Degradation of cinnamate via beta-oxidation to benzoate by a defined,syntrophic consortium of anaerobic bacteria

A syntrophic consortium was enriched in a basal medium containing cinnamate as the carbon and energy source. It was found to consist of three morphologically distinct microbes, viz., a short, rod-shaped, non-motile bacterium with distinctly pointed ends, Papillibacter cinnamivorans; a rod-shaped, motile bacterium with rounded ends, Syntrophus sp.; and a methanoarchaeon, Methanobacterium sp. This methanogen was then replaced by a collection strain of Methanobacterium formicicum. A syntrophic interdependency of the three partners of the consortium was observed during growth on cinnamate. In the presence of bromoethanesulfonic acid (BESA), cinnamate was transformed to benzoate, whereas under methanogenic conditions without BESA, cinnamate was first transformed to benzoate via beta-oxidation and subsequently completely degraded into acetate, CH(4), and CO(2). Papillibacter cinnamivorans was responsible for benzoate production from cinnamate, whereas a syntrophic association between Syntrophus sp. and the methanogen degraded benzoate to acetate, CH(4), and CO(2). A new anaerobic degradation pathway of cinnamate into benzoate via beta-oxidation by a pure culture of P. cinnamivorans is proposed.     

Uptake of phenol by the phenol-metabolizing bacteria of a stable,strictly anaerobic consortium

Phenol was absorbed unspecifically by active and by inactivated cells of a strictly anaerobic, phenol-degrading consortium to reach about twice the concentration of the medium. The absorption was temperature-dependent. A Q₁₀ of 1.7 was determined, indicating that accumulation was due to diffusion or facilitated diffusion and not to an active transport process. At increasing phenol concentration in the medium, concentrated cell suspensions adsorpted phenol proportionally until saturation was reached at about 25 nmol phenol/mg cell dry weight. At a phenol concentration in the medium of 2 mm, the washed cell pellet contained 3.5 mm phenol. Under conditions that allowed phenol metabolism (presence of CO₂), [¹⁴C]4-hydroxybenzoyl-coenzyme A and [¹⁴C]4-hydroxybenzoate were found as early intermediates of [U-¹⁴C]phenol degradation for the first time. [¹⁴C]Benzoate was excreted stoichiometrically if phenol degradation to acetate was prevented by H₂. Absolutely no ¹⁴C-label was found in the phenylphosphate peak after HPLC separation, which excluded phosphorylation of phenol during uptake or during degradation in the cells. Correspondence to: J. Winter     

Degradation of benzoate by an anaerobic consortium and some properties of a hydrogenotrophic methanogen and sulfate-reducing bacterium in the consortium

A highly simplified anaerobic consortium which was able to degrade benzoate under mesophilic conditions was obtained from digested sludge acclimatized with benzoate. It converted 5 mM benzoate to methane quantitatively within 3 weeks in the absence of any organic nutrients under an N₂/CO₂ atmosphere. Degradation of benzoate was strictly inhibited by hydrogen. The consortium consisted of at least three microorganisms including an autofluorescent irregular coccus which was identified as Methanogenium sp., a short rod which did not autofluoresce and was considered to be a benzoate degrader, and a filamentous bacterium apparently classified as Methanothrix (= “Methanosaeta”. When sulfate was added to the medium, the methanogens were readily replaced by a sulfate-reducing bacterium, probably belonging to the genus Desulfovibrio, which had still remained in very low number in the consortium in the absence of sulfate, and benzoate was stoichiometrically converted to acetate without methanogenesis. Of various compounds which were expected to be intermediates in the benzoate degradation, only crotonate was degraded by concentrated cells of the consortium.     

Evidence for anaerobic syntrophic benzoate degradation threshold and isolation of the syntrophic benzoate degrader.

An anaerobic, motile, gram-negative, rod-shaped, syntrophic, benzoate-degrading bacterium, strain SB, was isolated in pure culture with crotonate as the energy source. Benzoate was degraded only in association with an H2-using bacterium. The kinetics of benzoate degradation by cell suspensions of strain SB in coculture with Desulfovibrio strain G-11 was studied by using progress curve analysis. The coculture degraded benzoate to a threshold concentration of 214 nM to 6.5 microM, with no further benzoate degradation observed even after extended incubation times. The value of the threshold depended on the amount of benzoate added and, consequently, the amount of acetate produced. The addition of sodium acetate, but not that of sodium chloride, affected the threshold value; higher acetate concentrations resulted in higher threshold values for benzoate. When a cell suspension that had reached a threshold benzoate concentration was reamended with benzoate, benzoate was used without a lag. The hydrogen partial pressure was very low and formate was not detected in cell suspensions that had degraded benzoate to a threshold value. The Gibbs free energy change calculations showed that the degradation of benzoate was favorable when the threshold was reached. These studies showed that the threshold for benzoate degradation was not caused by nutritional limitations, the loss of metabolic activity, or inhibition by hydrogen or formate. The data are consistent with a thermodynamic explanation for the existence of a threshold, but a kinetic explanation based on acetate inhibition may also account for the existence of a threshold.     

