Microbial Communities Associated with Anaerobic Benzene Degradation in a Petroleum-Contaminated Aquifer

Microbial community composition associated with benzene oxidation under in situ Fe(III)-reducing conditions in a petroleum-contaminated aquifer located in Bemidji, Minn., was investigated. Community structure associated with benzene degradation was compared to sediment communities that did not anaerobically oxidize benzene which were obtained from two adjacent Fe(III)-reducing sites and from methanogenic and uncontaminated zones. Denaturing gradient gel electrophoresis of 16S rDNA sequences amplified with bacterial or Geobacteraceae-specific primers indicated significant differences in the composition of the microbial communities at the different sites. Most notable was a selective enrichment of microorganisms in the Geobacter cluster seen in the benzene-degrading sediments. This finding was in accordance with phospholipid fatty acid analysis and most-probable-number–PCR enumeration, which indicated that members of the family Geobacteraceae were more numerous in these sediments. A benzene-oxidizing Fe(III)-reducing enrichment culture was established from benzene-degrading sediments and contained an organism closely related to the uncultivated Geobacter spp. This genus contains the only known organisms that can oxidize aromatic compounds with the reduction of Fe(III). Sequences closely related to the Fe(III) reducer Geothrix fermentans and the aerobe Variovorax paradoxus were also amplified from the benzene-degrading enrichment and were present in the benzene-degrading sediments. However, neither G. fermentans nor V. paradoxus is known to oxidize aromatic compounds with the reduction of Fe(III), and there was no apparent enrichment of these organisms in the benzene-degrading sediments. These results suggest that Geobacter spp. play an important role in the anaerobic oxidation of benzene in the Bemidji aquifer and that molecular community analysis may be a powerful tool for predicting a site’s capacity for anaerobic benzene degradation.     

Attack on Lignified Grass Cell Walls by a Facultatively Anaerobic Bacterium

A filamentous, facultatively anaerobic microorganism that attacked lignified tissue in forage grasses was isolated from rumen fluid with a Bermuda grass-containing anaerobic medium in roll tubes. The microbe, designated 7-1, demonstrated various colony and cellular morphologies under different growth conditions. Scanning electron microscopy revealed that 7-1 attacked lignified cell walls in aerobic and anaerobic culture. 7-1 predominately degraded tissues reacting positively for lignin with the chlorine-sulfite stain (i.e., sclerenchyma in leaf blades and parenchyma in stems) rather than the more resistant acid phloroglucinol-positive tissues (i.e., lignified vascular tissue and sclerenchyma ring in stems), although the latter tissues were occasionally attacked. Turbidimetric tests showed that 7-1 in anaerobic culture grew optimally at 39°C at a pH of 7.4 to 8.0. Tests for growth on plant cell wall carbohydrates showed that 7-1 grew on xylan and pectin slowly in aerobic cultures but not with pectin and only slightly with xylan in anaerobic culture. 7-1 was noncellulolytic as shown by filter paper tests. The microbe used the phenolic acids sinapic, ferulic, and p-coumaric acids as substrates for growth; the more highly methoxylated acids were used more effectively.     

Spirochaeta aurantia, a Pigmented, Facultatively Anaerobic Spirochete

A strain of Spirochaeta aurantia was isolated from mud by a procedure involving migration of the organisms through cellulose ester filter discs (0.3-mum pore diameter) onto the surface of culture plates. The helical cells measured 0.3 by 10 to 20 mum during exponential growth. Electron microscopy showed the presence of two subterminally inserted axial fibrils partially overlapping in a 1-2-1 arrangement. An outer envelope, exhibiting a polygonal substructure, was observed. The spirochete grew either aerobically or anaerobically, with aerobic yields of 9.8 x 10(8) cells per ml and anaerobic yields of 3.0 x 10(8) cells per ml. The organism used carbohydrates, but not amino acids, as energy sources. Amino acids served as sole nitrogen sources, whereas inorganic ammonium salts did not. The presence of biotin and thiamine in the medium was required for growth. Growing cells fermented maltose mainly to carbon dioxide, hydrogen, ethyl alcohol, and acetic acid. Small amounts of formic and lactic acids, acetoin, and diacetyl were produced. Cells of S. aurantia growing aerobically produced a yellow-orange pigment. Chemical analysis indicated that the pigment was carotenoid in nature, its main component being lycopene or a similar compound. S. aurantia is not closely related to the leptospires, since it lacks both the hemolytic antigen and the hooked ends typical of the latter organisms. Furthermore, the guanine plus cytosine content in the deoxyribonucleic acid of S. aurantia (66.8 moles%) differs drastically from that of leptospires.     

