Growth of thermophilic acetogenic bacteria on methoxylated aromatic acids

Abstract The methoxylated aromatic acids vanillate and syringate supported the growth of Clostridium thermoaceticum and Clostridium thermoautotrophicum in an undefined culture medium (U); p -hydroxybenzoate or U did not support growth. Growth was proportional to the number of O -methyl residues on the aromatic ring and to the concentration of the methoxylated aromatic acid in U. Protein profiles, obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoretic (SDS-PAGE) analysis of cell extracts from vanillate, syringate, methanol, and glucose cultures of C. thermoaceticum , indicated differential gene expression between O -methyl aromatic-, methanol- and glucose-grown cells.     

Metabolism of One-Carbon Compounds by the Ruminal Acetogen Syntrophococcus sucromutans

Syntrophococcus sucromutans is the predominant species capable of O demethylation of methoxylated lignin monoaromatic derivatives in the rumen. The enzymatic characterization of this acetogen indicated that it uses the acetyl coenzyme A (Wood) pathway. Cell extracts possess all the enzymes of the tetrahydrofolate pathway, as well as carbon monoxide dehydrogenase, at levels similar to those of other acetogens using this pathway. However, formate dehydrogenase could not be detected in cell extracts, whether formate or a methoxyaromatic was used as electron acceptor for growth of the cells on cellobiose. Labeled bicarbonate, formate, [1-¹⁴C] pyruvate, and chemically synthesized O-[methyl-¹⁴C]vanillate were used to further investigate the catabolism of one-carbon (C₁) compounds by using washed-cell preparations. The results were consistent with little or no contribution of formate dehydrogenase and pointed out some unique features. Conversion of formate to CO₂ was detected, but labeled formate predominantly labeled the methyl group of acetate. Labeled CO₂ readily exchanged with the carboxyl group of pyruvate but not with formate, and both labeled CO₂ and pyruvate predominantly labeled the carboxyl group of acetate. No CO₂ was formed from O demethylation of vanillate, and the acetate produced was position labeled in the methyl group. The fermentation pattern and specific activities of products indicated a complete synthesis of acetate from pyruvate and the methoxyl group of vanillate.     

Selective isolation of Acetobacterium woodii on methoxylated aromatic acids and determination of growth yields

Anaerobic enrichments with methoxylated aromatic compounds as substrates (vanillate, syringate, trimethoxycinnamate) were inoculated from freshwater mud and sewage sludge samples. In 12 out of 16 cultures the same type of rod-shaped, motile bacteria was selectively enriched. Two strains, NZva16 and NZva24, were isolated in pure culture and recognized as Acetobacterium woodii by comparison with the type strain (DSM 1030).All three Acetobacterium strains were able to grow with all 10 of the tested aromatic compounds containing methoxyl groups. In the presence of bicarbonate, these substrates were used as sole organic electron donors and carbon sources. UV-absorption spectra revealed that the aromatic rings were not degraded, and that the corresponding hydroxy derivatives of the methoxylated compounds were formed. The only further fermentation product formed was acetate. When equimolar concentrations of the methoxylated benzoic acid derivatives were applied, the growth yields were proportional to the number of methoxyl groups per molecule. Methoxyl groups or methanol were metabolized by homoacetate fermentation: in the presence of bicarbonate 4 mol of acetate. In case of the methoxylated cinnamic acid derivatives less acetate was formed and the corresponding hydroxy derivatives of phenylpropionic acid appeared as a result of the double bond reduction in the acrylate side chain. In comparison to the benzoate derivatives with the same number of methoxyl groups, higher growth yields were obtained with the cinnamate derivatives.     