The taxonomy of obligate anaerobic bacteria

Studies on the chemotaxonomy of obligate anaerobic bacteria have been made. The combination of gas chromatography and mass spectrometry with computer-assisted analysis, permitting the multicomponent analysis of all products of bacterial metabolism and bacterial cell components, has been shown to be a research method, quite suitable for such studies. The chromatographic profiles of the end products of metabolism in anaerobic cultures of different age have been found to differ not in the set and number of peaks indicating various metabolites, but only in the concentration of metabolites, increasing in the process of prolonged incubation. The authors believe that the national microbiological "library" of the chromatographic profiles of anaerobic organisms should be created and the album of typing chromatographic profiles should be published; besides, data on new profiles should regularly appear in magazines.     

Anaerobic carboxylation of phenol to benzoate: use of deuterated phenols revealed carboxylation exclusively in the C4-position

Summary [U-D]Phenol and [4-D]phenol were used to rule out carboxylation of phenol in the C1-position by a strictly anaerobic, defined mixed culture. By mass spectrometric analysis of deuterated phenol species and of benzoate, which were formed from [U-D]phenol by D/H-exchange or by carboxylation from cell suspensions, it was shown that only one deuterium (D) from the aromatic nucleus was replaced with a least 97% efficiency. This excluded benzoate synthesis by carboxylation in the C1-position of phenol. Finally, carboxylation in the para-position of phenol was demonstrated with [4-D]phenol by gas chromatography/mass spectroscopy of the products. Since direct measurement of phenol carboxylase activity was impossible due to a very active interfering decarboxylase activity, the optimal pH range and ion strength, as well as the requirement of cations in crude cell-free extracts was characterized by means of D/H-exchange from deuterated phenol.Dedicated to Prof. R. S. Wolfe on the occasion of his 70th birthday Offsprint requests to: J. Winter     

Synthesis and function of polyhydroxyalkanoates in anaerobic syntrophic bacteria

Abstract Anaerobic syntrophic bacteria degrade fatty acids and some aromatic compounds which are important intermediates in the degradation of organic matter in methanogenic environments. Several of the described syntrophic species produce poly-β-hydroxyalkanoate (PHA) suggesting that the synthesis and use of PHA is important in their physiology. In the fatty acid-degrading, syntrophic bacterium, Syntrophomonas wolfei , PHA is made during exponential phase of growth and used after growth has stopped and substrate levels are low. Altering the carbon to nitrogen ratio of the medium does not affect the amount of PHA made or its monomeric composition. It is hypothesized that PHA serves as an endogenous energy source for syntrophic bacteria when the concentrations of hydrogen or acetate are too high for the degradation of the growth substrate to be thermodynamically favorable. In S. wolfei , PHA is synthesized by two routes, the direct incorporation of 3-ketoacyl-coenzyme A (CoA) generated in β-oxidation without cleavage of a C-C bond, and by the condensation and subsequent reduction of two acetyl-CoA molecules. Genes that encode for the synthesis of PHA in S. wolfei have been cloned into Escherichia coli in order to understand the molecular mechanisms that regulate PHA synthesis.     

Anaerobic degradation of benzoate to methane by a microbial consortium

A stabilized consortium of microbes which anaerobically degraded benzoate and produced CH₄ was established by inoculation of a benzoate-mineral salts medium with sewage sludge; the consortium was routinely subcultured anaerobically in this medium for 3 years. Acetate, formate, H₂ and CO₂ were identified as intermediates in the overall conversion of benzoate to CH₄ by the culture. Radioactivity was equally divided between the CH₄ and CO₂ from the degradation of uniformly ring-labeled [¹⁴C]benzoate. The methyl group of acetate was stoichiometrically converted to CH₄. Acetate, cyclohexanecarboxylate, 2-hydroxycyclohexanecarboxylate, o-hydroxybenzoic acid and pimelic acid were converted to CH₄ without a lag suggesting that benzoate was degraded by a reductive pathway. Addition of o-chlorobenzoate inhibited benzoate degradation but not acetate degradation or methane formation. Two methanogenic organisms were isolated from the mixed culture, neither organism was able to degrade benzoate, showing that the methanogenic bacteria served as terminal organisms of a metabolic food chain composed of several organisms. Removal of intermediates by the methanogenic bacteria provided thermodynamically favorable conditions for benzoate degradation.     

