The effect of biomass density on cellulose solubilisation rates

The aim of this work was to compare the impact of inoculation density on the rate of cellulose hydrolysis by a rumen derived culture with that of a microbial enrichment from an organic waste anaerobic digester. The results showed a linear relationship between the mass of biomass at the start of the first order degradation phase (Xo) and the first order hydrolysis rate (r) for both rumen inoculated and leachate inoculated cellulose digestions and that the slopes of these relationships were not distinguishable. This suggested that differences in the microbial community, media and other environmental factors had a lesser impact on the hydrolysis rate compared to the effect of the number of cells in the system. This could be of great importance to industrial applications of anaerobic digestion technologies as it suggested that if cells densities in the waste treatment digesters could be boosted to match those seen in the rumen, then the rates of the cellulose hydrolysis would rise.     

Concurrent microscopic observations and activity measurements of cellulose hydrolyzing and methanogenic populations during the batch anaerobic digestion of crystalline cellulose

This study compares process data with microscopic observations from an anaerobic digestion of organic particles. As the first part of the study, this article presents detailed observations of microbial biofilm architecture and structure in a 1.25-L batch digester where all particles are of an equal age. Microcrystalline cellulose was used as the sole carbon and energy source. The digestions were inoculated with either leachate from a 220-L anaerobic municipal solid waste digester or strained rumen contents from a fistulated cow. The hydrolysis rate, when normalized by the amount of cellulose remaining in the reactor, was found to reach a constant value 1 day after inoculation with rumen fluid, and 3 days after inoculating with digester leachate. A constant value of a mass specific hydrolysis rate is argued to represent full colonization of the cellulose surface and first-order kinetics only apply after this point. Additionally, the first-order hydrolysis rate constant, once surfaces were saturated with biofilm, was found to be two times higher with a rumen inoculum, compared to a digester leachate inoculum. Images generated by fluorescence in situ hybridization (FISH) probing and confocal laser scanning microscopy show that the microbial communities involved in the anaerobic biodegradation process exist entirely within the biofilm. For the reactor conditions used in these experiments, the predominant methanogens exist in ball-shaped colonies within the biofilm.     

Effect of biomass concentration and inoculum source on the rate of anaerobic cellulose solubilization

This study determines cellulose solubilization kinetics from controlled batch digestions and shows the effect of inoculum biomass concentrations. Separate measurements and analyzes were performed for sessile biomass (biofilms) and planktonic biomass (free suspensions). Experiments were conducted using either leachate enriched on cellulose or rumen fluid as inoculum to assess if the effect of biomass concentration was consistent for microbial populations from different source environments. All batch digestions were fitted to a first-order kinetic model (R² ranging from 0.94 to 0.99). Regression analysis used to compare the first-order hydrolysis rate showed that the first-order hydrolysis rate was most strongly correlated with the concentration of sessile biomass rather than with the concentration of total or planktonic biomass. The correlation between solubilization rate and sessile biomass was statistically the same for the rumen and leachate inoculated reactors indicating that at low concentration ratios of inoculum to cellulose, the rate of cellulose solubilization is dependant primarily on sessile biomass concentration rather than the species profile of the cellulolytic community.     

Testing the Efficiency of Biofilms Formed on Natural Substratum,Coyonoxtle (Opuntia Imbricata), for the Anaerobic Degradation of Aromatic Compounds

In this study, previously developed anaerobic microbial consortia capable of degrading aromatic compounds were used to develop biofilms on a natural material, coyonoxtle (Opuntia imbricata), which is abundantly available in North Mexico. The developed biofilms were evaluated for their efficiency in the biodegradation of different aromatic compounds, viz., phenol, catechol, 4‐aminobenzoic acid and p‐phenylenediamine in batch reactors. It was observed that in reactors with biofilms a more than 90 % COD removal and a concomitant production of methane could be obtained. But the rate of COD removal and methane production varied depending upon the type of biofilm used. Rumen‐derived biofilms demonstrated a lag phase of 7 to 14 days, whereas sludge‐derived biofilms exhibited a lag phase of more than three weeks. Between the biofilms from two sources, rumen‐derived biofilms showed a higher COD removal and methane production than sludge‐derived biofilms. When biofilm reactors were compared with reactors containing freely suspended consortia, it was evident that both rumen– and sludge‐derived biofilm reactors exhibited a two‐fold higher COD removal and methane production. Based on the results obtained, it can be concluded that coyonoxtle has the potential for use as a substratum.     

