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Title: Energetics of end product excretion in anaerobic bacteria and the metabolism of fatty acids by Syntrophomonas wolfei. Progress report, January 31-December 15, 1985

Abstract

This work addresses the metabolism of fatty acids and the energetics of growth of the anaerobic, syntrophic, fatty acid-degrading bacterium, Syntrophomonas wolfei. S. wolfei degrades C/sub 4/ to C/sub 8/ straight chain fatty acids to acetate and H/sub 2/ or acetate, propionate and H/sub 2/; isoheptanoate is degraded to isovalerate, acetate, and H/sub 2/. S. wolfei can not use any common bacterial energy source that will allow it to grow in pure culture. A significant breakthrough in the cultivation of S. wolfei was achieved. Long term (3 months) incubation of S. wolfei cocultures in medium with crotonate selects for a population of S. wolfei cells that can use this compound. These cultures contain large numbers of S. wolfei cells and very few cells of the methanogen. Pure cultures of S. wolfei do not use butyrate. However, when pure cultures of S. wolfei are incubated in the presence of H/sub 2/-using bacteria, butyrate is degraded to acetate and H/sub 2/. These data show that the cells present in the pure cultures are in fact S. wolfei. Growth of S. wolfei with crotonate is faster and much higher cell densities are obtained. Thus, large amounts of cell material will be available formore » biochemical studies. 3 refs.« less

Authors:
Publication Date:
Research Org.:
Oklahoma Univ., Norman (USA)
OSTI Identifier:
6407976
Report Number(s):
DOE/ER/13053-4
ON: DE86004532
DOE Contract Number:
AS05-83ER13053
Resource Type:
Technical Report
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; 59 BASIC BIOLOGICAL SCIENCES; METHANOGENIC BACTERIA; CULTIVATION TECHNIQUES; MONOCARBOXYLIC ACIDS; METABOLISM; ACETIC ACID; ANAEROBIC CONDITIONS; BIOLOGICAL ADAPTATION; BUTYRIC ACID; CROTONIC ACID; EXCRETION; GAS CHROMATOGRAPHY; SYMBIOSIS; BACTERIA; CARBOXYLIC ACIDS; CHROMATOGRAPHY; CLEARANCE; MICROORGANISMS; ORGANIC ACIDS; ORGANIC COMPOUNDS; SEPARATION PROCESSES; 090122* - Hydrocarbon Fuels- Preparation from Wastes or Biomass- (1976-1989); 550700 - Microbiology; 550500 - Metabolism

Citation Formats

McInerney, M.J.. Energetics of end product excretion in anaerobic bacteria and the metabolism of fatty acids by Syntrophomonas wolfei. Progress report, January 31-December 15, 1985. United States: N. p., 1985. Web.
McInerney, M.J.. Energetics of end product excretion in anaerobic bacteria and the metabolism of fatty acids by Syntrophomonas wolfei. Progress report, January 31-December 15, 1985. United States.
McInerney, M.J.. 1985. "Energetics of end product excretion in anaerobic bacteria and the metabolism of fatty acids by Syntrophomonas wolfei. Progress report, January 31-December 15, 1985". United States. doi:.
@article{osti_6407976,
title = {Energetics of end product excretion in anaerobic bacteria and the metabolism of fatty acids by Syntrophomonas wolfei. Progress report, January 31-December 15, 1985},
author = {McInerney, M.J.},
abstractNote = {This work addresses the metabolism of fatty acids and the energetics of growth of the anaerobic, syntrophic, fatty acid-degrading bacterium, Syntrophomonas wolfei. S. wolfei degrades C/sub 4/ to C/sub 8/ straight chain fatty acids to acetate and H/sub 2/ or acetate, propionate and H/sub 2/; isoheptanoate is degraded to isovalerate, acetate, and H/sub 2/. S. wolfei can not use any common bacterial energy source that will allow it to grow in pure culture. A significant breakthrough in the cultivation of S. wolfei was achieved. Long term (3 months) incubation of S. wolfei cocultures in medium with crotonate selects for a population of S. wolfei cells that can use this compound. These cultures contain large numbers of S. wolfei cells and very few cells of the methanogen. Pure cultures of S. wolfei do not use butyrate. However, when pure cultures of S. wolfei are incubated in the presence of H/sub 2/-using bacteria, butyrate is degraded to acetate and H/sub 2/. These data show that the cells present in the pure cultures are in fact S. wolfei. Growth of S. wolfei with crotonate is faster and much higher cell densities are obtained. Thus, large amounts of cell material will be available for biochemical studies. 3 refs.},
doi = {},
journal = {},
number = ,
volume = ,
place = {United States},
year = 1985,
month = 1
}

