Attachment Behavior of Shewanella putrefaciens onto Magnetite under Aerobic and Anaerobic Conditions

In this study, batch experiments were used to characterize attachment behavior of Shewanella putrefaciens strain 200R to ferrihydrite and magnetite. Attachment was quantified in batch experiments with a 0.01 M NaNO ₃ solution as a function of pH (ranging from 3 to 10), sorbed anion (PO₄ ^{3 ?} ), and growth conditions (aerobic vs. anaerobic). Electrophoretic mobility data was collected for S. putrefaciens cells and magnetite grains and used as a means to interpret the role of electrostatic interaction in attachment studies. Little difference in attachment behavior was observed as a function of growth conditions or surface treatments. The exception was at pH ranging from 2 to 4, under anaerobic conditions, where increased attachment was measured on magnetite surfaces with sorbed PO₄ ^{3 ?} . This increased attachment was attributed to development of Fe-PO₄ surface complexes or secondary mineral phases, resulting in altered surface interactions between cell and mineral surfaces. Attachment was irreversible and increased with time under anaerobic conditions even under elevated pH conditions unfavourable to electrostatic interactions between cells and mineral surfaces. These results suggest that electrophoretic mobility data in this system is not a good predictor of attachment behavior, while surface charge development via protonation and deprotonation of surface functional groups is consistent with experimental attachment data. In this study, S. putrefaciens appears to utilize polymers or pili to remain attached to Fe-oxides and this process may facilitate Fe reduction on these surfaces. Results from this study underscore the need for quantitative bulk measurements of microbial attachment to accurately predict partitioning of dissimilatory iron reducing bacteria between solution and solid phases.     

Phenotypic Characterization of Shewanella oneidensis MR-1 under Aerobic and Anaerobic Growth Conditions by Using Fourier Transform Infrared Spectroscopy and High-Performance Liquid Chromatography Analyses

Shewanella oneidensis is able to conserve energy for growth by reducing a wide variety of terminal electron acceptors during anaerobic respiration, including several environmentally hazardous pollutants. This bacterium employs various electron transfer mechanisms for anaerobic respiration, including cell-bound reductases and secreted redox mediators. The aim of this study was to develop rapid tools for profiling the key metabolic changes associated with these different growth regimes and physiological responses. Initial experiments focused on comparing cells grown under aerobic and anaerobic conditions. Fourier transform infrared (FT-IR) spectroscopy with cluster analysis showed that there were significant changes in the metabolic fingerprints of the cells grown under these two culture conditions. FT-IR spectroscopy clearly differentiated cells of S. oneidensis MR-1 cultured at various growth points and cells grown using different electron acceptors, resulting in different phenotypic trajectories in the cluster analysis. This growth-related trajectory analysis is applied successfully for the first time, here with FT-IR spectroscopy, to investigate the phenotypic changes in contrasting S. oneidensis cells. High-performance liquid chromatography (HPLC) was also used to quantify the concentrations of flavin compounds, which have been identified recently as extracellular redox mediators released by a range of Shewanella species. The partial least-squares regression (PLSR) multivariate statistical technique was combined with FT-IR spectroscopy to predict the concentrations of the flavins secreted by cells of S. oneidensis MR-1, suggesting that this combination could be used as a rapid alternative to conventional chromatographic methods for analysis of flavins in cell cultures. Furthermore, coupling of the FT-IR spectroscopy and HPLC techniques appears to offer a potentially useful tool for rapid characterization of the Shewanella cell metabolome in various process environments.Shewanella oneidensis, a Gram-negative dissimilatory metal-reducing bacterium, is able to conserve energy for growth by reducing a variety of terminal electron acceptors during aerobic and anaerobic respiration (24), including several environmentally hazardous pollutants (1). The terminal electron acceptors used during anaerobic respiration vary, ranging from nitrate, fumarate, trimethylamine N-oxide (TMAO), dimethyl sulfoxide (DMSO), and sulfur compounds to fuel cell anodes and various metals and metalloids, including insoluble metal oxides (5, 33, 34).There has been intense interest in the versatile metabolism of this bacterium and its potential to respire and bioremediate toxic environmental chemicals and metals, such as U(VI) and Cr(VI) (25, 43), under anoxic conditions. Riboflavin (RF), flavin mononucleotide (FMN), and flavin-adenine dinucleotide (FAD) were identified as the dominant electron shuttles secreted by a diversity of Shewanella cells and shown to mediate extracellular reduction of insoluble Fe(III) minerals and organic molecules, including azo dyes (23, 40). Field and Brady tested riboflavin and found that its presence during anaerobic reduction of azo dyes improves the overall kinetics of the reduction process (9). von Canstein et al. recently found that Shewanella cells are able to secrete flavins, FAD, FMN, and riboflavin as extracellular redox mediators and quantified these using high-performance liquid chromatography-mass spectrometry (HPLC-MS) (44). The experiments showed that the production profiles of these three chemicals were different under anaerobic and aerobic regimes, and this can be used to distinguish the two metabolic pathways with oxygen or fumarate as electron acceptors.Although we are gaining a deeper understanding of the genetic and biochemical basis of the diverse respiration pathways of this organism, supported by the recent availability of the complete genome sequence (14), complementary metabolomic approaches have not been used to identify or quantify the metabolic changes expected with major physiological shifts in this organism under contrasting growth regimes. Fourier transform infrared (FT-IR) spectroscopy was chosen for this study, as this method offers the advantage of minimal sample preparation, as well as being rapid, nondestructive, readily automatable, relatively inexpensive, and quantitative, compared with other metabolic profiling techniques (8, 12, 15, 35). The infrared absorbance spectra generated by FT-IR spectroscopy have been used to identify specific biochemical features and also provide a global biochemical “fingerprint” for mixed, complex samples (11, 27) in many research areas (10, 11). For instance, FT-IR spectroscopy has proved sensitive enough for analysis of the chemical composition of a single strain of Escherichia coli after exposure to ionic liquids (6), for functional genomics screening (22), for measuring abiotic perturbations in algae (39), and for characterization of microbial degradation pathways (16). We believe that this technique offers considerable potential for rapid differentiation of metabolic changes of bacteria responding to contrasting growth regimes in natural and engineered environments, although this remains to be demonstrated.The FT-IR spectra generated from microorganisms have very complex profiles that can be related back to biochemical components which one would expect to be detected in the samples. While some subtle quantitative differences can be observed between the spectra, there are few, if any, qualitative differences to be seen, and so it is almost impossible to interpret these data with the naked eye (19, 20, 45). Therefore, multivariate statistical techniques are needed to model the relationship between the phenotypic changes occurring in this organism during growth with the observations recorded by FT-IR spectroscopy.In the present study, we investigated the ability of FT-IR spectroscopy to analyze the S. oneidensis MR-1 cell metabolome and distinguish significant metabolic changes associated with anaerobic and aerobic growth conditions. Analyses were conducted directly on the cells for the endometabolome in assessing the metabolic fingerprint. Both unsupervised and supervised learning methods (viz., principal component analysis [PCA] [21] and discriminant function analysis [DFA] [26]) were used to identify any differences between the FT-IR spectra from cells grown under the two regimes, and the contributions of a range of biomolecules were elucidated. In addition to metabolic fingerprinting of whole cells by FT-IR, key components of the extracellular metabolome (the so-called exometabolome, or metabolic footprint) were also quantified by HPLC analysis. Here, riboflavin, FMN, and FAD in culture supernatants were quantified, and partial least-squares regression (PLSR) was used to identify correlations between the cell''s FT-IR spectra and flavin concentrations assessed using HPLC data. These studies show for the first time that FT-IR analyses can be used for rapid identification of metabolic shifts in both the intracellular and the extracellular metabolomes of S. oneidensis MR-1 cultures.     

