An aerobic fixed-phase biofilm reactor system for the degradation of the low-molecular weight aromatic compounds occurring in the effluents of anaerobic digestors treating olive mill wastewaters

An aerobic co-culture, prepared by combining Ralstonia sp. LD35 and Pseudomonas putida DSM1868, was recently found to be capable of extensively degrading many of the hydroxylated and/or methoxylated benzoic, phenylacetic and 3-phenyl-2-propenoic acids occurring in the olive mill wastewaters (OMWs). In the perspective of developing a biotechnological process for the degradation of low-molecular weight (MW) aromatic compounds occurring in the effluents of anaerobic digestors treating OMWs, the capability of this bacterial co-culture of biodegrading a synthetic mix of the above mentioned compounds and the aromatic compounds of an anaerobic OMW-treatment plant effluent in the physiological state of immobilised cells was investigated. Two aerobic fixed-bed biofilm reactors were developed by immobilising the co-culture cells on Manville silica beads and on polyurethane foam cubes. Both supports were found to give rise to a microbiologically stable and biologically active biofilm. The two biofilm reactors were found to be similarly capable of rapidly and completely biodegrading the components of a synthetic mix of nine monocyclic aromatic acids typically present in OMWs and the low-MW aromatic compounds occurring in the anaerobic effluent in batch conditions. However, in the same conditions, the silica bead-packed reactor was found to be more effective in the removal of high-MW phenolic compounds from the anaerobic effluent with respect to the polyurethane cube-packed reactor. These results are encouraging in the perspective of using the co-culture as immobilized cells for developing a continuous biotechnological process for the post-treatment of effluents with low-MW aromatic compounds produced by anaerobic digestors treating OMWs.     

Characterization of four olive-mill-wastewater indigenous bacterial strains capable of aerobically degrading hydroxylated and methoxylated monocyclic aromatic compounds

Seven aerobic bacterial strains capable of degrading several of the monocyclic aromatic compounds occurring in the phenolic fraction of olive-mill wastewaters (OMWs) were isolated from an Italian OMW. The results of the 16S rDNA restriction analysis evidenced that these strains are distributed among four different groups. One strain of each group was taxonomically characterized by sequencing the amplified 16S rDNA, and the four strains were assigned to the genera Comamonas (strain AV1A), Ralstonia (strain AV5BG), Pseudomonas (strain AV2A) and Sphingomonas (strain AV6C). The four strains, when checked for the ability to degrade nine monocyclic aromatic compounds abundant in OMWs, were found to significantly metabolize five to eight of them, both as resting cells and growing cells. Specific enzyme analyses of the same selected strains showed: (1) the occurrence of O-demethylating activities towards four methoxylated mono-aromatic acids in three of the four studied strains (strains AV1A, AV5BG and AV6C), (2) ring-cleavage activity towards protocatechuic acid in all of the strains, and (3) a ring-cleavage activity towards catechol in strain AV6C. The isolates described here exhibit a biodegradation potential towards monocyclic aromatic compounds of OMWs that is markedly broader and higher than that displayed by other aerobic bacteria described previously. These features make them excellent candidates for removing the low-molecular-weight phenolic compounds persisting in the effluent following anaerobic digestion of OMWs.     

Characterization of Ligninolytic Bacteria and Analysis of Alkali-Lignin Biodegradation Products

Ligninolytic bacteria degrading lignin were isolates and identified, and their biodegradation mechanism of alkaline-lignin was investigated. Four strains with lignin degradation capability were screened and identified from the soil, straw, and silage based on their decolorizing capacity of aniline blue and colony size on alkaline-lignin medium. The degradation ratio of Bacillus aryabhattai BY5, Acinetobacter johnsonii LN2, Acinetobacter lwoffii LN4, and Micrococcus yunnanensis CL32 have been assayed using alkaline-lignin as the unique carbon source. Further, the Lip (lignin peroxidase) and Mnp (manganese peroxidase) activities of strains were investigated. Lip activity of A. lwoffii LN4 was highest after 72 h of incubation and reached 7151.7 U · l^–1. Mnp activity of M. yunnanensis CL32 was highest after 48 h and reached 12533 U · l^–1. The analysis of alkaline-lignin degradation products by GC-MS revealed that the strains screened could utilize aromatic esters compounds such as dibutyl phthalate (DBP), and decomposite monocyclic aromatic compounds through the DBP aerobic metabolic pathway. The results indicate that B. aryabhattai BY5, A. johnsonii LN2, A. lwoffii LN4, and M. yunnanensis CL32 have high potential to degrade alkaline-lignin, and might utilize aromatic compounds by DBP aerobic metabolic pathway in the process of lignin degradation.Key words: isolation, bacteria, alkali-lignin, biodegradation products     