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  • We developed methods to physically separate cells of the anaerobic, fatty acid degrade, Syntrophomonas wolfei, from cells of the hydrogen user by Percoll gradient centrifugation and to selectively lyse S. wolfei cells using lysozyme. These methods allowed the study of the physiology of S. wolfei without significant contamination. Fatty acids were degraded by the B-oxidation pathway using a coenzyme A (CoA) transferase activity to activate the fatty acid and substrate- level phosphorylation reactions to synthesize. The substrate specificity of the CoA transferase activity in the pure culture of S. wolfei differed from that found in the coculture suggesting that themore » ability to use crotonate resulted from an alteration of this enzyme. S. wolfei grown alone degraded crotonate in a manner similar to that of other crotonate-fermenting anaerobes, but the molar growth yields of S. wolfei were 2 to 3 times higher than those organisms. This suggests that the reduction of crotonyl-CoA to butyryl-CoA is energy yielding. S. wolfei contained a c-type cytochrome which may be involved in this reaction. S. wolfei synthesized large amounts of the storage polymer, poly-B-hydroxybutyrate.« less
  • We have studied the growth and metabolism of Syntrophomonas wolfei in pure culture with crotonate as the energy source. S. wolfei grows in crotonate mineral salts medium without rumen fluid with cobalamin, thymine, lipoic acid and biotin added. However, after four to six transfers in this medium, growth ceases, indicating that another vitamin is required. The chemically defined medium allows large batches of S. wolfei to be grown for enzyme purification. All the enzymes involved in the oxidation of crotonyl-CoA to acetate have been detected. The pure culture of S. wolfei or coculture of S. wolfei grown with crotonate containmore » high activities of a crotonate: acetyl-CoA CoA-transferase activity. This activity is not detected in cocultures grown with butyrate. Thus, we believe that the reason why S. wolfei can now grow with crotonate is that an alteration or mutation occurred which allows the organism to activate this crotonate. S. wolfei also makes small amounts of H/sub 2/ when grown in pure culture with crotonate. A methyl viologen-dependent hydrogenase activity was found. We have also demonstrated the production of H/sub 2/ from 3-hydroxybutyryl-CoA in cell-free extracts of S. wolfei by coupling H/sub 2/ production to CH/sub 4/ production with the addition of Methanobacterium bryantii and directly using a hydrogen electrode. These results clearly show that S. wolfei makes H/sub 2/. S. wolfei does not contain formate dehydrogenase or CO dehydrogenase activities.« less
  • The study of anaerobic hydrogen-producing syntrophic bacteria is important for several reasons. These bacteria degrade fatty acids which are important intermediates in anaerobic degradation and methanogenesis. The rate and extent of anaerobic degradation of complex polymeric materials often depends on the activity of these organisms. The production of H{sub 2} during anaerobic fatty acid degradation is energetically favorable only when H{sub 2} is maintained at a low level by another bacterium such as a H{sub 2}- using methanogen. Thus, the fatty acid-degrading syntrophic associations serve as excellent models to study the biochemical aspects of mutualism. The fatty acid-degrading syntrophic bacteriamore » are very slow growers since little free energy is released during fatty acid degradation. These bacteria must have very efficient energy conservation systems which are not understood at this time. Further study of these organisms will provide useful information on bioenergetics of living systems. We have chosen to study the metabolism and energetics of the anaerobic, syntrophic, fatty acid degrader, Syntrophomonas wolfei. This organism is the best characterized syntrophic bacterium and serves as an appropriate model organism.« less
  • Anaerobic syntrophic bacteria degrade fatty acids which are important intermediates in anaerobic degradation and methanogenesis. These bacteria grow very slowly and require the presence of a hydrogen/formate-using organism to degrade fatty acids. Thus, these bacteria serve as models to study the biochemical aspects of mutualism and the energetics of slow growing organisms. We developed methods to physically separate cells of the anaerobic, fatty acid degrader, Syntrophomonas wolfei, from cells of the hydrogen user by Percoll gradient centrifugation and to selectively lyse S. wolfei cells using lysozyme. These methods allowed the study of the physiology of S. wolfei without significant contaminationmore » by cellular components of the hydrogen user. We also obtained pure cultures of S. wolfei by adapting the organism to grow on crotonate. Fatty acids were degraded by the {beta}-oxidation pathway using a coenzyme A (CoA) transferase activity to activate the fatty acid and substrate-level phosphorylation reactions to synthesize adenosine-5{prime}-triphosphate (ATP). The substrate specificity of the CoA transferase activity in the pure culture of S. wolfei differed from that found in the coculture suggesting that the ability to use crotonate resulted from an alteration of this enzyme. S. wolfei grown alone degraded crotonate in a manner similar to that of other crotonate-fermenting anaerobes, but the molar growth yields of S. wolfei were 2 to 3 times higher than those organisms. This suggests that the reduction of crotonyl-CoA to butyryl-CoA is energy yielding. S. wolfei contained a c-type cytochrome which may be involved in this reaction. S. wolfei synthesized large amounts of the storage polymer, poly-{beta}-hydroxybutyrate (PHB). Radioisotopic incorporation experiments showed that PHB was synthesized directly from the {beta}-oxidation intermediate rather than from the condensation of two acetyl-CoA molecules.« less
  • The degradation of fatty acids is often the rate-limiting step in the complete anaerobic degradation of organic matter to CO/sub 2/ and CH/sub 4/. Syntrophomonas wolfei anaerobically degrades even-numbered fatty acids to acetate and H/sub 2/, odd-numbered fatty acids to acetate, propionate and H/sub 2/, and isoheptanoate to acetate, isovalerate and H/sub 2/ only when grown in co-culture with H/sub 2/-using bacteria such as methanogens. Methods to mass culture the S. wolfei - Methanospirillum hungatei co-culture have been developed and about 1 to 1.5 g (wet weight) of cells is obtained from a 10-liter culture in 4 to 6 weeks.more » S. wolfei cells were selective lyzed by lysozyme and the unlyzed cells of M. hungatei were removed by centrifugation. Methods have been developed to study the biochemistry of this important group of bacteria. Cell-free-extracts of S. wolfei have very high specific activities of the following beta-oxidation enzymes: acyl-coenzyme A(CoA) dehydrogenase, enoyl-CoA hydratase, L-3-hydroxy-acyl-CoA dehydrogenase and thiolase. The specific activity of these enzymes in S. wolfei is 5 to 168-fold higher than the specific activity of the respective enzyme in Escherichia coli extracts assayed under identical conditions. We have recently obtained preliminary evidence that the hydrogenase of S. wolfei is membrane-bound.« less