Archives of Microbiology

, Volume 150, Issue 3, pp 254–266 | Cite as

Eubacterium acidaminophilum sp. nov., a versatile amino acid-degrading anaerobe producing or utilizing H2 or formate

Description and enzymatic studies
  • U. Zindel
  • W. Freudenberg
  • M. Rieth
  • J. R. Andreesen
  • J. Schnell
  • F. Widdel
Original Papers


An obligately anaerobic, rod-shaped bacterium was isolated on alanine in co-culture with H2-scavenging Desulfovibrio and obtained in pure culture with glycine as sole fermentation substrate. The isolated strain, al-2, was motile by a polar to subpolar flagellum and stained Gram-positive. The guanine plus cytosine content of the DNA was 44.0 mol%. Strain al-2 grew in defined, reduced glycine media supplemented with biotin. The pure culture fermented 4 mol glycine to 3 mol acetate, 4 mol ammonia and 2 mol CO2. Under optimum conditions (34°C, pH 7.3), the doubling time on glycine was 60 min and the molar growth yield 7.6 g cell dry mass. Serine was fermented to acetate, ethanol, CO2, H2 and ammonia. In addition, betaine, sarcosine or creatine served as substrates for growth and acetate production if H2, formate or e.g. valine were added as H-donors. In pure culture on alanine under N2, strain al-2 grew very poorly and produced H2 up to a partial pressure of 3.6 kPa (0.035 atm). Desulfovibrio species, Methanospirillum hungatei and Acetobacterium woodii served as H2-scavengers that allowed good syntrophic growth on alanine. The co-cultures also grew on aspartate, leucine, valine or malate. Alanine and aspartate were stoichiometrically degraded to acetate and ammonia, whereas the reducing equivalents were recovered as H2S, CH4 or newly synthetized acetate, respectively. Growth of strain al-2 in co-culture with the hydrogenase-negative, formate-utilizing Desulfovibrio baarsii indicated that a syntrophy was also possible by interspecies formate transfer. Growth on glycine, or on betaine, sarcosine or creatine (plus H-donors) depended strictly on the addition of selenite (≥0.1 μM); selenite was not required for fermentation of serine, or for degradation of alanine, aspartate or valine by the co-cultures. Cell-free extracts of glycine-grown cells contained active glycine reductase, glycine decarboxylase and reversible methyl viologen-dependent formate dehydrogenase in addition to the other enzymes necessary for an oxidation to CO2. In all reactions NADP was the preferred H-carrier. Both formate and glycine could be synthesized from bicarbonate. Serine-grown cells did not contain serine hydroxymethyl transferase but serine dehydratase and other enzymes commonly involved in pyruvate metabolism to acetate, CO2 and H2. The enzymes involved in glycine metabolism were repressed during growth on serine. By its morphology and physiology, strain al-2 did not resemble described amino acid-degrading species. Therefore, the new isolate is proposed as type strain of a new species, Eubacterium acidaminophilum.