Cellulose Fermentation by a Rumen Anaerobic Fungus in Both the Absence and the Presence of Rumen Methanogens

The fermentation of cellulose by an ovine rumen anaerobic fungus in the absence and presence of rumen methanogens is described. In the monoculture, moles of product as a percentage of the moles of hexose fermented were: acetate, 72.7; carbon dioxide, 37.6; formate, 83.1; ethanol, 37.4; lactate, 67.0; and hydrogen, 35.3. In the coculture, acetate was the major product (134.7%), and carbon dioxide increased (88.7%). Lactate and ethanol production decreased to 2.9 and 19%, respectively, little formate was detected (1%), and hydrogen did not accumulate. Substantial amounts of methane were produced in the coculture (58.7%). Studies with [2-¹⁴C]acetate indicated that acetate was not a precursor of methane. The demonstration of cellulose fermentation by a fungus extends the range of known rumen organisms capable of participating in cellulose digestion and provides further support for a role of anaerobic fungi in rumen fiber digestion. The effect of the methanogens on the pattern of fermentation is interpreted as a shift in flow of electrons away from electron sink products to methane via hydrogen. The study provides a new example of intermicrobial hydrogen transfer and the first demonstration of hydrogen formation by a fungus.     

Homologous catalytic domains in a rumen fungal xylanase: evidence for gene duplication and prokaryotic origin

A cDNA (xynA), encoding xylanase A (XYLA), was isolated from a cDNA library, derived from mRNA extracted from the rumen anaerobic fungus, Neocallimastix patriciarum. Recombinant XYLA, purified from Escherichia coli harbouring xynA, had a M(r) of 53,000 and hydrolysed oat-spelt xylan to xylobiose and xylose. The enzyme did not hydrolyse any cellulosic substrates. The nucleotide sequence of xynA revealed a single open reading frame of 1821 bp coding for a protein of M(r) 66,192. The predicted primary structure of XYLA comprised an N-terminal signal peptide followed by a 225-amino-acid repeated sequence, which was separated from a tandem 40-residue C-terminal repeat by a threonine/proline linker sequence. The large N-terminal reiterated regions consisted of distinct catalytic domains which displayed similar substrate specificities to the full-length enzyme. The reiterated structure of XYLA suggests that the enzyme was derived from an ancestral gene which underwent two discrete duplications. Sequence comparison analysis revealed significant homology between XYLA and bacterial xylanases belonging to cellulase/xylanase family G. One of these homologous enzymes is derived from the rumen bacterium Ruminococcus flavefaciens. The homology observed between XYLA and a rumen prokaryote xylanase could be a consequence of the horizontal transfer of genes between rumen prokaryotes and lower eukaryotes, either when the organisms were resident in the rumen, or prior to their colonization of the ruminant. It should also be noted that Neocallimastix XYLA is the first example of a xylanase which consists of reiterated sequences. It remains to be established whether this is a common phenomenon in other rumen fungal plant cell wall hydrolases.     

Anaerobic fermentation: Microbes from ruminants

Fed-batch fermentation of biomass could provide a route for direct conversion of renewable resources to commercially significant chemicals. The ecosystem in the forestomach (rumen) of ruminants provides a highly reduced environment (oxidation-reduction potential of ?250 to ?450 mV) in which anaerobic bacteria directly utilize cellulose, hemicellulose, and other fermentable biomass constituents to produce acetate, butyrate, propionate, methane and carbon dioxide at pH 5.7 to 7.3. The cellulose fermentation in the rumen is impacted by the physically and chemically heterogeneous character of the insoluble substrate, as well as the properties of the mixed culture responsible for fibre hydrolysis and carbohydrate utilization. The rumen system provides an interesting case study in the context of possible process concepts for direct fermentation of biomass to commercially important chemicals such as acetate, propionate, succinate, lactate and ethanol. The role of the chemical and physical characteristics of the substrate, the microbes in the rumen system and the metabolic pathways of soluble carbohydrates are discussed in the context of cellulose and hemicellulose fermentation.     

