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Title: Evidence for an anaerobic syntrophic benzoate degradation threshold and isolation of the syntrophic benzoate degrader

Abstract

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 H{sub 2}-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 {mu}M, 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 causedmore » 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.« less

Authors:
; ;  [1]
  1. Univ. of Oklahoma, Norman, OK (United States)
Publication Date:
OSTI Identifier:
621395
DOE Contract Number:
FG05-89ER14003
Resource Type:
Journal Article
Resource Relation:
Journal Name: Applied and Environmental Microbiology; Journal Volume: 61; Journal Issue: 2; Other Information: PBD: Feb 1995
Country of Publication:
United States
Language:
English
Subject:
55 BIOLOGY AND MEDICINE, BASIC STUDIES; BIODEGRADATION; AROMATICS; POLLUTANTS; ACETATES; SODIUM COMPOUNDS; BENZOIC ACID; SALTS; ANAEROBIC CONDITIONS; BACTERIA

Citation Formats

Hopkins, B.T., McInerney, M.J., and Warikoo, V. Evidence for an anaerobic syntrophic benzoate degradation threshold and isolation of the syntrophic benzoate degrader. United States: N. p., 1995. Web.
Hopkins, B.T., McInerney, M.J., & Warikoo, V. Evidence for an anaerobic syntrophic benzoate degradation threshold and isolation of the syntrophic benzoate degrader. United States.
Hopkins, B.T., McInerney, M.J., and Warikoo, V. 1995. "Evidence for an anaerobic syntrophic benzoate degradation threshold and isolation of the syntrophic benzoate degrader". United States. doi:.
@article{osti_621395,
title = {Evidence for an anaerobic syntrophic benzoate degradation threshold and isolation of the syntrophic benzoate degrader},
author = {Hopkins, B.T. and McInerney, M.J. and Warikoo, V.},
abstractNote = {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 H{sub 2}-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 {mu}M, 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.},
doi = {},
journal = {Applied and Environmental Microbiology},
number = 2,
volume = 61,
place = {United States},
year = 1995,
month = 2
}
  • Acetate inhibited benzoate degradation by a syntrophic coculture of an anaerobic benzoate degrader (strain BZ-2) and Methanospirillum strain PM-1; the apparent K/sub i/ for acetate was approximately 40 mM. The addition of acetate resulted in a decrease in the hydrogen concentration in the coculture, indicating that phenomena related to interspecies hydrogen transfer affected this value and that the effect of acetate on the benzoate-degrading partner was probably greater than the apparent K/sub i/ for the coculture suggests.
  • A soluble benzoate-coenzyme A (CoA) ligase was purified from the phototrophic bacterium Rhodopseudomonas palustris. Synthesis of the enzyme was induced when cells were grown anaerobically in light with benzoate as the sole carbon source. Purification by chromatography successively on hydroxylapatite, phenyl-Sepharose, and hydroxylapatite yielded an electrophoretically homogeneous enzyme preparation with a specific activity of 25 ..mu..mol/min per mg of protein and a molecular weight of 60,000. The purified enzyme was insensitive to oxygen and catalyzed the Mg/sup 2 +/ ATP-dependent formation of acyl-CoA from carboxylate and free reduced CoA, with high specificity for benzoate and 2-fluorobenzoate. Apparent K/sub m/ valuesmore » of 0.6 to 2 ..mu..M for benzoate, 2 to 3 ..mu..M for ATP, and 90 to 120 ..mu..M for reduced CoA were determined. The reaction product, benzoyl-CoA, was an effective inhibitor of the ligase reaction. The kinetic properties of the enzyme match the kinetics of substrate uptake by whole cells and confirm a role for benzoate-CoA ligase in maintaining entry of benzoate into cells as well as in catalyzing the first step in the anaerobic degradation of benzoate by R. palustris.« less