Key words

Interspecies hydrogen transfer Interspecies formate transfer Alanine Serine Glycine fermentation Selenium Glycine reductase Sarcosine reduction Betaine reduction Eubacterium acidaminophilum 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Andreesen JR, El Ghazzawi E, Gottschalk G (1974) The effect of ferrous ions, tungstate and selenite on the level of formate dehydrogenase in Clostridium formicoaceticum and formate synthesis from CO2 during pyruvate fermentation. Arch Microbiol 96:103–118Google Scholar
  2. Badziong W, Thauer RK (1980) Vectorial electron transport in Desulfovibrio vulgaris (Marburg) growing on hydrogen plus sulfate as sole energy source. Arch Microbiol 125:167–174Google Scholar
  3. Balch WE, Fox GE, Magrum LJ, Woese CR, Wolfe RS (1979) Methanogens: reevaluation of a unique biological group. Microbiol Rev 43:260–296Google Scholar
  4. Barik S, Brulla WJ, Bryant MP (1985) PA-1, a versatile anaerobe obtained in pure culture, catabolizes benzenoids and other compounds in syntrophy with hydrogenotrophs, and P-2 plus Wolinella sp. degrades benzenoids. Appl Environ Microbiol 50:304–310Google Scholar
  5. Barker HA (1981) Amino acid degradation by anaerobic bacteria. Ann Rev Biochem 50:23–40Google Scholar
  6. Barnard GF, Akhtar M (1979) Mechanistic and stereochemical studies on the glycine reductase of Clostridium sticklandii. Eur J Biochem 99:593–603Google Scholar
  7. Bergmeyer HU (ed) (1985) Methoden der enzymatischen Analyse, 3rd edn. Verlag Chemie, WeinheimGoogle Scholar
  8. Beuscher HU, Andreesen JR (1984) Eubacterium angustum sp. nov., a Gram-positive anaerobic, non-sporeforming, obligate purine fermenting organism. Arch Microbiol 140:2–8Google Scholar
  9. Bentley CM, Dawes EA (1974) The energy-yielding reactions of Peptococcus prevotii, their behaviour on starvation and the role and regulation of threonine dehydratase. Arch Microbiol 100:363–387Google Scholar
  10. Britz ML, Wilkinson RG (1982) Leucine dissimilation to isovaleric and isocaproic acids by cell suspensions of amino acid fermenting anaerobes: the Stickland reaction revisited. Can J Microbiol 28:291–300Google Scholar
  11. Bryant MP (1972) Commentary on the Hungate technique for culture of anaerobic bacteria. Am J Clin Nutr 25:1324–1328Google Scholar
  12. Buckel W (1986) Substrate stereochemistry of the biotin-dependent sodium pump glutaconyl-CoA decarboxylase from Acidaminococcus fermentans. Eur J Biochem 156:259–263Google Scholar
  13. Cardon BP, Barker HA (1947) Amino acid fermentations by Clostridium propionicum and Diplococcus glycinophilus. Arch Biochem 12:165–180Google Scholar
  14. Cato EP, Johnson JL, Hash DE, Holdeman LV (1983) Synonymy of Peptococcus glycinophilus (Cardon & Barker 1946; Douglas 1957) with Peptostreptococcus micros (Prevot 1933; Smith 1957) and electrophoretic differentiation of Peptostreptococcus micros from Peptococcus magnus (Prevot 1933; Holdeman & Moore 1972). Int J Syst Bacteriol 33:207–210Google Scholar
  15. Champion AB, Rabinowitz JC (1977) Ferredoxin and formyltetrahydrofolate synthetase: comparative studies with Clostridium acidiurici, Clostridium cylindrosporum, and newly isolated anaerobic uric acid-fermenting strains. J Bacteriol 132:1003–1020Google Scholar
  16. Cline JD (1969) Spectrophotometric determination of hydrogen sulfide in natural waters. Limnol Oceanogr 14:454–458Google Scholar
  17. Cole SC, Kuwahara SS (1984) Acetylacetone method for glycine improved by use of ammonium citrate buffer. Clin Chem 30:1260–1261Google Scholar
  18. Diekert GB, Thauer RK (1978) Carbon monoxide oxidation by Clostridium thermoaceticum and Clostridium formicoaceticum. J Bacteriol 136:597–606Google Scholar
  19. Dürre P, Andreesen JR (1982) Selenium-dependent growth and glycine fermentation by Clostridium purinolyticum. J Gen Microbiol 128:1457–1466Google Scholar
  20. Dürre P, Andreesen JR (1983) Purine and glycine metabolism by purinolytic clostridia. J Bacteriol 154:192–199Google Scholar
  21. Dürre P, Spahr R, Andreesen JR (1983) Glycine fermentation via glycine reductase in Peptococcus glycinophilus and Peptococcus magnus. Arch Microbiol 134:127–135Google Scholar
  22. Eden G, Fuchs G (1983) Autotrophic CO2 fixation in Acetobacterium woodii. II. Demonstration of enzymes involved. Arch Microbiol 135:68–73Google Scholar
  23. Elsden SR, Hilton MG (1978) Volatile acid production from threonine, valine, leucine and isoleucine by clostridia. Arch Microbiol 117:165–172Google Scholar
  24. Elsden SR, Hilton MG (1979) Amino acid utilization patterns in clostridial taxonomy. Arch Microbiol 123:137–141Google Scholar