Photocatabolism of Aromatic Compounds by the Phototrophic Purple Bacterium Rhodomicrobium vannielii

The phototrophic purple non-sulfur bacterium Rhodomicrobium vannielii grew phototrophically (illuminated anaerobic conditions) on a variety of aromatic compounds (in the presence of CO₂). Benzoate was universally photocatabolized by all five strains of R. vannielii examined, and benzyl alcohol was photocatabolized by four of the five strains. Catabolism of benzyl alcohol by phototrophic bacteria has not been previously reported. Other aromatic substrates supporting reasonably good growth of R. vannielii strains were the methoxylated benzoate derivatives vanillate (4-hydroxy-3-methoxybenzoate) and syringate (4-hydroxy-3,5-dimethoxybenzoate). However, catabolism of vanillate and syringate led to significant inhibition of bacteriochlorophyll synthesis in R. vannielii cells, eventually causing cultures to cease growing. No such effect on photopigment synthesis in cells grown on benzoate or benzyl alcohol was observed. Along with a handful of other species of anoxygenic phototrophic bacteria, the ability of the species R. vannielii to photocatabolize aromatic compounds indicates that this organism may also be ecologically significant as a consumer of aromatic derivatives in illuminated anaerobic habitats in nature.     

Coexistence of two different O demethylation systems in lignin metabolism by Sphingomonas paucimobilis SYK-6: cloning and sequencing of the lignin biphenyl-specific O-demethylase (LigX) gene

Sphingomonas paucimobilis SYK-6 can grow on several dimeric model compounds of lignin as a carbon and energy source. It has O demethylation systems on three kinds of substrates: 5, 5'-dehydrodivanillic acid (DDVA), syringate, and vanillate. We previously reported the cloning of a gene involved in the tetrahydrofolate-dependent O demethylation of syringate and vanillate. In the study reported here, we cloned the gene responsible for DDVA O demethylation. Using nitrosoguanidine mutagenesis, a mutant strain, NT-1, which could not degrade DDVA but could degrade syringate and vanillate, was isolated and was used to clone the gene responsible for the O demethylation of DDVA by complementation. Sequencing analysis showed an open reading frame (designated ligX) of 1,266 bp in this fragment. The deduced amino acid sequence of LigX had similarity to class I type oxygenases. LigX was involved in O demethylation activity on DDVA but not on vanillate and syringate. DDVA O demethylation activity in S. paucimobilis SYK-6 cell extracts was inhibited by addition of the LigX polyclonal antiserum. Thus, LigX is an essential enzyme for DDVA O demethylation in SYK-6. S. paucimobilis SYK-6 has two O demethylation systems: one is an oxygenative demethylase system, and the other is a tetrahydrofolate-dependent methyltransferase system.     

Characterization of a CO-dependent O-demethylating enzyme system from the acetogen Clostridium thermoaceticum.

An inducible O-demethylating enzyme system was characterized from Clostridium thermoaceticum cultivated at the expense of syringate. Glucose and methanol, but not CO, partially repressed its expression. Induced whole cells catalyzed the carbon monoxide (CO)-dependent O demethylation of methoxylated aromatic compounds with the concomitant formation of acetate. Pyruvate and, to a lesser extent, H2-CO2 could replace CO in these reactions. KCN inhibited pyruvate-dependent activity but not the CO-dependent activity. The ATPase inhibitor N,N'-dicyclohexylcarbodiimide, the protonophore carbonyl cyanide m-chlorophenylhydrazone, and methyl viologen did not appreciably inhibit O demethylation by induced cells, whereas Triton X-100 was inhibitory. The enzyme system appeared to convert syringate sequentially to 5-hydroxyvanillate and gallate. The proposed overall reaction stoichiometry was as follows: syringate + 2CO + 2H2O----gallate + 2 acetates. Growth-supportive methoxylated aromatic compounds were O demethylated by syringate-cultivated cells and inhibitory to syringate O demethylation.     