The rumen anaerobic fungi

The anaerobic fungi represent a new group of organisms inhabiting the rumen ecosystem and possess a life cycle alternating between a motile flagellated form (zoospore) and a non-motile vegetative reproductive form (thallus). In vivo studies show extensive colonization of plant material suspended in the rumen indicating the fungi have a role in fiber digestion. Pure cultures of anaerobic fungi ferment cellulose to give lactate, acetate, CO₂ and H₂ as the major products. Ethanol and formate may also be produced. Fermentation of cellulose by the fungi in coculture with H₂-utilizing methanogens results in a shift in the fermentation pattern favouring the production of H₂ (utilized in the formation of CH₄) and acetate at the expense of the electron-sink products, lactate and ethanol. It is postulated that the methanogens in reducing the partial pressure of H₂, facilitate an increased passage of reducing equivalents towards the production of H₂ via a pyridine-nucleotide (PN)-linked hydrogenase reaction. H₂ is believed to be produced in microbodies of the fungi called hydrogenosomes which possess all of the enzymes necessary for this function including PN-linked hydrogenase. Absence of mitochondria and key electron transport components in these organisms indicate a dependence wholly on fermentative processes for growth. Anaerobic fungi also participate in hemicellulose and starch degredation but it is not yet clear whether they have a role in the degradation of lignin. Simple sugars (mono- and disaccharides) are readily utilized and their uptake is subject to similar regulatory constraints such as is found with other micro-organisms.Enzymological studies have revealed that anaerobic fungi release substantial amounts of endo-acting cellulase and protease, possibly giving them a competitive advantage over rumen bacteria in the degradation of plant structural material.     

Form and Function of Clostridium thermocellum Biofilms

The importance of bacterial adherence has been acknowledged in microbial lignocellulose conversion studies; however, few reports have described the function and structure of biofilms supported by cellulosic substrates. We investigated the organization, dynamic formation, and carbon flow associated with biofilms of the obligately anaerobic cellulolytic bacterium Clostridium thermocellum 27405. Using noninvasive, in situ fluorescence imaging, we showed biofilms capable of near complete substrate conversion with a characteristic monolayered cell structure without an extracellular polymeric matrix typically seen in biofilms. Cell division at the interface and terminal endospores appeared throughout all stages of biofilm growth. Using continuous-flow reactors with a rate of dilution (2 h⁻¹) 12-fold higher than the bacterium''s maximum growth rate, we compared biofilm activity under low (44 g/liter) and high (202 g/liter) initial cellulose loading. The average hydrolysis rate was over 3-fold higher in the latter case, while the proportions of oligomeric cellulose hydrolysis products lost from the biofilm were 13.7% and 29.1% of the total substrate carbon hydrolyzed, respectively. Fermentative catabolism was comparable between the two cellulose loadings, with ca. 4% of metabolized sugar carbon being utilized for cell production, while 75.4% and 66.7% of the two cellulose loadings, respectively, were converted to primary carbon metabolites (ethanol, acetic acid, lactic acid, carbon dioxide). However, there was a notable difference in the ethanol-to-acetic acid ratio (g/g), measured to be 0.91 for the low cellulose loading and 0.41 for the high cellulose loading. The results suggest that substrate availability for cell attachment rather than biofilm colonization rates govern the efficiency of cellulose conversion.     

Use of zoospore concentrations and life cycle parameters in determining the population of anaerobic fungi in the rumen ecosystem

An indirect approach to quantification of the fibrolytic anaerobic fungi in the rumen is described. A mathematical model of the life cycle of anaerobic fungi, based upon observations of the life histories and growth kinetics of these organisms in vitro and in vivo, is constructed and solved in the steady-state to determine the population of particle-attached (substrate-associated) fungal thalli from the concentration of free-swimming zoospores in rumen liquid. The values obtained are broadly consistent with ruminal observations and with observations on faecal populations, which assume that a significant proportion of fungi leaving the rumen (as cysts or spores) can ultimately be accounted for in the faeces.     