CO2-dependent fermentation of phenol to acetate, butyrate and benzoate by an anaerobic, pasteurised culture

Fermentative degradation of phenol was studied using a non-methanogenic, pasteurised enrichment culture containing two morphologically different bacteria. Phenol was fermented to benzoate, acetate and butyrate and their relative occurrence depended on the concentration of hydrogen. Proportionately more benzoate was formed with high initial levels of H2. The influence of PH2 on the fermentation pattern was studied both in dense cell suspensions and in growing cultures by addition of hydrogen. An increase in growth yield (OD578) was observed, compared to controls, as a consequence of phenol degradation; however, the increase was less in H2-amended treatments, in which most of the phenol ended up as benzoate. The degradation of phenol in the dense cell suspension experiments was dependent on CO2. Benzoate was not degraded when added as a substrate to the growing culture. This is, to our knowledge, the first report concerning the fermentative degradation of phenol to nonaromatic products.     

Degradation of phenol and phenolic compounds by a defined denitrifying bacterial culture

Phenol, a major pollutant in several industrial waste waters is often used as a model compound for studies on biodegradation. This study investigated the anoxic degradation of phenol and other phenolic compounds by a defined mixed culture of Alcaligenes faecalis and Enterobacter species. The culture was capable of degrading high concentrations of phenol (up to 600 mg/l) under anoxic conditions in a simple minimal mineral medium at an initial cell mass of 8 mg/l. However, the lag phase in growth and phenol removal increased with increase in phenol concentration. Dissolved CO₂ was an absolute requirement for phenol degradation. In addition to nitrate, nitrite and oxygen could be used as electron acceptors. The kinetic constants, maximum specific growth rate _max; inhibition constant, K _i and saturation constant, K _s were determined to be 0.206 h^–1, 113 and 15 mg phenol/l respectively. p-Hydroxybenzoic acid was identified as an intermediate during phenol degradation. Apart from phenol, the culture utilized few other monocyclic aromatic compounds as growth substrates. The defined culture has remained stable with consistent phenol-degrading ability for more than 3 years and thus shows promise for its application in anoxic treatment of industrial waste waters containing phenolic compounds.     

para-hydroxybenzoate as an intermediate in the anaerobic transformation of phenol to benzoate

Anaerobic phenol transformation was studied using a consortium which transformed phenol to benzoate without complete mineralization of benzoate. Products of monofluorophenol transformation indicated para-carboxylation. Phenol and benzoate were detected during para-hydroxybenzoate (p-OHB) degradation. p-OHB was detected in phenol-transforming cultures containing 6-hydroxynicotinic acid (6-OHNA), a structural analogue of p-OHB, or at elevated initial concentrations of phenol (greater than or equal to 5 mM), or benzoate (greater than or equal to 10 mM).     

Effect of fluorinated analogues of phenol and hydroxybenzoates on the anaerobic transformation of phenol to benzoate

The effects of fluorinated analogues on the anaerobic transformation of phenol to benzoate were examined. At 250 M 2- or 3-fluorophenol, phenol transformation was delayed. 2-Fluorophenol had no apparent effect on subsequent degradation of benzoate, but benzoate accumulated in the presence of 250 M 3-fluorophenol. In contrast, 4-fluorophenol at 2 mM had no effect on either phenol transformation or benzoate degradation. Phenol and 2-, or 3-fluorophenol were transformed simultaneously, but phenol was transformed more rapidly than either fluorophenol. Thus, fluorinated analogues of phenol did not prevent anaerobic transformation of phenol to benzoate. 2-Fluorophenol was converted to 3-fluorobenzoate, and phenol enhanced the rate and extent of its transformation. 3-Fluorophenol was transformed to 2-fluorobenzoate to a limited extent (3%) when phenol was present. 4-Fluorophenol was not transformed regardless of the presence of phenol. 3-Fluoro-4-hydroxybenzoate, a potential fluorinated intermediate product of para-carboxylation, was transformed rapidly to 2-fluorophenol and 3-fluorobenzoate, irrespective of the presence of phenol, indicating that both dehydroxylation and decarboxylation occurred. Initially, 2-fluorophenol and 3-fluorobenzoate were rapidly formed in an approximate molar ratio of 2 : 1. Once 3-fluoro-4-hydroxybenzoate was completely removed, the 2-fluorophenol, initially formed, was converted to 3-fluorobenzoate at a slower rate. Thus, phenol enhanced transformation of the fluorinated analogues, and the products of transformation suggested para-carboxylation. 3-Fluoro-2-hydroxybenzoate was not transformed in either the presence or absence of phenol, indicating that ortho-carboxylation did not occur.Abbreviations 3F4HB 3-fluoro-4-hydroxybenzoate - 3F2HB 3-fluoro-2-hydroxybenzoate (3-fluorosalicylate) Contribution No. 692, Environmental Research Laboratory, U.S. EPA, Gulf Breeze, FL. 32561, USA     