Aerobic and Anaerobic Catabolism of Vanillic Acid and Some Other Methoxy-Aromatic Compounds by Pseudomonas sp. Strain PN-1

Vanillic acid (4-hydroxy-3-methoxybenzoic acid) supported the anaerobic (nitrate respiration) but not the aerobic growth of Pseudomonas sp. strain PN-1. Cells grown anaerobically on vanillate oxidized vanillate, p-hydroxybenzoate, and protocatechuic acid (3,4-dihydroxybenzoic acid) with O₂ or nitrate. Veratric acid (3,4-dimethoxybenzoic acid) but not isovanillic acid (3-hydroxy-4-methoxybenzoic acid) induced cells for the oxic and anoxic utilization of vanillate, and protocatechuate was detected as an intermediate of vanillate breakdown under either condition. Aerobic catabolism of protocatechuate proceeded via 4,5-meta cleavage, whereas anaerobically it was probably dehydroxylated to benzoic acid. Formaldehyde was identified as a product of aerobic demethylation, indicating a monooxygenase mechanism, but was not detected during anaerobic demethylation. The aerobic and anaerobic systems had similar but not identical substrate specificities. Both utilized m-anisic acid (3-methoxybenzoic acid) and veratrate but not o- or p-anisate and isovanillate. Syringic acid (4-hydroxy-3,5-dimethoxybenzoic acid), 3-O-methylgallic acid (3-methoxy-4,5-dihydroxybenzoic acid), and 3,5-dimethoxybenzoic acid were attacked under either condition, and formaldehyde was liberated from these substrates in the presence of O₂. The anaerobic demethylating system but not the aerobic enzyme was also active upon guaiacol (2-methoxyphenol), ferulic acid (3-[4-hydroxy-3-methoxyphenyl]-2-propenoic acid), 3,4,5-trimethoxycinnamic acid (3-[3,4,5-trimethoxyphenyl]-2-propenoic acid), and 3,4,5-trimethoxybenzoic acid. The broad specificity of the anaerobic demethylation system suggests that it probably is significant in the degradation of lignoaromatic molecules in anaerobic environments.     

Metabolism of aromatic aldehydes as cosubstrates by the acetogen Clostridium formicoaceticum

When the acetogen Clostridium formicoaceticum was cultivated on mixtures of aromatic compounds (e.g., 4-hydroxybenzaldehyde plus vanillate), the oxidation of aromatic aldehyde groups occurred more rapidly than did O-demethylation. Likewise, when fructose and 4-hydroxybenzaldehyde were simultaneously provided as growth substrates, fructose was utilized only after the aromatic aldehyde group was oxidized to the carboxyl level. Aromatic aldehyde oxidoreductase activity was constitutive (activities approximated 0.8 U mg^–1), and when pulses of 4-hydroxybenzaldehyde were added during fructose-dependent growth, the rate at which fructose was utilized decreased until 4-hydroxybenzaldehyde was consumed. Although 4-hydroxybenzaldehyde inhibited the capacity of cells to metabolize fructose, lactate or gluconate were consumed simultaneously with 4-hydroxybenzaldehyde, and lactate or aromatic compounds lacking an aldehyde group were utilized concomitantly with fructose. These results demonstrate that (1) aromatic aldehydes can be utilized as cosubstrates and have negative effects on the homoacetogenic utilization of fructose by C. formicoaceticum, and (2) the consumption of certain substrates by this acetogen is not subject to catabolite repression by fructose. Received: 14 May 1998 / Accepted: 7 August 1998     

Anaerobic c(1) metabolism of the o-methyl-C-labeled substituent of vanillate

The O-methyl substituents of aromatic compounds constitute a C(1) growth substrate for a number of taxonomically diverse anaerobic acetogens. In this study, strain TH-001, an O-demethylating obligate anaerobe, was chosen to represent this physiological group, and the carbon flow when cells were grown on O-methyl substituents as a C(1) substrate was determined by C radiotracer techniques. O-[methyl-C]vanillate (4-hydroxy-3-methoxy-benzoate) was used as the labeled C(1) substrate. The data showed that for every O-methyl carbon converted to [C]acetate, two were oxidized to CO(2). Quantitation of the carbon recovered in the two products, acetate and CO(2), indicated that acetate was formed in part by the fixation of unlabeled CO(2). The specific activity of C in acetate was 70% of that in the O-methyl substrate, suggesting that only one carbon of acetate was derived from the O-methyl group. Thus, it is postulated that the carboxyl carbon of the product acetate is derived from CO(2) and the methyl carbon is derived from the O-methyl substituent of vanillate. The metabolism of O-[methyl-C]vanillate by strain TH-001 can be described as follows: 3CH(3)OC(7)H(5)O(3) + CO(2) + 4H(2)O --> CH(3)COOH + 2CO(2) + 10H + 10e + 3HOC(7)H(5)O(3).     