Dissimilatory Reduction of Inorganic Sulfur by Facultatively Anaerobic Marine Bacteria

THREE STRAINS, SELECTED FROM A LARGE NUMBER OF NEWLY ISOLATED, FACULTATIVELY ANAEROBIC MARINE BACTERIA, REDUCED INORGANIC SULFUR COMPOUNDS OTHER THAN SULFATE ANAEROBICALLY IN DEFINED CULTURE MEDIA IN THE FOLLOWING DIFFERENT PATTERNS: (i) sulfite and thiosulfate were reduced to sulfide, and tetrathionate was reduced to thiosulfate; (ii) tetrathionate was reduced to thiosulfate only; or (iii) thiosulfate was reduced to sulfide only when pyruvate was the substrate. Comparison of anaerobic growth in the presence or absence of inorganic sulfur compounds indicated true dissimilatory reductions.     

Anaerobic Benzene Degradation in Petroleum-Contaminated Aquifer Sediments after Inoculation with a Benzene-Oxidizing Enrichment

Sediments from the sulfate-reduction zone of a petroleum-contaminated aquifer, in which benzene persisted, were inoculated with a benzene-oxidizing, sulfate-reducing enrichment from aquatic sediments. Benzene was degraded, with apparent growth of the benzene-degrading population over time. These results suggest that the lack of benzene degradation in the sulfate-reduction zones of some aquifers may result from the failure of the appropriate benzene-degrading sulfate reducers to colonize the aquifers rather than from environmental conditions that are adverse for anaerobic benzene degradation.     

Cultivation of Anaerobic and Facultatively Anaerobic Bacteria from Spacecraft-Associated Clean Rooms