  25. Eneroth P, Lindstedt G (1965) Thin-layer chromatography of betaines and other compounds related to carnitine. Arch Biochem 10:479–485Google Scholar
  26. Frenkel EP, Kitchens RL (1981) Acetyl-CoA synthetase from baker's yeast. Meth Enzymol 71:317–324Google Scholar
  27. Fryer TF, Mead GC (1979) Development of a selective medium for the isolation of Clostridium sporogenes and related organisms. J Appl Bacteriol 47:425–431Google Scholar
  28. Genthner BR, Davis CL, Bryant MP (1981) Features of rumen and sewage sludge strains of Eubacterium limosum, a methanol- and H2-CO2-utilizing species. Appl Environ Microbiol 42:12–19Google Scholar
  29. Giesel H, Machacek G, Bayerl J, Simon H (1981) On the formation of 3-phenylpropionate and the different stereo-chemical course of the reduction of cinnamate by Clostridium sporogenes and Peptostreptococcus anaerobius. FEBS Lett 123:107–110Google Scholar
  30. Gottschalk G, Andreesen JR, Hippe H (1981) The genus Clostridium (nonmedical aspects). In: Starr MP, Stolp H, Trüper HG, Balows A, Schlegel HG (eds) The prokaryotes, vol II. Springer, Berlin Heidelberg New York, pp 1767–1803Google Scholar
  31. Gregersen T (1978) Rapid method for distinction of Gram-negative from Gram-positive bacteria. Eur J Appl Microbiol Biotechnol 5:123–127Google Scholar
  32. Higgins CF, Cairney J, Stirling DA, Sutherland L, Booth IR (1987) Osmotic regulation of gene expression: ionic strength as an intracellular signal. TIBS 12:339–344Google Scholar
  33. Kado CI, Liu ST (1981) Rapid procedure for detection and isolation of large and small plasmids. J Bacteriol 145:1365–1373Google Scholar
  34. Klein SM, Sagers RD (1962) Intermediary metabolism of Diplococcus glycinophilus. II. Enzymes of the acetate-generating system. J Bacteriol 83:121–126Google Scholar
  35. Klein SM, Sagers RD (1967) Glycine metabolism. III. A flavin-linked dehydrogenase associated with the glycine cleavage system in Peptococcus glycinophilus. J Biol Chem 242:297–300Google Scholar
  36. Laanbroek HJ, Lambers JT, De Vos WM, Veldkamp H (1978) L-Aspartate fermentation by a free-living Campylobacter species. Arch Microbiol 117:109–114Google Scholar
  37. Lang E, Lang H (1972) Specific colour reaction for the direct identification of formic acid. Fresenius Z Anal Chem 260:8–10Google Scholar
  38. Lebertz H (1984) Selenabhängiger Glycin-Stoffwechsel bei anaeroben Bakterien und vergleichende Untersuchungen zur Glycin-Reduktase und zur Glycin-Decarboxylase. PhD thesis, Universität GöttingenGoogle Scholar
  39. Levering PR, Binnema DJ, van Dijken JP, Harder W (1981) Enzymatic evidence for a simultaneous operation of two one-carbon assimilation pathways during growth of Arthrobacter P1 on choline. FEMS Microbiol Lett 12:19–25Google Scholar
  40. Mead GC (1971) The amino acid-fermenting Clostridia. J Gen Microbiol 67:47–56Google Scholar
  41. Möller B, Oßmer R, Howard BH, Gottschalk G, Hippe H (1984) Sporomusa, a new genus of Gram-negative anaerobic bacteria including Sporomusa sphaeroides spec. nov. and Sporomusa ovata spec. nov. Arch Microbiol 139:388–396Google Scholar
  42. Möller B, Hippe H, Gottschalk G (1986) Degradation of various amino compounds by mesophilic clostridia. Arch Microbiol 145:85–90Google Scholar
  43. Moench TT, Zeikus JG (1983) An improved preparation method for a titanium (III) media reductant. J Microbiol Meth 1:199–202Google Scholar
  44. Moore WEC, Holdeman-Moore LV (1986) Genus Eubacterium. In: Sneath PAH (ed) Bergey's manual of systematic bacteriology, vol 2. Williams & Wilkins, Baltimore London, pp 1353–1373Google Scholar
  45. Nagase M, Matsuo T (1982) Interaction between amino-acid-degrading bacteria and methanogenic bacteria in anaerobic digestion. Biotechnol Bioeng 24:2227–2239Google Scholar
  46. Nanninga HJ, Gottschal JC (1985) Amino acid fermentation and hydrogen transfer in mixed cultures. FEMS Microbiol Ecol 31:261–269Google Scholar
  47. Nanninga HJ, Drent WJ, Gottschal JC (1986) Major differences between glutamate-fermenting species isolated from chemostat enrichments at different dilution rates. FEMS Microbiol Ecol 38:321–329Google Scholar
  48. Naumann E, Hippe H, Gottschalk G (1983) Betaine: new oxidant in the Stickland reaction and methanogenesis from betaine and L-alanine by a Clostridium sporogenes — Methanosarcina barkeri co-culture. Appl Environ Microbiol 45:474–483Google Scholar