Formation of dimethylsulfide and methanethiol from methoxylated aromatic compounds and inorganic sulfide by newly isolated anaerobic bacteria

Formation of gas and of methylated sulfur compounds was observed in anaerobic enrichment cultures with methoxylated aromatic compounds as substrates. Via direct dilution of mud samples in defined reduced media supplemented with trimethoxybenzoate or syringate two new strains of anaerobic homoacetogenic bacteria (strain TMBS4 and strain SA2) were obtained in pure culture. Both strains produced dimethylsulfide and methanethiol during growth on methoxylated aromatic compounds. Growth tests and determination of stoichiometries demonstrated that the volatile sulfur compounds were formed from the methyl group at the aromatic ring and the sulfide added as reducing agent to the medium (R = aromatic residue): 2 R - O - CH₃ + H₂ S 2 R - OH + (CH₃)₂SDimethylsulfide was the major organic sulfur compound formed, whereas methanethiol appeared only as intermediate in small quantities. The isolates grew also with trihydroxybenzenes such as gallate, phloroglucinol, or pyrogallol without formation of methylated sulfur compounds. The aromatic compounds were degraded to acetate. The freshwater strain TMBS4 also fermented pyruvate. Other aliphatic or aromatic compounds were not utilized. External electron acceptors (sulfate, nitrate, fumarate) were not reduced. Both strains were mesophilic and formed rod-shaped, non-motile, Gram-negative cells. Spore formation was not observed. Tentatively, both isolates can be affiliated to the genus Pelobacter.Abbreviations TMB 3,4,5-trimethoxybenzoate - MT methanethiol - DMS dimethylsulfide     

Utilization of aromatic compounds by phototrophic purple nonsulfur bacteria

Biodegradation of aromatic compounds byRhodopseudomonas blastica andRhodospirillum rubrum appears to be lacking in the literature. The above species grew phototrophically (illuminated anaerobic conditions) on a variety of organic compounds. They were found to degrade benzoate, benzyl alcohol, 4-hydroxy-3,5-dimethoxybenzoate (Syringate) and 4-hydroxy-3-methoxybenzoate (vanillate). The ability of the above species to photocatabolize aromatic compounds indicates that these organisms may be ecologically significant as scavengers of aromatic derivatives in illuminated anaerobic habitats in nature.     

Rapid and simple method for the most-probable-number estimation of arsenic-reducing bacteria.

A rapid and simple most-probable-number (MPN) procedure for the enumeration of dissimilatory arsenic-reducing bacteria (DARB) is presented. The method is based on the specific detection of arsenite, the end product of anaerobic arsenate respiration, by a precipitation reaction with sulfide. After 4 weeks of incubation, the medium for the MPN method is acidified to pH 6 and sulfide is added to a final concentration of about 1 mM. The brightly yellow arsenic trisulfide precipitates immediately and can easily be scored at arsenite concentrations as low as 0.05 mM. Abiotic reduction of arsenate upon sulfide addition, which could yield false positives, apparently produces a soluble As-S intermediate, which does not precipitate until about 1 h after sulfide addition. Using the new MPN method, population estimates of pure cultures of DARB were similar to direct cell counts. MPNs of environmental water and sediment samples yielded DARB numbers between 10(1) and 10(5) cells per ml or gram (dry weight), respectively. Poisoned and sterilized controls showed that potential abiotic reductants in environmental samples did not interfere with the MPN estimates. A major advantage is that the assay can be easily scaled to a microtiter plate format, enabling analysis of large numbers of samples by use of multichannel pipettors. Overall, the MPN method provides a rapid and simple means for estimating population sizes of DARB, a diverse group of organisms for which no comprehensive molecular markers have been developed yet.     