In the course of this biodiversity study, the cultivable microbial community of European spacecraft-associated clean rooms and the Herschel Space Observatory located therein were analyzed during routine assembly operations. Here, we focused on microorganisms capable of growing without oxygen. Anaerobes play a significant role in planetary protection considerations since extraterrestrial environments like Mars probably do not provide enough oxygen for fully aerobic microbial growth. A broad assortment of anaerobic media was used in our cultivation strategies, which focused on microorganisms with special metabolic skills. The majority of the isolated strains grew on anaerobic, complex, nutrient-rich media. Autotrophic microorganisms or microbes capable of fixing nitrogen were also cultivated. A broad range of facultatively anaerobic bacteria was detected during this study and also, for the first time, some strictly anaerobic bacteria (Clostridium and Propionibacterium) were isolated from spacecraft-associated clean rooms. The multiassay cultivation approach was the basis for the detection of several bacteria that had not been cultivated from these special environments before and also led to the discovery of two novel microbial species of Pseudomonas and Paenibacillus.The major issue of planetary protection is to prevent the contamination of extraterrestrial environments by terrestrial biomolecules and life forms. Furthermore, reverse contamination of Earth by extraterrestrial material is also a fundamental concern (1). In order not to affect or even to confound future life detection missions on celestial bodies, which are of interest for their chemical and biological evolution, spacecraft are constructed in so-called clean rooms and are subject to severe cleaning processes and microbiological controls before launch (9). Therefore, these clean rooms are considered extreme environments for microorganisms (47).Detailed planetary protection protocols for missions to Mars were designed for the Viking missions, which were launched in 1975, and about 7,000 samples were taken from the two Viking spacecraft during prelaunch activities in order to determine the cultivable microbial load (37). Besides human-associated bacteria (pathogens and opportunistic pathogens), which were predominant among the microbes detected in these samples, aerobic spore-forming microorganisms (Bacillus) were found frequently on spacecraft and within the facilities.Spores are the resting states of bacteria and are often highly resistant to heat, desiccation, and other abiotic stresses. These multiresistance properties of such spore-forming microorganisms make them perfect candidates for surviving a space flight, and thus, the main focus of attention has been on them. Furthermore, only the detection of aerobic spore-forming bacteria is currently included in space agencies'' planetary protection protocols for the quantitative determination of microbial burden on spacecraft.The presence of extraordinarily (UV-) resistant spores in spacecraft facilities has been reported (31), but it also has been proven that vegetative microbial cells (e.g., Deinococcus radiodurans and Halobacterium sp. strain NRC-1) can resist very harsh conditions, such as extreme doses of (UV and ionizing) radiation and desiccation (8, 11). Recent culture-based and molecular studies have shown that the microbial diversity on spacecraft and within the clean rooms is extraordinarily high and does include extremotolerant bacteria and even archaea (25, 30).The atmospheres of most planets and bodies within the reach of human exploration contain only traces of oxygen (Mars contains 0.13%), probably not enough to support terrestrial aerobic life as we know it (26, 44). Even though Mars'' surface is highly oxidizing and radiation exposed, the Martian subsurface, as well as those of other planets and bodies (like, e.g., Titan), has been discussed as an anaerobic biotope for possible life (4, 40).Therefore, the lack of studies of the existence of anaerobically growing microorganisms in spacecraft-associated clean rooms is quite surprising. One possible reason for this discrepancy might be that the cultivation of anaerobes is challenging. Already in 1969, Hungate published a method for the cultivation of strictly anaerobic methanogenic Archaea (20). Although this technique has undergone a few simplifications during past decades, the cultivation of anaerobes requires specialized and expensive equipment (e.g., anaerobic glove boxes and gas stations), practical experience, and skills in specific methodology. Nevertheless, by the application of anaerobic cultivation strategies, many fascinating microorganisms—such as Nanoarchaeum equitans, the first representative of the new archaeal phylum Nanoarchaeota, or Thermotoga maritima, a hyperthermophilic bacterium growing at up to 90°C (17, 18)—have successfully been isolated from diverse and sometimes extreme biotopes.Generally, there are different types of anaerobic organisms. Facultative anaerobes (like Escherichia coli) are able to adapt their metabolism and can grow under conditions with or without oxygen but prefer aerobic conditions. Aerotolerant anaerobes do not need oxygen for their growth and show no preference, and strict anaerobes (e.g., methanogens) never require oxygen for their reproduction and metabolism. Even more, obligate (strict) anaerobes can be growth inhibited or even killed by oxygen.The presence of anaerobic microorganisms (enriched using the BD GasPak system) in surface samples from U.S. clean rooms has rarely been reported. Members of the facultatively anaerobic genera Paenibacillus and Staphylococcus have been isolated in the course of a study about extremotolerant microorganisms (25). During molecular surveys of U.S. clean rooms, the 16S rRNA genes from strictly anaerobic microorganisms, such as the spore-forming genus Clostridium, have already been detected (29). Nevertheless, the cultivation of these microbes has not yet been successful.With the ExoMars mission impending, the European Space Agency (ESA) is organizing and funding a biodiversity study of the ESA''s clean rooms and the spacecraft therein. The microbiology of these special environments is characterized in detail by a combination of standard procedures, new cultivation approaches, and molecular methods that shall illuminate the presence of planetary protection-relevant microorganisms in these facilities. At the date of sampling, all the clean rooms harbored the Herschel Space Observatory, a spacecraft to be launched together with the Planck satellite in spring 2009, as of this writing. Herschel will be fitted with the largest mirror ever built for a space mission (3.5 m in diameter), and its main goal will be the exploration of the cold universe, i.e., the formation and evolution of proto-galaxies (35). The Herschel Space Observatory does not demand planetary protection requirements, but all clean rooms were in a fully operating state during the construction work. This gave us the opportunity to sample the microbial diversity in these extreme environments without bioburden control but under strict contamination-controlled conditions, with respect to particulates and molecular contamination.This paper presents the results from our attempts to isolate anaerobic and facultatively anaerobic microorganisms from samples of spacecraft and surfaces in European spacecraft-associated clean rooms. For this purpose, we have successfully applied Hungate technology for anaerobic culturing and used an assortment of noncommercial media for the cultivation of a broad variety of microorganisms. Besides the capability of anaerobic growth, many of our isolates revealed special physiological capacities (e.g., nitrogen fixation and autotrophic metabolism) that might be relevant for further planetary protection considerations.     