  • When grown in the absence of added sulfate, cocultures of Desulfovibrio desulfuricans or Desulfovibrio vulgaris with Methanobrevibacter smithii (Methanobacterium ruminantium), which uses H2 and CO2 for methanogenesis, degraded lactate, with the production of acetate and CH4. When D. desulfuricans or D. vulgaris was grown in the absence of added sulfate in coculture with Methanosarcina barkeri (type strain), which uses both H2-CO2 and acetate for methanogenesis, lactate was stoichiometrically degraded to CH4 and presumably to CO2. During the first 12 days of incubation of the D. desulfuricans-M. barkeri coculture, lactate was completely degraded, with almost stoichiometric production of acetate and CH4.more » Later, acetate was degraded to CH4 and presumably to CO2. In experiments in which 20 mM acetate and 0 to 20 mM lactate were added to D. desulfuricans-M. barkeri cocultures, no detectable degradation of acetate occurred until the lactate was catabolized. The ultimate rate of acetate utilization for methanogenesis was greater for those cocultures receiving the highest levels of lactate. A small amount of H2 was detected in cocultures which contained D. desulfuricans and M. barkeri until after all lactate was degraded. The addition of H2, but not of lactate, to the growth medium inhibited acetate degradation by pure cultures of M. barkeri. Pure cultures of M. barkeri produced CH4 from acetate at a rate equivalent to that observed for cocultures containing M. barkeri. Inocula of M. barkeri grown with H2-CO2 as the methanogenic substrate produced CH4 from acetate at a rate equivalent to that observed for acetate-grown inocula when grown in a rumen fluid-vitamin-based medium but not when grown in a yeast extract-based medium. The results suggest that H2 produced by the Desulfovibrio species during growth with lactate inhibited acetate degradation by M. barkeri. (Refs. 26).« less
  • Benzoate degradation by an anaerobic, syntrophic bacterium, strain SB, in coculture with Desulfovibrio strain G-11 reached a threshold value which depended on the amount of acetate added, and ranged from about 2.5 to 29.9 {mu}M. Increasing acetate concentrations also uncompetitively inhibited benzoate degradation. The apparent V{sub max} and K{sub m} for benzoate degradation decreased with increasing acetate concentration, but the benzoate degradation capacity (V{sub max}/K{sub m}) of cell suspensions remained comparable. The addition of an acetate-using bacterium to cocultures after the threshold was reached resulted in the degradation of benzoate to below the detection limit. Mathematical simulations showed that themore » benzoate threshold was not predicted by the inhibitory effect of acetate on benzoate degradation kinetics. With nitrate instead of sulfate as the terminal electron acceptor, no benzoate threshold was observed in the presence of 20 mM acetate even though the degradation capacity was lower with nitrate than with sulfate. When strain SB was grown with a hydrogen-using partner that had a 5-fold lower hydrogen utilization capacity, a 5 to 9-fold lower the benzoate degradation capacity was observed compared to SB/G-11 cocultures. The Gibb`s free energy for benzoate degradation was less negative in cell suspensions with threshold compared to those without threshold. These studies showed that the threshold was not a function of the inhibition of benzoate degradation capacity by acetate, or the toxicity of the undissociated form of acetate. Rather a critical or minimal Gibb`s free energy may exist where thermodynamic constraints preclude further benzoate degradation.« less
  • Rhodopseudomonas palustris grows anaerobically on structurally diverse aromatic compounds and has served as a model organism in studies of anaerobic aromatic compound degradation. The authors examined {Delta}-1-chca degradation by intact cells of R. paulustris and reexamined enzymatic activities proposed to be involved in the conversion of {Delta}-1-chca to a ring cleavage product. The study confirmed the proposed degradation pathway illustrated. Profiles of intracellular metabolites formed during short-term incubations of whole cells indicated that benzoate grown and {Delta}-1-chc grown cells degrade benzoate by the same route. 27 refs., 6 figs., 3 tabs.