  49. Pfennig N, Widdel F (1981) Ecology and physiology of some anaerobic bacteria from the microbial sulfur cycle. In: Bothe H, Trebst A (eds) Biology of inorganic nitrogen and sulfur. Springer, Berlin Heidelberg New York, pp 169–177Google Scholar
  50. Rabinowitz JC, Pricer WE (1962) Formyltetrahydrofolate synthetase. I. Isolation and crystallization of the enzyme. J Biol Chem 237:2898–2902Google Scholar
  51. Stadtman TC (1970) Glycine reductase system (Clostridium). Meth Enzymol 17A:959–966Google Scholar
  52. Stams AJM, Hansen TA (1984) Fermentation of glutamate and other compounds by Acidaminobacter hydrogenoformans gen. nov. sp. nov., an obligate anaerobe isolated from black mud. Studies with pure cultures and mixed cultures with sulfate-reducing and methanogenic bacteria. Arch Microbiol 137:329–337Google Scholar
  53. Stouthamer AH (1979) The search for correlation between theoretical and experimental growth yields. In: Quayle JR (ed) Microbial Biochem, Intern Rev Biochem, vol 21. University Park Press, Baltimore, pp 1–47Google Scholar
  54. Tanaka H, Stadtman TC (1979) Selenium-dependent clostridial glycine reductase. Purification and characterization of the two membrane-associated protein components. J Biol Chem 254:447–452Google Scholar
  55. Tanner RS, Stackebrandt E, Fox GE, Woese CR (1981) A phylogenetic analysis of Acetobacterium woodii, Clostridium barkeri, Clostridium lituseburense, Eubacterium limosum, and Eubacterium tenue. Curr Microbiol 5:35–38Google Scholar
  56. Taylor RT, Weissbach H (1965) Radioactive assay for serine transhydroxymethylase. Arch Biochem 13:80–84Google Scholar
  57. Thauer RK (1973) CO2 reduction to formate in Clostridium acidiurici. J Bacteriol 114:443–444Google Scholar
  58. Thauer RK, Rupprecht E, Jungermann K (1970) Separation of 14C-formate from CO2 fixation metabolites by isoionic-exchange chromatography. Anal Biochem 38:461–468Google Scholar
  59. Thauer RK, Jungermann K, Decker K (1977) Energy conservation in chemotrophic anaerobic bacteria. Bacteriol Rev 41:100–180Google Scholar
  60. Uyeda K, Rabinowitz JC (1967) Metabolism of formiminoglycine. Formiminotetrahydrofolate cyclodeaminase. J Biol Chem 242:24–31Google Scholar
  61. Waber LJ, Wood HG (1979) Mechanism of acetate synthesis from CO2 by Clostridium acidiurici. J Bacteriol 140:468–478Google Scholar
  62. Wagner R, Andreesen JR (1977) Differentiation between Clostridium acidiurici and Clostridium cylindrosporum on the basis of specific metal requirements for formate dehydrogenase formation. Arch Microbiol 114:219–224Google Scholar
  63. Wallace RJ (1986) Catabolism of amino acids by Megasphaera elsdenii LCI. Appl Environ Microbiol 51:1141–1143Google Scholar
  64. Weatherburn MW (1967) Phenol-hypochlorite reaction for determination of ammonia. Anal Chem 39:971–974Google Scholar
  65. Whiteley HR (1957) Fermentation of amino acids by Micrococcus aerogenes. J Bacteriol 74:324–330Google Scholar
  66. Widdel F (1988) Microbiology ecology of sulfate- and sulfur-reducing bacteria. In: Zehnder AJB (ed) Biology of anaerobic microorganisms. John Wiley & Sons, New York, pp 469–585Google Scholar
  67. Widdel F, Pfennig N (1981) Studies on dissimilatory sulfate-reducing bacteria that decompose fatty acids. I. Isolation of new sulfate-reducing bacteria enriched with acetate from saline environments. Description of Desulfobacter postgatei gen. nov., sp. nov. Arch Microbiol 129:395–400Google Scholar
  68. Widdel F, Pfennig N (1984) Dissimilatory sulfate- or sulfur-reducing bacteria. In: Krieg NR, Holt JG (ed) Bergey's manual of systematic bacteriology, vol 1. Williams & Wilkins, Baltimore London, pp 663–679Google Scholar
  69. Wildenauer FX, Winter J (1986) Fermentation of isoleucine and arginine by pure and syntrophic cultures of Clostridium sporogenes. FEMS Microbiol Ecol 38:373–379Google Scholar
  70. Winter J, Schindler F, Wildenauer FX (1987) Fermentation of alanine and glycine by pure and syntrophic cultures of Clostridium sporogenes. FEMS Microbiol Ecol 45:153–161Google Scholar
  71. Wolin MJ (1982) Hydrogen transfer in microbial communities. In: Bull AT, Slater JH (ed) Microbial interactions and communities, vol 1. Academic Press, London New York, pp 323–356Google Scholar

Copyright information

© Springer-Verlag 1988

Authors and Affiliations

  • U. Zindel
    • 1
  • W. Freudenberg
    • 1
  • M. Rieth
    • 1
  • J. R. Andreesen
    • 1
  • J. Schnell
    • 2
  • F. Widdel
    • 2
  1. 1.Institut für MikrobiologieUniversität GöttingenGöttingenFederal Republic of Germany
  2. 2.Fakultät für BiologieUniversität KonstanzKonstanz 1Federal Republic of Germany

Personalised recommendations