Anaerobic Benzene Oxidation by Geobacter Species

The abundance of Geobacter species in contaminated aquifers in which benzene is anaerobically degraded has led to the suggestion that some Geobacter species might be capable of anaerobic benzene degradation, but this has never been documented. A strain of Geobacter, designated strain Ben, was isolated from sediments from the Fe(III)-reducing zone of a petroleum-contaminated aquifer in which there was significant capacity for anaerobic benzene oxidation. Strain Ben grew in a medium with benzene as the sole electron donor and Fe(III) oxide as the sole electron acceptor. Furthermore, additional evaluation of Geobacter metallireducens demonstrated that it could also grow in benzene-Fe(III) medium. In both strain Ben and G. metallireducens the stoichiometry of benzene metabolism and Fe(III) reduction was consistent with the oxidation of benzene to carbon dioxide with Fe(III) serving as the sole electron acceptor. With benzene as the electron donor, and Fe(III) oxide (strain Ben) or Fe(III) citrate (G. metallireducens) as the electron acceptor, the cell yields of strain Ben and G. metallireducens were 3.2 × 10⁹ and 8.4 × 10⁹ cells/mmol of Fe(III) reduced, respectively. Strain Ben also oxidized benzene with anthraquinone-2,6-disulfonate (AQDS) as the sole electron acceptor with cell yields of 5.9 × 10⁹ cells/mmol of AQDS reduced. Strain Ben serves as model organism for the study of anaerobic benzene metabolism in petroleum-contaminated aquifers, and G. metallireducens is the first anaerobic benzene-degrading organism that can be genetically manipulated.     

Anaerobic Degradation of Benzene, Toluene, Ethylbenzene, and Xylene Compounds by Dechloromonas Strain RCB

Dechloromonas strain RCB has been shown to be capable of anaerobic degradation of benzene coupled to nitrate reduction. As a continuation of these studies, the metabolic versatility and hydrocarbon biodegradative capability of this organism were investigated. The results of these revealed that in addition to nitrate, strain RCB could alternatively degrade benzene both aerobically and anaerobically with perchlorate or chlorate [(per)chlorate] as a suitable electron acceptor. Furthermore, with nitrate as the electron acceptor, strain RCB could also utilize toluene, ethylbenzene, and all three isomers of xylene (ortho-, meta-, and para-) as electron donors. While toluene and ethylbenzene were completely mineralized to CO₂, strain RCB did not completely mineralize para-xylene but rather transformed it to some as-yet-unidentified metabolite. Interestingly, with nitrate as the electron acceptor, strain RCB degraded benzene and toluene concurrently when the hydrocarbons were added as a mixture and almost 92 μM total hydrocarbons were oxidized within 15 days. The results of these studies emphasize the unique metabolic versatility of this organism, highlighting its potential applicability to bioremediative technologies.     

Anaerobic Degradation of m-Cresol by a Sulfate-Reducing Bacterium

m-Cresol metabolism under sulfate-reducing conditions was studied with a pure culture of Desulfotomaculum sp. strain Groll. Previous studies with a sulfate-reducing consortium indicated that m-cresol was degraded via an initial para-carboxylation reaction. However, 4-hydroxy-2-methylbenzoic acid was not degraded by strain Groll, and no evidence for ring carboxylation of m-cresol was found. Strain Groll readily metabolized the putative metabolites of a methyl group oxidation pathway, including 3-hydroxybenzyl alcohol, 3-hydroxybenzaldehyde, 3-hydroxybenzoic acid, and benzoic acid. Degradation of these compounds preceded and inhibited m-cresol decay. 3-Hydroxybenzoic acid was detected in cultures that received either m-cresol or 3-hydroxybenzyl alcohol, and trace amounts of benzoic acid were detected in m-cresol-degrading cultures. Therefore, we propose that strain Groll metabolizes m-cresol by a methyl group oxidation pathway which is an alternate route for the catabolism of this compound under sulfate-reducing conditions.     

Anaerobic Naphthalene Degradation by a Sulfate-Reducing Enrichment Culture

Anaerobic naphthalene degradation by a sulfate-reducing enrichment culture was studied by substrate utilization tests and identification of metabolites by gas chromatography-mass spectrometry. In substrate utilization tests, the culture was able to oxidize naphthalene, 2-methylnaphthalene, 1- and 2-naphthoic acids, phenylacetic acid, benzoic acid, cyclohexanecarboxylic acid, and cyclohex-1-ene-carboxylic acid with sulfate as the electron acceptor. Neither hydroxylated 1- or 2-naphthoic acid derivatives and 1- or 2-naphthol nor the monoaromatic compounds ortho-phthalic acid, 2-carboxy-1-phenylacetic acid, and salicylic acid were utilized by the culture within 100 days. 2-Naphthoic acid accumulated in all naphthalene-grown cultures. Reduced 2-naphthoic acid derivatives could be identified by comparison of mass spectra and coelution with commercial reference compounds such as 1,2,3,4-tetrahydro-2-naphthoic acid and chemically synthesized decahydro-2-naphthoic acid. 5,6,7,8-Tetrahydro-2-naphthoic acid and octahydro-2-naphthoic acid were tentatively identified by their mass spectra. The metabolites identified suggest a stepwise reduction of the aromatic ring system before ring cleavage. In degradation experiments with [1-¹³C]naphthalene or deuterated D₈-naphthalene, all metabolites mentioned derived from the introduced labeled naphthalene. When a [¹³C]bicarbonate-buffered growth medium was used in conjunction with unlabeled naphthalene, ¹³C incorporation into the carboxylic group of 2-naphthoic acid was shown, indicating that activation of naphthalene by carboxylation was the initial degradation step. No ring fission products were identified.     