Continuous detoxification, transformation, and degradation of nitrophenols in upflow anaerobic sludge blanket (UASB) reactors

The anaerobic transformation and degradation of nitrophenols by granular sludge was investigated in upflow anaerobic sludge blanket (UASB) reactors continuously fed with a volatile fatty acid (VFA) mixture as the primary substrate. During the start-up, subtoxic concentrations of 2-nitrophenol (2-NP), 4-nitrophenol (4-NP), and 2, 4-dinitrophenol (2, 4-DNP) were utilized. 4-NP and 2, 4-DNP were readily converted to the corresponding aromatic amine; whereas 2-NP was converted to nonaromatic products via intermediate formation of 2-aminophenol (2-AP). These conversions led to a dramatic detoxification of the mononitrophenols because the reactors treated the nitrophenolics at the concentrations which were over 25 times higher than those that caused severe inhibition. VFA removal efficiencies greater than 99% were achieved in both reactors at loading rates greater than 11.4 g COD per liter of reactor volume per day even at volumetric loading of mononitrophenols up to 910 mg/L . d.The sludges obtained from each of the reactors at the end of the continuous experiments were assayed for their specific nitrophenol reducing activity in the presence of different primary substrates. Reduction rates of 45 and 26 mg/g volatile suspended solids per day were observed for 2-NP and 4-NP, respectively, when utilizing the VFA mixture as primary substrate. Hydrogen, an interspecies-reduced compound, and substrates that provide interspecies-reducing equivalents-such as butyrate, propionate, and ethanol stimulated nitrophenol reduction, whereas acetate and methanol did not. Anaerobic batch biodegradability tests with the 2-NP-adapted sludge revealed that its corresponding aromatic amine, 2-AP, was degraded to methane at a specific rate of 14.5 mg/g VSS . d. Acetate was observed to be the major intermediate during 2-AP degradation in the presence of a specific methanogenic inhibitor 2-bromoethanesulfonate. The results of this study indicate that UASB reactors can be applied to rapidly detoxify and, under certain circumstances, degrade nitroaromatic compounds. (c) 1996 John Wiley & Sons, Inc.     

Microbial consortia for the aerobic degradation of aromatic compounds in olive oil mill effluent

Aerobic consortia that grow on olive oil mill effluent (OOME) were obtained by enrichment. Several cultures were capable of metabolizing monoaromatic compounds, supplied as the sole carbon source at 2 g L^–1. Some consortia degraded mixtures of seven aromatics (4 g L^–1) after 1 week of incubation at 32°C. The consortia were also active against monoaromatics of the undiluted OOME. This reduced the inhibitory effect of phenolic compounds prior to the anaerobic digestion of OOME at batch scale. No inhibition of the anaerobic microbial populations was noticed with treated OOME. From the most active consortium, nine different bacterial strains were isolated and shown to grow on simple aromatic compounds. Removal of 50% of the initial chemical oxygen demand and degradation of almost all of the simple aromatics in undiluted OOME was obtained with reconstituted bacterial mixtures. A slight reduction in colouration was due to adsorption of coloured compounds to bacterial cells. Presumably, the consortia could not reduce and degrade the coloured compounds in OOME.     

Biodegradation of Oil Tank Bottom Sludge using Microbial Consortia

We present a rationale for the selection of a microbial consortia specifically adapted to degrade toxic components of oil refinery tank bottom sludge (OTBS). Sources such as polluted soils, petrochemical waste, sludge from refinery-wastewater plants, and others were used to obtain a collection of eight microorganisms, which were individually tested and characterized to analyze their degradative capabilities on different hydrocarbon families. After initial experiments using mixtures of these strains, we developed a consortium consisting of four microorganisms (three bacteria and one yeast) selected in the basis of their cometabolic effects, emulsification properties, colonization of oil components, and degradative capabilities. Although the specific contribution each of the former parameters makes is not clearly understood, the activity of the four-member consortium had a strong impact not only on linear alkane degradation (100%), but also on the degradation of cycloalkanes (85%), branched alkanes (44%), and aromatic and sulphur–aromatic compounds (31–55%). The effectiveness of this consortium was significantly superior to that obtained by individual strains, commercial inocula or an undefined mixture of culturable and non-culturable microorganisms obtained from OTBS-polluted soil. However, results were similar when another consortium of four microorganisms, previously isolated in the same OTBS-polluted soil, was assayed.     