Identification, detection, and spatial resolution of Clostridium populations responsible for cellulose degradation in a methanogenic landfill leachate bioreactor

An anaerobic landfill leachate bioreactor was operated with crystalline cellulose and sterile landfill leachate until a steady state was reached. Cellulose hydrolysis, acidogenesis, and methanogenesis were measured. Microorganisms attached to the cellulose surfaces were hypothesized to be the cellulose hydrolyzers. 16S rRNA gene clone libraries were prepared from this attached fraction and also from the mixed fraction (biomass associated with cellulose particles and in the planktonic phase). Both clone libraries were dominated by Firmicutes phylum sequences (100% of the attached library and 90% of the mixed library), and the majority fell into one of five lineages of the clostridia. Clone group 1 (most closely related to Clostridium stercorarium), clone group 2 (most closely related to Clostridium thermocellum), and clone group 5 (most closely related to Bacteroides cellulosolvens) comprised sequences in Clostridium group III. Clone group 3 sequences were in Clostridium group XIVa (most closely related to Clostridium sp. strain XB90). Clone group 4 sequences were affiliated with a deeply branching clostridial lineage peripherally associated with Clostridium group VI. This monophyletic group comprises a new Clostridium cluster, designated cluster VIa. Specific fluorescence in situ hybridization (FISH) probes for the five groups were designed and synthesized, and it was demonstrated in FISH experiments that bacteria targeted by the probes for clone groups 1, 2, 4, and 5 were very abundant on the surfaces of the cellulose particles and likely the key cellulolytic microorganisms in the landfill bioreactor. The FISH probe for clone group 3 targeted cells in the planktonic phase, and these organisms were hypothesized to be glucose fermenters.     

A novel identified Pseudomonas aeruginosa,which exhibited nitrate- and nitrite-dependent methane oxidation abilities,could alleviate the disadvantages caused by nitrate supplementation in rumen fluid fermentation

After the occurrence of nitrate-dependent anaerobic methane oxidation (AMO) in rumen fluid culture was proved, the organisms that perform the denitrifying anaerobic methane oxidizing (DAMO) process in the rumen of dairy goat were investigated by establishing two enrichment culture systems, which were supplied with methane as the sole carbon source and NaNO₃ or NaNO₂ as the electron acceptor. Several Operational Taxonomic Units (OTU) belonging to Proteobacteria became dominant in the two enrichment systems. The identified Pseudomonas aeruginosa, which was isolated from the NaNO₂ enrichment system, could individually perform a whole denitrifying anaerobic methane oxidizing process. Further in vitro rumen fermentation showed that supplementation with the isolated P. aeruginosa could reduce methane emissions, alleviate the nitrite accumulation and prevent the decrease in propionic acid product caused by nitrate supplementation.     

Axenic Cultivation of the Cellulolytic Flagellate Trichomitopsis termopsidis (Cleveland) from the Termite Zootermopsis*

SYNOPSIS. Trichomitopsis termopsidis (Cleveland), a cellulolytic hindgut symbiote of the termite Zootermopsis, has been cultivated axenically under anaerobic conditions. The medium consists of cellulose, reduced glutathione, fetal calf serum, yeast extract, and autoclaved rumen fluid or autoclaved rumen bacteria, in a buffered salt solution the composition of which is based on an analysis of Zootermopsis hindgut fluid. The hindgut contents of surface-sterilized termites were inoculated into anaerobic buffer-containing cellulose and serum. Repeated passages yielded mixed cultures of T. termopsidis and termite hindgut bacteria. Flagellates were then inoculated into complete medium containing antibiotics, and after 2 passages, axenic cultures of T. termopsidis were obtained. Various nutritional supplements, including clarified rumen fluid or heat-killed bacteria of several known species failed to support the growth of T. termopsidis when substituted for autoclaved rumen fluid. The flagellates did not grow when any of several carbohydrates were substituted for cellulose. Electron microscopy of flagellates from axenic cultures revealed that cellulose particles and partially digested bacteria were present in food vacuoles. No endosymbiotic bacteria were present in the cytoplasm indicating that T. termopsidis does not depend on living prokaryotes for cellulose digestion. The results suggest that T. termopsidis possesses the enzyme cellulase.     