Anaerobic Degradation of p-Xylene by a Sulfate-Reducing Enrichment Culture

A strictly anaerobic enrichment culture was obtained with p-xylene as organic substrate and sulfate as electron acceptor from an aquifer at a former gasworks plant contaminated with aromatic hydrocarbons. p-Xylene was completely oxidized to CO₂. The enrichment culture depended on Fe(II) in the medium as a scavenger of the produced sulfide. 4-Methylbenzylsuccinic acid and 4-methylphenylitaconic acid were identified in supernatants of cultures indicating that degradation of p-xylene was initiated by fumarate addition to one of the methyl groups. Therefore, p-xylene degradation probably proceeds analogously to toluene degradation by Thauera aromatica or anaerobic degradation pathways for o- and m-xylene.     

Degradation of 4-Chlorobenzoate by Facultatively Alkalophilic Arthrobacter sp. Strain SB8

A facultative alkalophile capable of utilizing 4-chlorobenzoate (4-CBA), strain SB8, was isolated from soil with an alkaline medium (pH 10.0) containing the haloaromatic compound as the carbon source. The strain, identified as an Arthrobacter sp., showed rather extensive 4-CBA-degrading ability. 4-CBA utilization by the strain was possible in the alkaline medium containing up to 10 g of the compound per liter. The 4-CBA-dechlorinating activity of resting cells was almost completely uninhibited by substrate concentrations up to 150 mM. The bacterium dehalogenated 4-CBA in the initial stage of the degradation and metabolized the compound via 4-hydroxybenzoate and protocatechuate. O₂ was needed for 4-CBA dechlorination by resting cells but not by cell extracts. O₂ was inhibitory to the 4-CBA dechlorination activity of cell extracts. These facts suggest dechlorination of 4-CBA by halide hydrolysis and an energy requirement for the transport of 4-CBA into cells.     

Anaerobic Degradation of 2-Methylnaphthalene by a Sulfate-Reducing Enrichment Culture

Anaerobic degradation of 2-methylnaphthalene was investigated with a sulfate-reducing enrichment culture. Metabolite analyses revealed two groups of degradation products. The first group comprised two succinic acid adducts which were identified as naphthyl-2-methyl-succinic acid and naphthyl-2-methylene-succinic acid by comparison with chemically synthesized reference compounds. Naphthyl-2-methyl-succinic acid accumulated to 0.5 μM in culture supernatants. Production of naphthyl-2-methyl-succinic acid was analyzed in enzyme assays with dense cell suspensions. The conversion of 2-methylnaphthalene to naphthyl-2-methyl-succinic acid was detected at a specific activity of 0.020 ± 0.003 nmol min⁻¹ mg of protein⁻¹ only in the presence of cells and fumarate. We conclude that under anaerobic conditions 2-methylnaphthalene is activated by fumarate addition to the methyl group, as is the case in anaerobic toluene degradation. The second group of metabolites comprised 2-naphthoic acid and reduced 2-naphthoic acid derivatives, including 5,6,7,8-tetrahydro-2-naphthoic acid, octahydro-2-naphthoic acid, and decahydro-2-naphthoic acid. These compounds were also identified in an earlier study as products of anaerobic naphthalene degradation with the same enrichment culture. A pathway for anaerobic degradation of 2-methylnaphthalene analogous to that for anaerobic toluene degradation is proposed.     