Two distinct pathways for anaerobic degradation of aromatic compounds in the denitrifying bacterium Thauera aromatica strain AR-1

Denitrifying bacteria degrade many different aromatic compounds anaerobically via the well-described benzoyl-CoA pathway. We have shown recently that the denitrifiers Azoarcus anaerobius and Thauera aromatica strain AR-1 use a different pathway for anaerobic degradation of resorcinol (1,3-dihydroxybenzene) and 3,5-dihydroxybenzoate, respectively. Both substrates are converted to hydroxyhydroquinone (1,2,4-trihydroxybenzene). In the membrane fraction of T. aromatica strain AR-1 cells grown with 3,5-dihydroxybenzoate, a hydroxyhydroquinone-dehydrogenating activity of 74 nmol min(-1)(mg protein)-1 was found. This activity was significantly lower in benzoate-grown cells. Benzoate-grown cells were not induced for degradation of 3,5-dihydroxybenzoate, and cells grown with 3,5-dihydroxybenzoate degraded benzoate only at a very low rate. With a substrate mixture of benzoate plus 3,5-dihydroxybenzoate, the cells showed diauxic growth. Benzoate was degraded first, while complete degradation of 3,5-dihydroxybenzoate occurred only after a long lag phase. The 3,5-dihydroxybenzoate-oxidizing and the hydroxyhydroquinone-dehydrogenating activities were fully induced only during 3,5-dihydroxybenzoate degradation. Synthesis of benzoyl-CoA reductase appeared to be significantly lower in 3,5-dihydroxybenzoate-grown cells as shown by immunoblotting. These results confirm that T. aromatica strain AR-1 harbors, in addition to the benzoyl-CoA pathway, a second, mechanistically distinct pathway for anaerobic degradation of aromatic compounds. This pathway is inducible and subject to catabolite repression by benzoate.     

Proteome analysis of Pseudomonas sp. K82 biodegradation pathways

Pseudomonas sp. K82 is a soil bacterium that can degrade and use monocyclic aromatic compounds including aniline, 3-methylaniline, 4-methylaniline, benzoate and p-hydroxybenzoate as its sole carbon and energy sources. In order to understand the impact of these aromatic compounds on metabolic pathways in Pseudomonas sp. K82, proteomes obtained from cultures exposed to different substrates were displayed by two-dimensional gel electrophoresis and were compared to search for differentially induced metabolic enzymes. Column separations of active fractions were performed to identify major biodegradation enzymes. More than thirty proteins involved in biodegradation and other types of metabolism were identified by electrospray ionization-quadrupole time of flight mass spectrometry. The proteome analysis suggested that Pseudomonas sp. K82 has three main metabolic pathways to degrade these aromatic compounds and induces specific metabolic pathways for each compound. The catechol 2,3-dioxygenase (CD2,3) pathway was the major pathway and the catechol 1,2-dioxygenase (beta-ketoadipate) pathway was the secondary pathway induced by aniline (aniline analogues) exposure. On the other hand, the catechol 1,2-dioxygenase pathway was the major pathway induced by benzoate exposure. For the degradation of p-hydroxybenzoate, the protocatechuate 4,5-dioxygenase pathway was the major degradation pathway induced. The nuclear magnetic resonance analysis of substrates demonstrated that Pseudomonas sp. K82 metabolizes some aromatic compounds more rapidly than others (benzoate > p-hydroxybenzoate > aniline) and that when combined, p-hydroxybenzoate metabolism is repressed by the presence of benzoate or aniline. These results suggest that proteome analysis can be useful in the high throughput study of bacterial metabolic pathways, including that of biodegradation, and that inter-relationships exist with respect to the metabolic pathways of aromatic compounds in Pseudomonas sp. K82.     

Hemicellulose-degrading bacteria and yeasts from the termite gut

Termites play a major role in the recycling of photosynthetically fixed carbon. With the aid of their symbiotic intestinal flora, they are able to degrade extensively wood constituents such as cellulose and hemicellulose. Nevertheless, the microbial species involved in the degradation of hemicelluloses are poorly defined. The purpose of this paper was to examine the microflora involved in hemicellulose degradation. Different aerobic and facultatively anaerobic bacteria and yeasts were isolated using xylan, arabinogalactan and carboxymethylcellulose as substrates. Gram-positive isolates belonged to the genera Bacillus, Paenibacillus, Streptomyces or the actinobacteria group, while the Gramnegative strains were assigned to the genera Pseudomonas, Acinetobacter, Ochrobactrum , and to genera belonging to the family Enterobacteriaceae. The spectrum and activity of xylan- and arabinogalactan-hydrolysing glycosidases of these new isolates, together with additional bacterial strains originally obtained from enrichments with aromatic compounds were determined.     