Anaerobic degradation of methoxylated aromatic compounds by Clostridium methoxybenzovorans and a nitrate-reducing bacterium Thauera sp. strain Cin3,4

The coupling of growth of the o-demethylating bacterium, Clostridium methoxybenzovorans SR3, with a nitrate-reducing bacterium able to degrade aromatic compounds, Thauera sp. Cin3,4, allowed complete mineralization of poorly oxidizable methoxylated aromatic compounds such as vanillate, isovanillate, vanilline, anisate, ferulate and veratrate. C. methoxybenzovorans o-demethylated these aromatic compounds to their corresponding hydroxylated derivatives and fermented the side chains to acetate and butyrate. The hydroxylated compounds and the fermentation end-products in the C. methoxybenzovorans spent growth medium were then completely metabolized to CO₂ on inoculation with the Thauera strain. Kinetic studies with veratrate indicated that C. methoxybenzovorans initially o-demethylated the substrate to vanillate and then further to protocatechuate together with the production of acetate and butyrate from the demethylated side chains. Protocatechuate, acetate and butyrate were then utilized as a carbon source by the Thauera strain aerobically or anaerobically in the presence of nitrate. The results therefore suggest that mono- or dimethoxylated aromatic compounds can be completely mineralized by coupling the growth of a fermentative bacterium with a nitrate-reducing bacterium, and a metabolic pathway for this is proposed.     

Rapid and Simple Method for the Most-Probable-Number Estimation of Arsenic-Reducing Bacteria

A rapid and simple most-probable-number (MPN) procedure for the enumeration of dissimilatory arsenic-reducing bacteria (DARB) is presented. The method is based on the specific detection of arsenite, the end product of anaerobic arsenate respiration, by a precipitation reaction with sulfide. After 4 weeks of incubation, the medium for the MPN method is acidified to pH 6 and sulfide is added to a final concentration of about 1 mM. The brightly yellow arsenic trisulfide precipitates immediately and can easily be scored at arsenite concentrations as low as 0.05 mM. Abiotic reduction of arsenate upon sulfide addition, which could yield false positives, apparently produces a soluble As-S intermediate, which does not precipitate until about 1 h after sulfide addition. Using the new MPN method, population estimates of pure cultures of DARB were similar to direct cell counts. MPNs of environmental water and sediment samples yielded DARB numbers between 10¹ and 10⁵ cells per ml or gram (dry weight), respectively. Poisoned and sterilized controls showed that potential abiotic reductants in environmental samples did not interfere with the MPN estimates. A major advantage is that the assay can be easily scaled to a microtiter plate format, enabling analysis of large numbers of samples by use of multichannel pipettors. Overall, the MPN method provides a rapid and simple means for estimating population sizes of DARB, a diverse group of organisms for which no comprehensive molecular markers have been developed yet.     