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

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

Anaerobic solid state fermentation of cellulosic substrates with possible application to cellulase production

Summary A solid state fermentation process was developed for the conversion of straw and cellulose under anaerobic conditions by a mixed culture of cellulolytic and methanogenic organisms. The bioconversion rate and efficiency were compared under mesophilic (35° C) and thermophilic (55° C) conditions. Cellulolytic activity was assayed in terms of sugar and overall soluble organic matter (chemical oxygen demand, COD) production. Maximum conversion rates were obtained under thermophilic conditions, i.e. 8.4 g and 14.2 g COD/kg·d, respectively, when wheat straw and cellulose were used as substrates. The cellulolytic activity of the reactor contents (23% dry matter), measured under substrate excess conditions, amounted to 50 g COD/kg·d. As a comparison, the activity of rumen contents (15% dry matter) measured by the same assay amounted to 150 g COD/kg·d. The anaerobic cellulases appeared to be substrate bound. This and the relative low activity levels attained, limit the perspectives of producing cellulase enzymes by this type of process.     

Ureolytic bacteria in sheep rumen.

Estimates were made of the numbers of viable bacteria in the rumens of sheep receiving different rations. Representative colonies were isolated and tested for urease production. Some urease-positive isolates were characterized and identified. The ureolytic activities of the urease-producing isolates were determined and compared with the activity of rumen fluid. The rations fed to the sheep did not exert a significant influence on the relative numbers of the urease-producting organisms in the rumen. No obligately anaerobic ureolytic bacteria were found. All urease-positive isolates were facultatively anaerobic, Gram-positive, catalase-positive cocci. Out of ten isolates, nine were identified as Staphylococcus saprophyticus and one as Micrococcus varians. The total urease activity of the different isolates based on the lowest numbers in which they were present in the rumen, compared favourably with the urease activity of rumen fluid. The facultatively anaerobic Gram-positive cocci were probably responsible for a large proportion of the urease activity of the rumen fluid. Conditions prevailing in the rumen were found to be conducive to urease production by the isolates tested.     

Fatty acid analysis and spoilage potential of biofilms from two breweries

AIM: The microbial composition of biofilms from different locations of beer bottling plants were compared based on fatty acid profiles and correlated with the product-spoiling potential of these biofilms. METHODS AND RESULTS: The whole cell fatty acid profiles of 78 biofilms from bottling plants of two breweries were analysed. About half of the lipid profiles were dominated by oleic and linoleic acid, which refer to a high proportion of yeasts. In addition, more than half of all samples contained dimethylacetals indicating the presence of strictly anaerobic bacteria. Typical fatty acids for potentially beer-spoiling genera were detected in three biofilms. The majority of the biofilms contained no beer-spoiling organisms, as shown by inoculation experiments in beer. CONCLUSIONS: Biofilms from different locations of bottling plants were different with respect to their microbial composition. Potentially product-spoiling populations could be detected in a small number of samples. SIGNIFICANCE AND IMPACT OF THE STUDY: Biofilms on industrial plants can be characterized by a fast and cultivation-independent method with respect to overall microbial composition and presence of potentially product-spoiling micro-organisms.     

