The impact of grazing by soil flagellates Heteromita globosa on aerobic biodegradation of benzene by Pseudomonas strain PS+ was examined in batch culture. Growth of H. globosa on these bacteria obeyed Monod kinetics (mu(max), 0.17 +/- 0.03 h(-1); K(s), 1.1 +/- 0.2 x 10(7) bacteria mL(-1)) and was optimal at a bacteria/ flagellate ratio of 2000. Carbon mass balance showed that 5.2% of total [ring-U-(14)C]benzene fed to bacteria was subsequently incorporated into flagellate biomass. Growth-inhibiting concentrations (IC50) of alkylbenzenes (benzene, toluene, ethylbenzene) were inversely related with their octanol/ water partitioning coefficients, and benzene was least toxic for bacteria and flagellates with IC50 values of 4392 (+/- 167) microM and 2770 (+/- 653) microM, respectively. The first-order rate constant for benzene degradation (k1, 0.48 +/- 0.12 day(-1)) was unaffected by the presence or absence of flagellates in cultures. However, the rate of benzene degradation by individual bacteria averaged three times higher in the presence of flagellates (0.73 +/- 0.13 fmol cell(-1) h(-1)) than in their absence (0.26 +/- 0.03 fmol cell(-1) h(-1)). Benzene degradation also coincided with higher levels of dissolved oxygen and a higher rate of nitrate reduction in the presence of flagellates (p < 0.02). Grazing by flagellates may have increased the availability of dissolved oxygen to a smaller surviving population of bacteria engaged in the aerobic reactions initiating benzene degradation. In addition, flagellates may also have increased the rate of nitrate reduction through the excretion of acetate as an additional electron donor for these bacteria. Indeed, acetate was shown to progressively accumulate in cultures where flagellates grazed on heat-killed bacteria. This study provided evidence that grazing flagellates stimulate bacterial degradation of alkylbenzenes and provide a link for carbon cycling to consumers at higher trophic levels. This may have important implications for bioremediation processes.
An exopolymer (slime)-producing soil bacterium Pseudomonas sp. (strain PS؉) rapidly clogged sand-filled columns supplied with air-saturated artificial groundwater containing glucose (500 mg liter ؊1 ) as a sole carbon source and nitrate (300 mg liter ؊1 ) as an alternative electron acceptor. After 80 days of operation under denitrifying conditions, the effective porosity and saturated hydraulic conductivity (permeability) of sand in these columns had fallen by 2.5-and 26-fold, respectively. Bacterial biofilms appeared to induce clogging by occluding pore spaces with secreted exopolymer, although there may also have been a contribution from biogas generated during denitrification. The bacterivorous soil flagellate Heteromita globosa minimized reductions in effective porosity (1.6-fold) and permeability (13-fold), presumably due to grazing control of biofilms. Grazing may have limited growth of bacterial biomass and hence the rate of exopolymer and biogas secretion into pore spaces. Evidence for reduction in biogas production is suggested by increased nitrite efflux from columns containing flagellates, without a concomitant increase in nitrate consumption. There was no evidence that flagellates could improve flow conditions if added once clogging had occurred (60 days). Presumably, bacterial biofilms and their secretions were well established at that time. Nevertheless, this study provides evidence that bacterivorous flagellates may play a positive role in maintaining permeability in aquifers undergoing remediation treatments.
o-Xylene is one of the most difficult-to-degrade environmental pollutants. We report here Rhodococcus genes mediating oxygenation in the first step of o-xylene degradation. Rhodococcus opacus TKN14, isolated from soil contaminated with o-xylene, was able to utilize o-xylene as the sole carbon source and to metabolize it to o-methylbenzoic acid. A cosmid library from the genome of this strain was constructed in Escherichia coli. A bioconversion analysis revealed that a cosmid clone incorporating a 15-kb NotI fragment had the ability to convert o-xylene into o-methylbenzyl alcohol. The sequence analysis of this 15-kb region indicated the presence of a gene cluster significantly homologous to the naphthalene-inducible dioxygenase gene clusters (nidABCD) that had been isolated from Rhodococcus sp. strain I24. Complementation studies, using E. coli expressing various combinations of individual open reading frames, revealed that a gene (named nidE) for rubredoxin (Rd) and a novel gene (named nidF) encoding an auxiliary protein, which had no overall homology with any other proteins, were indispensable for the methyl oxidation reaction of o-xylene, in addition to the dioxygenase iron-sulfur protein genes (nidAB). Regardless of the presence of NidF, the enzyme composed of NidABE was found to function as a typical naphthalene dioxygenase for converting naphthalene and various (di)methylnaphthalenes into their corresponding cis-dihydrodiols. All the nidABEF genes were transcriptionally induced in R. opacus TKN14 by the addition of o-xylene to a mineral salt medium. It is very likely that these genes are involved in the degradation pathways of a wide range of aromatic hydrocarbons by Rhodococcus species as the first key enzyme. o-, m-and p-xylenes, which are included in petroleum, are used as solvents in the production of chemicals and drugs, as thinners for paints and enamels, and as intermediates for the synthesis of numerous chemical compounds. They are therefore widespread environmental contaminants in groundwater and soil (4,11,27). In m-and p-xylenes, numerous bacteria that are able to utilize them as the sole carbon and energy source have been isolated, and details of their catabolic pathways have been well documented. The most elucidated examples are the catabolism of toluene, m-xylene, and p-xylene with the enzymes encoded on a transmissible TOL plasmid (pWW0) from Pseudomonas putida mt-2 (38, 39). Xylene monooxygenase is the first enzyme on this degradation pathway (14, 34). This enzyme consists of two polypeptide subunits encoded by the xylM and xylA genes. XylA, the NADH acceptor reductase component (34), has been characterized as an electron transport protein transferring reducing equivalents from NADH to XylM, which is the hydroxylase component located in the membrane (18). Toluene and m-and p-xylenes are, respectively, metabolized with XylMA to benzyl alcohol and m-and p-methylbenzyl alcohol. These respective alcohols are further metabolized to benzoate and m-and p-toluates via their corresponding aldehydes b...
Soils contaminated with o-xylene were more difficult to bioremediate than those contaminated with other BTEX hydrocarbons (benzene, toluene, ethylbenzene, m-xylene and p-xylene). In order to identify microorganisms responsible for o-xylene degradation in soil, microbial community structure analyses were carried out with two soil samples in the presence of o-xylene and mineral nutrients. In two different soil samples, Rhodococcus opacus became abundant. We were also able to isolate o-xylene degrading Rhodococcus species from these soil samples. A primer set was developed to specifically detect a cluster of this Rhodococcus group including isolated Rhodococcus strains, Rhodococcus opacus and Rhodococcus koreensis. The growth of this bacterial group in an o-xylene-contaminated soil was followed by competitive PCR (cPCR). The decrease in o-xylene clearly paralleled the growth of the Rhodococcus group.
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