Degradation of methoxylated aromatic acids by Pseudomonas putida

J.E. TURNER AND N. ALLISON. 1995. A newly-isolated strain of Pseudomonas putida (HVA-1) utilized homovanillic acid as sole carbon and energy source. Homovanillate-grown bacteria oxidized homovanillate and homoprotocatechuate but monohydroxylated and other methoxylated phenylacetic acids were oxidized poorly; methoxy-substituted benzoates were not oxidized. Extracts of homovanillate-grown cells contained homoprotocatechuate 2,3-dioxygenase but the primary homovanillate-degrading enzyme could not be detected. No other methoxylated phenylacetic acid supported growth of the organism but vanillate was utilized as a carbon and energy source. When homovanillate-grown cells were used to inoculate media containing vanillate a 26 h lag period occurred before growth commenced. Vanillate-grown bacteria oxidized vanillate and protocatechuate but no significant oxygen uptake was obtained with homovanillate and other phenylacetic acid derivatives. Analysis of pathway intermediates revealed that homovanillate-grown bacteria produced homoprotocatechuate, formaldehyde and the ring-cleavage product 5-carboxymethyl 2-hydroxymuconic semialdehyde (CHMS) when incubated with homovanillate but monohydroxylated or monomethoxylated phenylacetic acids were not detected. These results suggest that homovanillate is degraded directly to the ring-cleavage substrate homoprotocatechuate by an unstable but highly specific demethylase and then undergoes extradiol cleavage to CHMS. It would also appear that the uptake/degradatory pathways for homovanillate and vanillate in this organism are entirely separate and independently controlled. If stabilization of the homovanillate demethylase can be achieved, there is potential for exploiting the substrate specificity of this enzyme in both medical diagnosis and in the paper industry.     

Bacteria isolated from the duodenum, ileum, and cecum of young chicks.

Facultatively anaerobic and strictly anaerobic bacteria colonizing the intestinal tracts of 14-day-old chicks fed a corn-based diet were enumerated, isolated, and identified. Colony counts from anaerobic roll tubes (rumen fluid medium) or aerobic plates (brain heart infusion agar) recovered from homogenates of the duodenum, upper and lower ileum, and cecum varied appreciably among samples from individual birds. Anaerobic and aerobic counts from the duodenum and ileum were similar. Anaerobic counts were highest from the cecum (0.7 X 10(11) to 1.6 X 10(11)/g of dry tissue) and exceeded aerobic plate counts by a factor of at least 10(2). Facultatively anaerobic groups (Streptococcus, Staphylococcus, Lactobacillus, and Escherichia coli) comprised the predominant flora of the duodenum and ileum, although large numbers of anaerobes (9 to 39% of the small intestine isolates), represented by species of Eubacterium, Propionibacterium, Clostridium, Gemmiger, and Fusobacterium, were also recovered. Strict anaerobes (anaerobic gram-positive cocci, Eubacterium, Clostridium Gemmiger, Fusobacterium, and Bacteriodes) made up nearly the entire microbial population of the cecum. Scanning electron microscopy of the intestinal epithelia of chicks revealed populations of microbes on the duodenal, ileal, and cecal mucosal surfaces.     

H(2)-CO(2)-Dependent Anaerobic O-Demethylation Activity in Subsurface Sediments and by an Isolated Bacterium

The ability of microorganisms in sediments from the Atlantic Coastal Plain to biodegrade methoxylated aromatic compounds was examined. O-demethylation activity was detected in deep (121- and 406-m) sediments, as well as in the surface soil. A syringate-demethylating consortium, containing at least three types of bacteria, was enriched from a deep-sediment sample in a medium containing syringate as the sole organic carbon source and with a N(2)-CO(2) atmosphere. An isolate which demethylated syringate was obtained from the enrichment on an agar medium incubated under a H(2)-CO(2) but not a N(2)-CO(2) or N(2) atmosphere. O demethylation of syringate of this isolate was dependent on the presence of both H(2) and CO(2) in the gas phase. The metabolism of syringate occurred in a sequential manner: methylgallate accumulated transiently before it was converted to gallate. Mass balance analysis suggests that the stoichiometry of the reaction in this isolate proceeds in accordance with the following generalized equation: C(7)H(3)O(3)(OCH(3))(n) + nHCO(3) + nH(2) --> C(7)H(3)O(3)(OH)(n) + nCH(3)COO + nH(2)O.     