Degradation of hydroquinone,gentisate, and benzoate by a fermenting bacterium in pure or defined mixed culture

From a methanogenic fixed-bed reactor fed with hydroquinone as sole energy and carbon source, a rodshaped bacterium was isolated in pure culture which could degrade hydroquinone and gentisate (2,5-dihydroxybenzoate). In syntrophic coculture with either Desulfovibrio vulgaris or Methanospirillum hungatei, also benzoate could be degraded. Other substrates such as sugars, fatty acids, alcohols, and cyclohexane derivatives were not degraded. Sulfate, sulfite, or nitrate were not used as external electron acceptor. The isolate was a Gram-negative, non-motile, nonsporeforming strict anaerobe; the guanine-plus-cytosine content of the DNA was 53.2±1.0 mol%. In pure culture, hydroquinone was degraded to acetate and benzoate, probably via an intermediate carboxylation. In syntrophic mixed cultures, all three substrates were converted completely to acetate. Phenol was never detected as a fermentation product.     

Degradation of phenol under meso- and thermophilic, anaerobic conditions

Based on the results of preliminary studies on phenol degradation under mesophilic conditions with a mixed methanogenic culture, we proposed a degradation pathway in which phenol is fermented to acetate: Part of the phenol is reductively transformed to benzoate while the rest is oxidised, forming acetate as end product. According to our calculations, this should result in three moles of phenol being converted to two moles of benzoate and three moles of acetate (3 phenol + 2 CO2 + 3 H2O --> 3 acetate + 2 benzoate): To assess the validity of our hypothesis concerning the metabolic pathway, we studied the transformation of phenol under mesophilic and thermophilic conditions in relation to the availability of hydrogen. Hence, methanogenic meso- and thermophilic cultures amended with phenol were run with or without an added over-pressure of hydrogen under methanogenic and non-methanogenic conditions. Bromoethanesulfonic acid (BES) was used to inhibit methanogenic activity. In the mesophilic treatments amended with only BES, about 70% of the carbon in the products found was benzoate. During the course of phenol transformation in these BES-amended cultures, the formation pattern of the degradation products changed: Initially nearly 90% of the carbon from phenol degradation was recovered as benzoate, whereas later in the incubation, in addition to benzoate formation, the aromatic nucleus degraded completely to acetate. Thus, the initial reduction of phenol to benzoate resulted in a lowering of H2 levels, giving rise to conditions allowing the degradation of phenol to acetate as the end product. Product formation in bottles amended with BES and phenol occurred in accordance with the hypothesised pathway; however, the overall results indicate that the degradation of phenol in this system is more complex. During phenol transformation under thermophilic conditions, no benzoate was observed and no phenol was transformed in the BES-amended cultures. This suggests that the sensitivity of phenol transformation to an elevated partial pressure of H2 is higher under thermophilic conditions than under mesophilic ones. The lack of benzoate formation could have been due to a high turnover of benzoate or to a difference in the phenol degradation pathway between the thermophilic and mesophilic cultures.     

Sulfide removal from livestock biogas by Azospirillum-like anaerobic phototrophic bacteria consortium

Biogas production from anaerobic biodegradation of livestock waste is a potential source of renewable energy. In addition to methane, biodegradation of this high-strength waste also produces sulfide that must be removed in order to prevent costly corrosive impacts on infrastructure. In this work, an anaerobic, phototrophic microbial community enriched from the native population in a swine waste lagoon was evaluated for its potential to remove sulfide from swine waste biogas. Batch experiments with the consortium attained removal efficiencies greater than 97% for sulfide concentrations above 1200 ppm. 16S rRNA gene sequencing revealed that the dominant population was most closely related to the isolate Azospirillum strain C5 (similarity index of 99%). Photomicrograph of the enriched consortium revealed the presence of cells with intracellular globules resembling sulfur storage. The enrichment of Azospirillum-like and the concomitant sulfide consumption suggest that this microorganism played an important role in sulfide removal in the bioreactor.     

