with cellobiose-utilizing isolates (70 and 35%, respectively, were CMCase positive). All CMCase positive, cellobiose-utilizing ruminal bacteria could grow on β-glucan. CMCase activity was not strongly correlated with cellulose utilization, and less than 15% of the CMCase positive isolates grew on ball-milled cellulose. Based on these results, the cell-associated CMCases of ruminal bacteria provide a mechanism for utilizing water soluble β-glucans from cereal grains.

Comparison of Staining Techniques for Scanning Electron Microscopic Detection of Ultrastructural Protuberances on Cellulolytic Bacteria

Cell surface protuberances found on cellulolytic bacteria, but not on noncellulolytic bacteria, can be detected by scanning electron microscopy. Cationized ferritin typically has been used as a stain to increase the microscopic resolution of these protuberances; however, as a cation it binds only to negatively charged molecules. Thus, binding of cationized ferritin to cell surface molecules can be affected by the cell's physiological state. We incubated the noncellulolytic bacterium, Clostridium beijerinckii, in different media at various temperatures to obtain cells with different growth rates and physiological states. Staining of these cells with cationized ferritin showed that slower growing cells exhibited more protuberant structures than faster growing cells. This provided a clear correlation of ultrastructural protuberances with physiological changes associated with growth rate. On the other hand, cells stained with osmium tetroxide exhibited no protuberant structures regardless of growth rate. Because various cations are known to induce aggregation of surface proteins on some cellulolytic Clostridia, we incubated the cellulolytic bacterium, Clostridium cellulovorans, in media containing glucose, cellobiose, or cellulose. Ultrastructural protuberances were evident on all cells stained with cationized ferritin, but extensive protuberances were detected only on cells grown in cellulose and stained with osmium tetroxide. For cells stained with cationized ferritin, the presence of ultrastructural protuberances was correlated with growth rate rather than induction of cellulolytic systems. By contrast, cells stained with osmium tetroxide showed a clear correlation between protuberant structures and cellulolytic activity.

Physiological interactions between a mesophilic cellulolytic Clostridium and a non-cellulolytic bacterium

A mesophilic cellulolytic bacterium ( Clostridium strain C7) capable of N 2 fixation and a non-cellulolytic bacterium ( Klebsiella strain W1), both isolated from freshwater environments rich in decaying plant material, were co-cultured in a chemically defined, vitamin-deficient medium containing cellulose as the carbon and energy source. In the co-culture, an extracellular cellulase complex produced by the Clostridium hydrolyzed cellulose to soluble sugars that served as fermentable substrates for the Klebsiella. In turn, the Klebsiella excreted growth factors, identified as biotin and p-aminobenzoic acid, which were required by the Clostridium. Furthermore, demonstration of NH 4 +-repressible acetylene reduction by co-cultures growing in medium lacking combined nitrogen showed that the Clostridium fixed N 2, thus allowing growth of the Klebsiella, which was not a nitrogen fixer. The mutualistic relationships observed in the co-cultures may be representative of interactions that take place in natural environments in which cellulose-containing plant materials are biodegraded.

Physiological interactions between a mesophilic cellulolyticClostridiumand a non-cellulolytic bacterium

A mesophilic cellulolytic bacterium (Clostridium strain C7) capable of N2 fixation and a non-cellulolytic bacterium (Klebsiella strain W1), both isolated from freshwater environments rich in decaying plant material, were co-cultured in a chemically defined, vitamin-deficient medium containing cellulose as the carbon and energy source. In the co-culture, an extracellular cellulase complex produced by the Clostridium hydrolyzed cellulose to soluble sugars that served as fermentable substrates for the Klebsiella. In turn, the Klebsiella excreted growth factors, identified as biotin and p-aminobenzoic acid, which were required by the Clostridium. Furthermore, demonstration of NH4+-repressible acetylene reduction by co-cultures growing in medium lacking combined nitrogen showed that the Clostridium fixed N2, thus allowing growth of the Klebsiella, which was not a nitrogen fixer. The mutualistic relationships observed in the co-cultures may be representative of interactions that take place in natural environments in which cellulose-containing plant materials are biodegraded.

Physiological interactions between a mesophilic cellulolytic Clostridium and a non-cellulolytic bacterium

Physiological interactions between a mesophilic cellulolytic Clostridium and a non-cellulolytic bacterium

Study of the relation between straw quality and its colonization by rumen micro-organisms

SummaryThe relation between the dry-matter degradability (DMD) of different cultivars of barley straw and their colonization by rumen micro-organisms was studied in incubations of straw with the mixed populationin situand with pure cultures of rumen bacteriain vitro.The total attached populationin situwas quantified by particle-associated i glutamate dehydrogenase (GDH) activity, while the cellulolytic population was assessed by bound carboxymethylcellulase (CMCase) activity. CMCase reached a peak between 24 and 36 h incubation, and the peak activity was highly correlated with DMD between 24 ) and 96 h. After 36 h, CMCase activity declined, particularly with the more degradable (varieties. GDH continued to increase after 36 h and had no relation to DMD. The adhesion of pure cultures of both cellulolytic and non-cellulolytic bacteria was measured i turbidimetrically in a 30 min incubation. No relation was found between DMD and the extent of adhesion by any ofRuminococcus flavefaciens, Bacteroides succinogenes,Butyrivibrio fibrisolvensorSelenomonas ruminantium.It was concluded the rate of degradation of straw is not determined by the initial rate of attachment of cellulolytic micro-organisms, but by the way in which colonization subsequently develops after attachment takes place

Endo-1, 4-β-glucanase and β-glucosidase of the ciliate Polyplastron multivesiculatum free of cellulolytic bacteria

To determine the contribution of protozoal activity to cellulose degradation, we maintained the ciliate Polyplastron multivesiculatum free of extracellular and intracellular cellulolytic bacteria. Control experiments to verify the absence of such bacteria were performed on cellular extracts, on filtrates, and on ciliates before lyophilization. The enzymatic activity was determined by viscometry and by determining the amount of reducing sugars produced. The enzyme was found to be an endo-1, 4- β-glucanase. Polyplastron multivesiculatum which was maintained free of extracellular and intracellular bacteria degraded soluble derivatives of cellulose and slightly degraded native cellulose. The activity was not due to intracellular bacterial cellulase, as ingested bacteria were lysed and digested by proteolytic enzymes of the protozoan. In addition, when preparing the filtrate solutions, P. multivesiculatum was maintained in culture for 5 days with Streptococcus faecalis (a facultatively anaerobic, noncellulolytic bacterium). Isoelectric focusing and chromatofocusing of lyophilized P. multivesiculatum that was free of cellulolytic bacteria yielded three protein fractions that degrade carboxymethylcellulose and hydroxyethylcellulose. Chromatofocusing also revealed the presence of two protein fractions with β-glucosidase activity.