Isolation and Partial Characterization of Bacteria in an Anaerobic Consortium That Mineralizes 3-Chlorobenzoic Acid

Abstract

A methanogenic consortium able to use 3-chlorobenzoic acid as its sole energy and carbon source was enriched from anaerobic sewage sludge. Seven bacteria were isolated from the consortium in monoor coculture. They included: one dechlorinating bacterium (strain DCB-1), one benzoate-oxidizing bacterium (strain BZ-2), two butyrate-oxidizing bacteria (strains SF-1 and NSF-2), two H2-consuming methanogens (Methanospirillum hungatei PM-1 and Methanobacterium sp. strain PM-2), and a sulfate-reducing bacterium (Desulfovibrio sp. strain PS-1). The dechlorinating bacterium (DCB-I) was a gram-negative, obligate anaerobe with a unique "collar" surrounding the cell. A medium containing rumen fluid supported minimal growth; pyruvate was the only substrate found to increase growth. The bacterium had a generation time of 4 to 5 days. 3-Chlorobenzoate was dechlorinated stoichiometrically to benzoate, which accumulated in the medium; the rate of dechlorination was ca. 0.1 pmol bacterium-' day-'. The benzoate-oxidizing bacterium (BZ-2) was a gram-negative, obligate anaerobe and could only be grown as a syntroph. Benzoate was the only substrate observed to support growth, and, when grown in coculture with M. hungatei, it was fermented to acetate and CH4. One butyrate-oxidizing bacterium (NSF-2) was a gram-negative, non-sporeforming, obligate anaerobe; the other (SF-1) was a gram-positive, sporeforming, obligate anaerobe. Both could only be grown as syntrophs. The substrates observed to support growth of both bacteria were butyrate, 2-DL-methylbutyrate, valerate, and caproate; isobutyrate supported growth of only the sporeforming bacterium (SF-1). Fermentation products were acetate and CH4 (from butyrate, isobutyrate, or caproate) or acetate, propionate, and CH4 (from 2-DLmethylbutyrate or valerate) when grown in coculture with M. hungatei. A mutualism among at least the dechlorinating, benzoate-oxidizing, and methane-forming members was apparently required for utilization of the 3-chlorobenzoate substrate. Many chlorinated organic compounds are not biodegraded under aerobic conditions, often because the chlorine substitutions prevent ring cleavage and thus subsequent dechlorination. Recently, however, certain chlorinated aromatic compounds have been shown to be dechlorinated in anaerobic habitats such as sediment, flooded soil, and digested sludge. These chemicals include chlorinated benzoates (5, 15, 16), chlorinated phenols (2, 3, 6, 12), and the pesticides diuron [3-(3,4-dichlorophenyl)-1,1-dimethylurea] (1), Techlofthalam [N-(2,3-dichlorophenyl)-3,4,5,6-tetrachlorophthalmic acid] (7), chloronitrofen (4-nitrophenyl-2,4,6-trichlorophenyl ether) (19), and 2,4,5-trichlorophenoxyacetic acid (17). In all of these cases, chlorine is removed from the aromatic ring before ring cleavage, which is in contrast to aerobic metabolism of chloroaromatic compounds. Thus, this reaction-a reductive dechlorination-is of particular interest because it has the potential for making some of the highly chlorinated, serious pollutants less persistent and...

Nitrogen fixation by reductively dechlorinating bacteria

“…We detected N 2 fixation capability in halorespiring anaerobes Desulfomonile tiedjei (Shelton and Tiedje, 1984), Sulphurospirillum multivorans (Scholz-Muramatsu et al, 1995), Desulfovibrio dechloracetivorans (Sun et al, 2000) and Desulfitobacterium dehalogenans (Utkin et al, 1994), which were isolated from distinct habitats and represent Gram-negative and Gram-positive bacteria. Partial nifH fragments (approximate 390 bp) were amplified using degenerate primers (Mehta et al, 2003) corresponding to the conserved regions of nifH.…”

Microbial breakdown of halogenated aromatic pesticides and related compounds

“…Suflita et al [257] enriched a stable 3-chlorobenzoate-utilizing consortium from sewage sludge. Seven bacteria were isolated from this consortium, which included a dehalogenating bacterium, strain DCB-1 [265]. This Gram-negative, obligately anaerobic, sulfate-reducing bacterium was proposed as a new species, Desulfomonile tiedjei [266].…”

“…This Gram-negative, obligately anaerobic, sulfate-reducing bacterium was proposed as a new species, Desulfomonile tiedjei [266]. Complete mineralization of 3-chlorobenzoate required mutualism among the dechlorinating, benzoate-oxidizing and methanogenic members of the consortium [265], and a defined consortium was constructed by combining the key organisms isolated from the enrichment culture, the dechlorinating bacterium, a benzoate degrader and a methanogen [267]. Reducing power required for dechlorination was obtained from the hydrogen produced in the acetogenic oxidation of benzoate, suggesting that the consortium operated as a food web [267].…”

Kinetics of two complementary hydrogen sink reactions in a defined 3-chlorobenzoate degrading methanogenic co-culture

“…1. All organisms have been isolated by Shelton in our laboratory [2]. The dechlorinating organism, strain DCB-1, was recently named Desulfomonile tiedjei…”

“…In the absence of nitrate and sulfate the anaerobic degradation of benzoate is a hydrogenogenic process: three tool of H 2 are produced per mol of benzoate catabolized (Eqn. 1), although some formate may be produced in place of some H E [1,2].…”