Rumen Fungi and Forage Fiber Degradation

The role of anaerobic rumen fungi in in vitro forage fiber degradation was determined in a two forage × two inoculum source × five treatment factorial design. Forages used as substrates for rumen microorganisms were Coastal bermuda grass and alfalfa; inoculum sources were rumen fluid samples from a steer fed Coastal bermuda grass hay or alfalfa hay; treatments were whole rumen fluid (WRF), WRF plus streptomycin (0.2 mg/ml of rumen fluid) and penicillin (1.25 mg/ml of fluid), WRF plus cycloheximide (0.5 mg/ml of fluid), WRF plus streptomycin, penicillin, and cycloheximide, and McDougall buffer. Populations of fungi as shown by sporangial development were greater on bermuda grass leaves than on alfalfa leaflets regardless of inoculum source. However, endogenous fungal populations were greater from the alfalfa hay inoculum. Cycloheximide inhibited the fungi, whereas streptomycin and penicillin, which inhibit bacterial populations, resulted in an increase in numbers of sporangia in the alfalfa inoculum, suggesting an interaction between bacteria and fungi. Bacteria (i.e., WRF plus cycloheximide) were equal to the total population in degrading dry matter, neutral-detergent fiber (NDF), acid-detergent fiber (ADF), and cellulose for both inocula and both forages. Degradation of dry matter, NDF, ADF, and cellulose by anaerobic fungi (i.e., WRF plus streptomycin and penicillin) was less than that due to the total population or bacteria alone. However, NDF, ADF, and cellulose digestion was 1.3, 2.4, and 7.9 percentage units higher, respectively, for bermuda grass substrate with the alfalfa versus bermuda grass inoculum, suggesting a slight benefit by rumen fungi. No substantial loss of lignin (72% H₂SO₄ method) occurred due to fungal degradation. The most active fiber-digesting population in the rumen was the bacteria, even when streptomycin and penicillin treatment resulted in an increase in rumen fungi over untreated WRF. The development of large numbers of sporangia on fiber may not indicate a substantial role as digesters of forage.     

Propionate Formation from Cellulose and Soluble Sugars by Combined Cultures of Bacteroides succinogenes and Selenomonas ruminantium

Succinate is formed as an intermediate but not as a normal end product of the bovine rumen fermentation. However, numerous rumen bacteria are present, e.g., Bacteroides succinogenes, which produce succinate as a major product of carbohydrate fermentation. Selenomonas ruminantium, another rumen species, produces propionate via the succinate or randomizing pathway. These two organisms were co-cultured to determine if S. ruminantium could decarboxylate succinate produced by B. succinogenes. When energy sources used competitively by both species, i.e. glucose or cellobiose, were employed, no succinate was found in combined cultures, although a significant amount was expected from the numbers of Bacteroides present. The propionate production per S. ruminantium was significantly greater in combined than in single S. ruminantium cultures, which indicated that S. ruminantium was decarboxylating the succinate produced by B. succinogenes. S. ruminantium, which does not use cellulose, grew on cellulose when co-cultured with B. succinogenes. Succinate, but not propionate, was produced from cellulose by B. succinogenes alone. Propionate, but no succinate, accumulated when the combined cultures were grown on cellulose. These interspecies interactions are models for the rumen ecosystem interactions involved in the production of succinate by one species and its decarboxylation to propionate by a second species.     

Electricity generation from cellulose by rumen microorganisms in microbial fuel cells

In microbial fuel cells (MFCs) bacteria generate electricity by mediating the oxidation of organic compounds and transferring the resulting electrons to an anode electrode. The objective of this study was to test the possibility of generating electricity with rumen microorganisms as biocatalysts and cellulose as the electron donor in two-compartment MFCs. The anode and cathode chambers were separated by a proton exchange membrane and graphite plates were used as electrodes. The medium in the anode chamber was inoculated with rumen microorganisms, and the catholyte in the cathode compartment was ferricyanide solution. Maximum power density reached 55 mW/m(2) (1.5 mA, 313 mV) with cellulose as the electron donor. Cellulose hydrolysis and electrode reduction were shown to support the production of current. The electrical current was sustained for over 2 months with periodic cellulose addition. Clarified rumen fluid and a soluble carbohydrate mixture, serving as the electron donors, could also sustain power output. Denaturing gradient gel electrophoresis (DGGE) of PCR amplified 16S rRNA genes revealed that the microbial communities differed when different substrates were used in the MFCs. The anode-attached and the suspended consortia were shown to be different within the same MFC. Cloning and sequencing analysis of 16S rRNA genes indicated that the most predominant bacteria in the anode-attached consortia were related to Clostridium spp., while Comamonas spp. abounded in the suspended consortia. The results demonstrated that electricity can be generated from cellulose by exploiting rumen microorganisms as biocatalysts, but both technical and biological optimization is needed to maximize power output.