Degradation of organic contaminants found in organic waste

In recent years, great interest has arisen in recycling of the waste created by modern society. A common way of recycling the organic fraction is amendment on farmland. However, these wastes may contain possible hazardous components in small amounts, which may prevent their use in farming. The objective of our study has been to develop biological methods by which selected organic xenobiotic compounds can be biotransformed by anaerobic or aerobic treatment. Screening tests assessed the capability of various inocula to degrade two phthalates di-n-butylphthalate, and di(2-ethylhexyl)phthalate, five polycyclic aromatic hydrocarbons, linear alkylbenzene sulfonates and three nonylphenol ethoxylates under aerobic and anaerobic conditions. Under aerobic conditions, by selecting the appropriate inoculum most of the selected xenobiotics could be degraded. Aerobic degradation of di(2-ethylhexyl)phthalate was only possible with leachate from a landfill as inoculum. Anaerobic degradation of some of the compounds was also detected. Leachate showed capability of degrading phthalates, and anaerobic sludge showed potential for degrading, polycyclic aromatic hydrocarbons, linear alkylbenzene sulfonates and nonyl phenol ethoxylates. The results are promising as they indicate that a great potential for biological degradation is present, though the inoculum containing the microorganisms capable of transforming the recalcitrant xenobiotics has to be chosen carefully.     

Biodegradation of the mixture of 2,4,6-trichlorophenol, 4-chlorophenol,and phenol by a defined mixed culture

Two new strains, Pseudomonas sp. TCP114 degrading 2,4,6-trichlorophenol (TCP) and Arthrobacter sp. CPR706 degrading 4-chlorophenol (4-CP), were isolated through a selective enrichment procedure. Both strains could also degrade phenol. The degradability of one component by a pure culture was strongly affected by the presence of other compounds in the medium. For example, when all three components (TCP, 4-CP, and phenol) were present in the medium, a pure culture of CPR706 could not degrade any of the components present. This restriction on degradability could be overcome by employing a defined mixed culture of the two strains. The mixed culture could degrade all three components in the mixture through cooperative activity. It was also demonstrated that the mixed culture could be immobilized by using calcium alginate for the semi-continuous degradation of the three-component mixture. Immobilization not only accelerates the degradation rate, but also enables reuse of the cell mass several times without losing the cells' degrading capabilities.     

Anaerobic degradation of non-substituted aromatic hydrocarbons

Aromatic hydrocarbons are among the most prevalent organic pollutants in the environment. Their removal from contaminated systems is of great concern because of the high toxicity effect on living organisms including humans. Aerobic degradation of aromatic hydrocarbons has been intensively studied and is well understood. However, many aromatics end up in habitats devoid of molecular oxygen. Nevertheless, anaerobic degradation using alternative electron acceptors is much less investigated. Here, we review the recent literature and very early progress in the elucidation of anaerobic degradation of non-substituted monocyclic (i.e. benzene) and polycyclic aromatic hydrocarbons (PAH such as naphthalene and phenanthrene). A focus will be on benzene and naphthalene as model compounds. This review concerns the microbes involved, the biochemistry of the initial activation and subsequent enzyme reactions involved in the pathway.     

Diversity and correlation of specific aromatic hydrocarbon biodegradation capabilities

This work investigated the biodegradation capabilitiesof indigenous microorganisms exposed to differentcombinations of aromatic hydrocarbons. Considerablediversity was found in the catabolic specificity of 55strains. Toluene was the most commonly degradedcompound, followed by p-xylene, m-xyleneand ethylbenzene. Strains capable of degradingo-xylene and benzene, which were theleast-frequently-degraded compounds, exhibited broaderbiodegradation capabilities. Kappa statistics showeda significant correlation between the abilities todegrade toluene and ethylbenzene, p-xylene andm-xylene, and p-xylene and o-xylene. The ability to degrade naphthalene was correlated tothe ability to degrade other alkylbenzenes, but notbenzene. In addition, the inability to degradebenzene was correlated to the inability to degradeo-xylene. Factorial analysis of variance showedthat biodegradation capabilities were generallybroader when aromatic hydrocarbons were fed asmixtures than when fed separately. Beneficialsubstrate interactions included enhanced degradationof benzene, p-xylene, and naphthalene whentoluene was present, and enhanced degradation ofnaphthalene by ethylbenzene. Such heuristicrelationships may be useful to predict biodegradationpatterns when bacteria are exposed to differentaromatic hydrocarbon mixtures.     

