Methanogenic fermentation of the naturally occurring aromatic amino acids by a microbial consortium.

Medium 2 was similar to Medium 1 except for the replacement of sodium lactate by sodium benzoate (0.75 9). Sterile culture flasks (500ml) were filled to the brim with freshly prepared sterile Medium 1 and 2 separately, inoculated with a suspension of freeze-dried Desulfovibrio N.C.I.B. 8399, capped with sterile Suba-Seal rubber stoppers and incubated by total immersion in a water bath at 37°C. Growth was evident in Medium 1 within 2-3 days by the appearance of black iron pyrites; a drop of this culture on a microscope slide revealed motile bacteria of the expected size and form. The flask containing Medium 2 showed no evidence of growth for 3 months; shortly afterwards it was noticed that the precipitate in this culture had also turned black. Inoculating a few drops of this culture into fresh Medium 2 resulted in growth now starting within 3-4 days with production of ferrous sulphide and concomitant utilization of benzoate as estimated by spectrophotometry (Williams & Evans, 1975). Initially, it was thought that Desulfovibrio N.C.I.B. 8399 had become adapted for the metabolism of benzoate. Smears of the culture fluid however revealed two types of motile bacteria-the vibrio and a rod-present. The contaminant was separated in pure culture and identified as a strain of Pseudomonas aeruginosa (see the acknowledgements). Neither organism alone metabolized the benzoate in Medium 2; when put together, growth with utilization of this substrate ensued. The benzoate disappeared significantly faster when sodium lactate (1 g/litre) was also added to Medium 2. Thioglycollate, yeast extract and the preservation of strict anaerobiosis were all essential for the phenomenon to occur. At this stage it was decided to apply the methods developed by Balba & Evans (1977a,b) and Balba (1978) for the identification of benzoate metabolites in anaerobic cultures: thtse included t.1.c. and g.1.c. of neutral and aciddiethyl ether extracts of culture fluid after suitable treatments. The Pseudomonas aeruginosa strain grew aerobically in benzoate or p-hydroxybenzoate mineral-salts medium using the ortho (i.e. intradiol) pathway of metabolism. Nitrate would not serve as terminal electron acceptor for anaerobic benzoate dissimilation in the medium used by Williams & Evans (1975). When pyruvate or fumarate (2g/litre) were substituted for nitrate in the latter medium, the bacterium flourished anaerobically and benzoate was utilized; their replacement by succinate or lactate (2g/litre) gave poor growth and benzoate hardly disappeared. Examination of the mixed culture (i.e. Desulfovibrio vulgaris N.C.I.B. 8399 + Pseudomonas aeruginosa strain) growing in Medium 2, revealed the simultaneous disappearance of benzoate with the formation of cyclohexanol among the products in the neutral-ether extract and cyclohexane carboxylate in the acid-ether extract; these are known intermediates in the reductive pathway of benzoate metabolism. This syntrophic association of a facultative pseudomonad together with Desulfovibrio vulgaris N.C.I.B. 8399 accomplishes the utilization of benzoate through anaerobic sulphate respiration. It seems likely that the Desulfovibrio produces initially small quantities of organic acids capable of acting as electron acceptors for the facultative organism to attack the benzoate-the chain of events would then become autocatalytic. Whether this association is of wide occurrence among sulphate-reducers remains to be demonstrated.