Determination of mercury and organomercurial resistance in obligate anaerobic bacteria

A methodology for determining the minimum inhibitory concentration of inorganic and organomercurial compounds for obligate anaerobic bacteria is described. A wide variation in the susceptibility of anaerobic clinical and sewage isolates was observed. Isolates of Bacteroides ruminicola and Clostridium perfringens resistant to mercury were examined for their plasmid content and ability to demonstrate inducible resistance. None of the resistant anaerobes contained any plasmids, while resistant facultative isolates from the same source contained several plasmids. In 24 h, resistant strains of clostridia and Bacteroides volatilized 20 and 43% of the 203Hg2+ added to cultures, while Escherichia coli R100 and a sewage isolate of Enterobacter cloacae volatilized 63 and 27%, respectively, of the added 203Hg2+. Attempts to induce mercury resistance in the aerobic isolates were successful, but no induction was seen in the anaerobes. Thus, mercury resistance in these anaerobic isolates was neither inducible nor plasmid mediated.     

Fermentative degradation of monohydroxybenzoates by defined syntrophic cocultures

From anaerobic freshwater enrichment cultures with 3-hydroxybenzoate as sole substrate, a slightly curved rod-shaped bacterium was isolated in coculture with Desulfovibrio vulgaris as hydrogen scavenger. The new isolate degraded only 3-hydroxybenzoate or benzoate, and depended on syntrophic cooperation with a hydrogenoxidizing methanogen or sulfate reducer. 3-Hydroxybenzoate was degraded via reductive dehydroxylation to benzoate. With 2-hydroxybenzoate (salicylate), short coccoid rods were enriched from anaerobic freshwater mud samples, and were isolated in defined coculture with D. vulgaris. This isolate also fermented 3-hydroxybenzoate or benzoate in obligate syntrophy with a hydrogen-oxidizing anaerobe. The new isolates were both Gram-negative, non-sporeforming strict anaerobes. They fermented hydroxybenzoate or benzoate to acetate, CO₂, and, presumably, hydrogen which was oxidized by the syntrophic partner organism. With hydroxybenzoates, but not with benzoate, Acetobacterium woodii could also serve as syntrophic partner. Other substrates such as sugars, alcohols, fatty or amino acids were not fermented. External electron acceptors such as sulfate, sulfite, nitrate, or fumarate were not reduced. In enrichment cultures with 4-hydroxybenzoate, decarboxylation to phenol was the initial step in degradation which finally led to acetate, methane and CO₂.     

Sugarcane cellulose utilization by a defined microbial consortium

Microorganisms isolated from diverse environmental sources were initially screened for carboxymethylcellulase activity. Nine strains that grew at elevated temperatures and which presented the highest activity were characterized further. Culture supernatants were assayed for potentiation of the enzymatic activity and, based on these results, consortia of four or nine microorganisms were tested for their capacity to grow on, and degrade a sugarcane leaf substrate. As predicted by the supernatant mixes, both consortia assayed were capable of degrading the cellulosic substrate provided. The group comprising of four strains was as efficient as the mix of all nine strains.     

Modeling simultaneous hexavalent chromium reduction and phenol degradation by a defined coculture of bacteria

Based on the kinetics of Cr(VI) reduction by Escherichia coli ATCC 33456 and phenol degradation by Pseudomonas putida DMP-1, a mathematical model is developed to describe simultaneous Cr(VI) reduction and phenol degradation in the coculture of the two species. The developed model incorporates the toxicity effects of Cr(VI) and phenol on phenol degradation and Cr(VI) reduction in the coculture. The model illustrates the inhibitory effects of phenol on Cr(VI) reduction and Cr(VI) toxicity toward phenol degradation. The model also reveals the recoveries of the activities of the repressed bacterial cells with continuous Cr(VI) reduction and phenol degradation in the coculture. The model is capable of predicting simultaneous Cr(VI) reduction and phenol degradation within a broad range of Cr(VI) and phenol concentrations and under an appropriate composition of populations. However, the model simulates lower concentrations of phenol than experimental observations once Cr(VI) is reduced to a low level (<7 mg/L). The model simulation for Cr(VI) also deviates from experimental data when P. putida is outnumbered by E. coli by a ratio of 1:5. (c) 1995 John Wiley & Sons, Inc.     

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.