Anaerobic and aerobic metabolism of diverse aromatic compounds by the photosynthetic bacterium Rhodopseudomonas palustris.

The purple nonsulfur photosynthetic bacterium Rhodopseudomonas palustris used diverse aromatic compounds for growth under anaerobic and aerobic conditions. Many phenolic, dihydroxylated, and methoxylated aromatic acids, as well as aromatic aldehydes and hydroaromatic acids, supported growth of strain CGA001 in both the presence and absence of oxygen. Some compounds were metabolized under only aerobic or under only anaerobic conditions. Two other strains, CGC023 and CGD052, had similar anaerobic substrate utilization patterns, but CGD052 was able to use a slightly larger number of compounds for growth. These results show that R. palustris is far more versatile in terms of aromatic degradation than had been previously demonstrated. A mutant (CGA033) blocked in aerobic aromatic metabolism remained wild type with respect to anaerobic degradative abilities, indicating that separate metabolic pathways mediate aerobic and anaerobic breakdown of diverse aromatics. Another mutant (CGA047) was unable to grow anaerobically on either benzoate or 4-hydroxybenzoate, and these compounds accumulated in growth media when cells were grown on more complex aromatic compounds. This indicates that R. palustris has two major anaerobic routes for aromatic ring fission, one that passes through benzoate and one that passes through 4-hydroxybenzoate.     

微生物降解石油烃的功能基因研究进展

微生物对石油烃的降解在自然衰减去除土壤和地下水石油烃污染的过程中发挥了重要作用。微生物通过其产生的一系列酶来利用和降解这类有机污染物,其中,编码关键降解酶的基因称为功能基因。功能基因可作为生物标志物用于分析环境中石油烃降解基因的多样性。因此,研究石油降解功能基因是分析土著微生物群落多样性、评价自然衰减潜力与构建基因工程菌的重要基础。本文主要介绍了烷烃和芳香烃在有氧和无氧条件下的微生物降解途径,重点总结了烷烃和芳香烃降解的主要功能基因及其作用,包括参与羟化作用的单加氧酶和双加氧酶基因、延胡索酸加成反应的琥珀酸合酶基因以及中心中间产物的降解酶基因等。     

Degradation of natural and synthetic polyesters under anaerobic conditions

Often, degradability under anaerobic conditions is desirable for plastics claimed to be biodegradable, e.g. in anaerobic biowaste treatment plants, landfills and in natural anaerobic sediments. The biodegradation of the natural polyesters poly(beta-hydroxybutyrate) (PHB), poly(beta-hydroxybutyrate-co-11.6%-beta-hydroxyvalerate) (PHBV) and the synthetic polyester poly(epsilon-caprolactone) (PCL) was studied in two anaerobic sludges and individual polyester degrading anaerobic strains were isolated, characterized and used for degradation experiments under controlled laboratory conditions. Incubation of PHB and PHBV films in two anaerobic sludges exhibited significant degradation in a time scale of 6-10 weeks monitored by weight loss and biogas formation. In contrast to aerobic conditions, PHB was degraded anaerobically more rapidly than the copolyester PHBV, when tested with either mixed cultures or a single strained isolate. PCL tends to degrade slower than the natural polyesters PHB and PHBV. Four PHB and PCL degrading isolates were taxonomically identified and are obviously new species belonging to the genus Clostridium group I. The depolymerizing enzyme systems of PHB and PCL degrading isolates are supposed to be different. Using one isolated strain in an optimized laboratory degradation test with PHB powder, the degradation time was drastically reduced compared to the degradation in sludges (2 days vs. 6-10 weeks).     

Anaerobic degradation of dehydrodiisoeugenol by rumen bacteria

The degradation of dehydrodiisoeugenol (DDIE) by cow rumen bacteria was studied under strictly anaerobic conditions. After two days of cultivation, about 23% of DDIE (1.2 mM) was degraded to volatile fatty acids (VFA) such as acetic acid, propionic acid and butyric acid. The aromatic intermediates were vanillic acid, 5-methylvanillin and 3-methyl-4-hydroxybenzaldehyde, which suggested that the coumaran ring in DDIE was cleaved during degradation. These results indicate that the rumen anaerobes can degrade this lignin-related dimer to monoaromatic compounds and VFA.     