Anaerobic Degradation of Flavonoids by Clostridium orbiscindens

An anaerobic, quercetin-degrading bacterium was isolated from human feces and identified as Clostridium orbiscindens by comparative 16S rRNA gene sequence analysis. The organism was tested for its ability to transform several flavonoids. The isolated C. orbiscindens strain converted quercetin and taxifolin to 3,4-dihydroxyphenylacetic acid; luteolin and eriodictyol to 3-(3,4-dihydroxyphenyl)propionic acid; and apigenin, naringenin, and phloretin to 3-(4-hydroxyphenyl)propionic acid, respectively. Genistein and daidzein were not utilized. The glycosidic bonds of luteolin-3-glucoside, luteolin-5-glucoside, naringenin-7-neohesperidoside (naringin), quercetin-3-glucoside, quercetin-3-rutinoside (rutin), and phloretin-2′-glucoside were not cleaved. Based on the intermediates and products detected, pathways for the degradation of the flavonol quercetin and the flavones apigenin and luteolin are proposed. To investigate the numerical importance of C. orbiscindens in the human intestinal tract, a species-specific oligonucleotide probe was designed and tested for its specificity. Application of the probe to fecal samples from 10 human subjects proved the presence of C. orbiscindens in 8 out of the 10 samples tested. The numbers ranged from 1.87 × 10⁸ to 2.50 × 10⁹ cells g of fecal dry mass⁻¹, corresponding to a mean count of 4.40 × 10⁸ cells g of dry feces⁻¹.     

Anaerobic Degradation of Biphenyl by the Facultative Anaerobic Strain Citrobacter freundii BS2211

Using a synthetic medium supplemented with biphenyl (a polycyclic aromatic hydrocarbon), a new bacterial strain of Citrobacter freundiiwas isolated from enrichment cultures containing soil and industrial wastewater samples of the Serpukhov Condenser Factory. This strain was found to be capable of degrading biphenyl under anaerobic conditions in the course of nitrate reduction. When the initial concentration of biphenyl in the culture medium equaled 150 mg/ml, the culture with a titer of 10⁹ cells/ml degraded up to 26–28% of biphenyl in 3 days (28°C). At 250 mg/ml, the culture with a titer of 10⁷ cells/ml degraded 15% of biphenyl in 21 days. Approximately 10% of the substrate consumed was utilized completely, whereas the remainder underwent transformation.     

Degradation of Alkyl Benzene Sulfonate by Pseudomonas Species

Pseudomonas sp. HK-1 showed a direct relation between the concentration of alkyl benzene sulfonate (ABS) supplied and cell yields. Since growth on ABS alone did not occur, it was necessary to correlate the total energy obtained by the cells to the ABS concentration when glucose was supplied in a limiting concentration. Several types of metabolic attack in addition to the sulfonate removal were noted: (i) side-chain utilization as indicated by the production of tertiarybutyl alcohol and isopropanol and (ii) ring metabolism as indicated by the presence of phenol, catechol, mandelic acid, benzyl alcohol, and benzoic acid in spent growth media. Utilization of ABS was greatly enhanced by the presence of phenol. This enhancement suggests co-metabolism and that limited concentrations of phenolic products derived from ABS must be accumulated to get active metabolism of the ABS molecule.     

Sulfurospirillum tamanensis sp. nov., a Facultatively Anaerobic Alkaliphilic Bacterium from a Terrestrial Mud Volcano

Microbiology - An alkaliphilic, facultatively anaerobic bacterium (strain T05bT) was isolated from a terrestrial mud volcano on the Taman Peninsula, Russia. The cells of the isolate were motile...     

Anaerobic Degradation of Cyanuric Acid, Cysteine, and Atrazine by a Facultative Anaerobic Bacterium

A facultative anaerobic bacterium that rapidly degrades cyanuric acid (CA) was isolated from the sediment of a stream that received industrial wastewater effluent. CA decomposition was measured throughout the growth cycle by using a high-performance liquid chromatography assay, and the concomitant production of ammonia was also measured. The bacterium used CA or cysteine as a major, if not the sole, carbon and energy source under anaerobic, but not aerobic, conditions in a defined medium. The cell yield was greatly enhanced by the simultaneous presence of cysteine and CA in the medium. Cysteine was preferentially used rather than CA early in the growth cycle, but all of the CA was used without an apparent lag after the cysteine was metabolized. Atrazine was also degraded by this bacterium under anaerobic conditions in a defined medium.     

Anaerobic Degradation of Benzene, Toluene, Ethylbenzene, and o-Xylene in Sediment-Free Iron-Reducing Enrichment Cultures

Monoaromatic hydrocarbons such as benzene, toluene, ethylbenzene, and xylene (BTEX) are widespread contaminants in groundwater. We examined the anaerobic degradation of BTEX compounds with amorphous ferric oxide as electron acceptor. Successful enrichment cultures were obtained for all BTEX substrates both in the presence and absence of AQDS (9,10-anthraquinone-2,6-disulfonic acid). The electron balances showed a complete anaerobic oxidation of the aromatic compounds to CO₂. This is the first report on the anaerobic degradation of o-xylene and ethylbenzene in sediment-free iron-reducing enrichment cultures.