Methyl chloride metabolism of the strictly anaerobic,methyl chloride-utilizing homoacetogen strain MC

The methyl chloride metabolism of the homoacetogenic, methyl chloride-utilizing strain MC was investigated with cell extracts and cell suspensions of the organism. Cell extracts were found to contain all enzyme activities required for the conversion of methyl chloride or of H₂ plus CO₂ to acetate. They catalyzed the dechlorination of methyl chloride with tetrahydrofolate as the methyl acceptor at a rate of 20 nmol/min × mg of cell protein. Also, the O-demethylation of vanillate with tetrahydrofolate could be measured at a rate of 40 nmol/min × mg. Different enzyme systems appeared to be responsible for the dehalogenation of CH₃Cl and for the O-demethylation of methoxylated aromatic compounds, since cells grown with methoxylated aromatic compounds exhibited a significantly lower activity of CH₃Cl conversion than methyl chloride grown cells and vice versa. In addition, ammonium thiocyanate (5 mM) completely inhibited CH₃Cl dechlorination, whereas the consumption of vanillate was not affected significantly. The data were taken to indicate, that the methyl chloride dehalogenation is catalyzed by a specific, inducible enzyme present in strain MC, and that tetrahydrofolate rather than the corrinoid-protein involved in acetate formation is the primary acceptor of the methyl group in the dechlorination reaction.     

Comparison of Two Methods for Enumeration of Anaerobe Numbers on Forages and Evaluation of Ethylene Oxide Treatment for Forage Sterilization

Experiments were conducted to (i) compare most-probable-number (MPN) procedures with roll tube procedures for enumeration of forage anaerobic bacteria and (ii) evaluate the efficacy of using ethylene oxide to sterilize wet herbage. Alfalfa, corn, and alfalfa-orchardgrass silages and alfalfa and orchardgrass herbages were analyzed for total anaerobic bacteria (medium pH, 6.8) and acid-tolerant anaerobic bacteria (medium pH, 4.5) by both roll tube and MPN procedures. No difference was found between the roll tube and MPN procedures for total bacteria; however, higher counts were obtained for acid-tolerant bacteria when the MPN procedure was used. Although MPN procedures require less time to obtain an estimate of bacterial numbers, isolation and identification of the microbial population is not possible. Alfalfa herbage was treated with ethylene oxide for 12, 24, or 36 h, incubated for 7 days at 37°C with or without addition of a bacterial inoculant, and analyzed for total bacteria by MPN procedures. Microbial growth after inoculation of ethylene oxide-treated herbage indicated that there was insufficient residual ethylene oxide to inhibit subsequent microbial growth. The results also indicated that 24 h was required to adequately sterilize fresh herbage. Thus, ethylene oxide can be used to sterilize wet herbage for use as a substrate for pure cultures of silage bacteria.     

Isolation from a methanogenic ferulate degrading consortium of an anaerobe that converts methoxyl groups of aromatic acids to volatile fatty acids

An anaerobic, non-motile, rod shaped bacterium is described which cleaves the phenylether bonds of methoxylated aromatic substrates to give the corresponding hydroxy aromatic derivatives and mixed volatile fatty acids, chain length, C₁, C₂ and C₄. The bacterium was isolated from an anaerobic digestor fed with contents from a wood fiber to alcohol fermentation plant, using anaerobic rolltube medium with ferulate as the carbon and energy source. Moles fatty acid produced per 100 mole of methoxyl group of aromatic substrate fermented were approximately: acetate, 14; butyrate, 18; and formate, 15. For the fermentation of equimolar amounts of methoxylated aromatic compounds, growth yields were proportional to the number of methoxylated groups per molecule, and the amount of cells per methoxyl group did not alter when phenylacrylate derivatives were used as substrates. The organism was unable to reduce the side-chain double bond of phenylacrylate derivatives. Coculture of the bacterium on ferulate with Methanospirillum hungatei, or Desulfovibrio in the presence of SO ₄ ⁼ resulted in no nett production of formate, and small quantities of methane and sulfide were produced respectively. The isolate utilized glucose, fructose, and lactate, but not methanol or H₂–CO₂ as growth substrates. Lactate, butyrate, acetate, formate and small quantities of H₂ were produced from glucose fermentation. No reduction of SO ₄ ⁼ or NO ₃ ^- occurred during fermentation of glucose or methoxylated aromatics and no growth occurred in the presence of oxygen.