Isolation and characterization of an anaerobic dehydrodivanillin-degrading bacterium

A novel, strictly anaerobic, gram-negative, non-spore-forming, fusiform, rod-shaped bacterium having high dehydrodivanillin (DDV)-degrading activity was isolated from cow ruminal fluid. This strain degraded a range of six main lignin-related compounds such as DDV, ferulic acid, dehydrodiisoeugenol, guaiacoxyacetic acid, vanillin, and veratrylglycerol-beta-guaiacyl ether to the extent of 14 to 83% within 2 days under strictly anaerobic conditions. As DDV degradation intermediates, three aromatic compounds (dehydrodivanillic acid, vanillic acid, and 5-carboxyvanillic acid) and two alicyclic compounds (cyclohexanecarboxylic acid and cyclohexanol) were detected by thin-layer, high-performance liquid, and gas chromatography and mass spectrometry. The addition of 1% glucose and peptone in a synthetic medium stimulated growth of the strain but slowed down DDV degradation. The presence of 0.1% yeast extract increased both cell growth and DDV degradation. The growth yield in defined medium was 151.5 g (dry weight) of cells per mol of DDV utilized. Characterization of the strain indicated that it was distinct from known Fusobacterium and Clostridium species. The bacterium was easily induced to form protoplasts after treatment with either penicillin or lysozyme. The frequencies of protoplast formation and regeneration in the strain were 94 and 18%, respectively.     

Isolation and characterization of an anaerobic dehydrodivanillin-degrading bacterium.

A novel, strictly anaerobic, gram-negative, non-spore-forming, fusiform, rod-shaped bacterium having high dehydrodivanillin (DDV)-degrading activity was isolated from cow ruminal fluid. This strain degraded a range of six main lignin-related compounds such as DDV, ferulic acid, dehydrodiisoeugenol, guaiacoxyacetic acid, vanillin, and veratrylglycerol-beta-guaiacyl ether to the extent of 14 to 83% within 2 days under strictly anaerobic conditions. As DDV degradation intermediates, three aromatic compounds (dehydrodivanillic acid, vanillic acid, and 5-carboxyvanillic acid) and two alicyclic compounds (cyclohexanecarboxylic acid and cyclohexanol) were detected by thin-layer, high-performance liquid, and gas chromatography and mass spectrometry. The addition of 1% glucose and peptone in a synthetic medium stimulated growth of the strain but slowed down DDV degradation. The presence of 0.1% yeast extract increased both cell growth and DDV degradation. The growth yield in defined medium was 151.5 g (dry weight) of cells per mol of DDV utilized. Characterization of the strain indicated that it was distinct from known Fusobacterium and Clostridium species. The bacterium was easily induced to form protoplasts after treatment with either penicillin or lysozyme. The frequencies of protoplast formation and regeneration in the strain were 94 and 18%, respectively.     

Substrate interactions during aerobic biodegradation of benzene

This study dealt with the interactions with benzene degradation of the following aromatic compounds in a mixed substrate: toluene, o-xylene, naphthalene, 1,4-dimethylnaphthalene, phenanthrene, and pyrrole. The experiment was performed as a factorial experiment with simple batch cultures. The effect of two different types of inocula was tested. One type of inoculum was grown on a mixture of aromatic hydrocarbons; the other was grown on a mixture of aromatic hydrocarbons and nitrogen-, sulfur-, and oxygen-containing aromatic compounds (NSO compounds), similar to some of the compounds identified in creosote waste. The culture grown on the aromatic hydrocarbons and NSO compounds was much less efficient in degrading benzene than the culture grown on only aromatic hydrocarbons. The experiments indicated that toluene- and o-xylene-degrading bacteria are also able to degrade benzene, whereas naphthalene-, 1,,4-dimethylnaphthalene-, and phenanthrene-degrading bacteria have no or very little benzene-degrading ability. Surprisingly, the stimulating effect of toluene and o-xylene was true only if the two compounds were present alone. In combination an antagonistic effect was observed, i.e., the combined effect was smaller than the sum from each of the compounds. The reason for this behavior has not been identified. Pyrrole strongly inhibited benzene degradation even at concentrations of about 100 to 200 micrograms/liter. Future studies will investigate the generality of these findings.