
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) Anaerobic transformations of the naturally occurring aromatic amino acids have been extensively studied in the rumen (Scott et al., 1964; Martin, 1973) , the gastro-intestinal tract of animals (Scheline, 1979 , Brown, 1977 and during the fermentation of farm slurry (Spoelstra, 1978) . Their products of metabolism by clostridia in pure culture have also been elucidated (Brot et al., 1965; Elsden et al., 1976) . Under all of these conditions the benzene ring remains intact (Balba & Evans, 1977~) . A primary inoculum from many anoxic environments (e.g. rumen liquor, sewage sludge, soils and anaerobic sediments) can, however, be adapted to accomplish the complete methanogenic fermentation of these amino acids in a mineral-salts medium supplemented by certain vitamins, by using an initial gas-phase mixture of composition CO,/H, (1 : 4). The bacterial consortia that developed were subcultured into fresh anaerobic mineralsalts medium (Ferry, 1974 ; 2 litres, 37OC) containing a single aromatic amino acid ( 2 m~) as substrate. The progress of the fermentations was monitored by culture sampling, and the gases produced were analysed by using the apparatus described by Balba (1978) . Culture fluids were extracted with diethyl ether under neutral and acidic conditions and these fractions were examined by t.1.c. and g.1.c. after suitable treatments as outlined by Balba & Evans (19776, 1979) . The results obtained are briefly reported as follows.

Phenylalanine: after reaching a steady state in C balance, the gas mixture consisted of methane (55%) and CO, (44%), corresponding to the empirical equation: 

The gaseous products contained approx. 50% of methane and CO,. Indol-3-ylacetate, anthranilate, salicylate, and benzoate were the main aromatic intermediates in the acid-ether extract, together with certain members of the benzoate reductive pathway (Balba & Evans, 19776) . Indole was present in the neutral-ether extract along with c yclohexanone.

From these preliminary results, tentative catabolic pathways for the methanogenesis of the aromatic amino acids in these consortia are depicted in Scheme 1; consolidation will require tracer studies analogous to those carried out with rumen liquor (c.f. Scott et al., 1964; Kristensen, 1974) . Metabolic transformations of the alanine side chain are similar to those occurring in the rumen (Scott et al., 1964) and in pure cultures of the clostridia (Elsden et al., 1976) ; a unique characteristic of the process is the complete utilization of the aromatic residues for cell maintenance and methanogenesis. The sequence of anaerobic enzymic reactions proposed to account for the intermediates detected, postulate the use of a molecule of water for hydroxylation, together with the usual addition andlor abstraction of hydrogen, and decarboxylation; e.g. replacement of the amino group of anthranilate by hydroxy group may occur by water addition across an imino double bond, followed by the elimination of NH,. The conversion ofp-cresol into phenol could be initiated by p-cresol methylhydroxylase, recently discovered in Pseudomonas pufida by Hopper (1976 Hopper ( , 1978 . Alternatively, phenol could arise directly from tyrosine through the action of a 8-elimination enzyme similar to that found in Escherichia coli phenologenes (Ichihara et al., 1956) and Closfridium tetanomorphum (Brot et al., 1965) .

Further progress in this field awaits the isolation in pure culture of the active members of these consortia. It is significant in this connection that the complete degradation of the carbohydrate monomers glucose and fructose into methane and CO, by syntrophic cultures of two bacterial species, Acetobacterium woodii and different strains of methanogens, e.g. Methanosarcina barkeri, has recently been demonstrated by Winter & Wolfe (1 979, 1980) . Carrot cells grown in suspension cultures can be induced to undergo a course of development that parallels zygotic embryogenesis. The inducing stimulus is the removal of auxin from the culture medium, after which typical development through the globular, heart and torpedo stages is observed. After about 5 days the structures germinate, i.e. root and shoot elongation occurs, and the seedlings can be removed from the culture medium and reared into fertile plants.

With repeated subculture, stock cells grown in the presence of auxin gradually lose the ability to form viable embryos once the auxin is removed. When such cultures are induced to undergo embryogenesis, the embryos formed abort before the end of the developmental sequence, usually before the torpedo stage ('naturally aborted embryos'). It was found that the addition, after 3 days, of auxin (2,4-dichlorophenoxyacetic acid) at 0 . 4 5~~ to healthy induced cultures, under minus-auxin conVol. 8 ditions, resulted in a cessation of embryo development morphologically similar to that seen in naturally aborting cultures.

Intercellular-space studies were undertaken to test the hypothesis that cessation of development is due to a breakdown of cellular communication, and that this might be made manifest as an increase in the intercellular space in aborted embryos.

Carrot-cell cultures were initiated and maintained, and embryogenesis was induced as described previously (Warren & Fowler, 1977) . Embryos were harvested by filtration on nylon mesh. The fractions collected were as follows: 3000-530pm (seedlings), 530-355pm (mature torpedos), 355-250pm (young torpedos) and 250-100pm (globular plus heart stages). The number of embryos per unit dry weight was approximately constant for viable and aborted embryos of a similar size. Embryos were experimentally aborted by the addition of OASp~-auxin to 3-day-old viable minus-auxin cultures.

Intercellular volume was estimated by measuring efRux of KNO, at l 0 C from preloaded embryos, by using an Orion nitrate electrode. For each determination approx. 0.2g wet wt. of embryos was placed in lOOml of a solution containing

{'paper_id': '8734183', 'title': 'Methanogenic fermentation of the naturally occurring aromatic amino acids by a microbial consortium', 's2_url': 'https://api.semanticscholar.org/CorpusID:8734183', 'source': 'S2ORC'}