Selenite and Tellurite Reduction by Shewanella oneidensis

Shewanella oneidensis MR-1 reduces selenite and tellurite preferentially under anaerobic conditions. The Se(0) and Te(0) deposits are located extracellularly and intracellularly, respectively. This difference in localization and the distinct effect of some inhibitors and electron acceptors on these reduction processes are taken as evidence of two independent pathways.     

Shewanella oneidensis MR-1 Uses Overlapping Pathways for Iron Reduction at a Distance and by Direct Contact under Conditions Relevant for Biofilms

We developed a new method to measure iron reduction at a distance based on depositing Fe(III) (hydr)oxide within nanoporous glass beads. In this “Fe-bead” system, Shewanella oneidensis reduces at least 86.5% of the iron in the absence of direct contact. Biofilm formation accompanies Fe-bead reduction and is observable both macro- and microscopically. Fe-bead reduction is catalyzed by live cells adapted to anaerobic conditions, and maximal reduction rates require sustained protein synthesis. The amount of reactive ferric iron in the Fe-bead system is available in excess such that the rate of Fe-bead reduction is directly proportional to cell density; i.e., it is diffusion limited. Addition of either lysates prepared from anaerobic cells or exogenous electron shuttles stimulates Fe-bead reduction by S. oneidensis, but iron chelators or additional Fe(II) do not. Neither dissolved Fe(III) nor electron shuttling activity was detected in culture supernatants, implying that the mediator is retained within the biofilm matrix. Strains with mutations in omcB or mtrB show about 50% of the wild-type levels of reduction, while a cymA mutant shows less than 20% of the wild-type levels of reduction and a menF mutant shows insignificant reduction. The Fe-bead reduction defect of the menF mutant can be restored by addition of menaquinone, but menaquinone itself cannot stimulate Fe-bead reduction. Because the menF gene encodes the first committed step of menaquinone biosynthesis, no intermediates of the menaquinone biosynthetic pathway are used as diffusible mediators by this organism to promote iron reduction at a distance. CymA and menaquinone are required for both direct and indirect mineral reduction, whereas MtrB and OmcB contribute to but are not absolutely required for iron reduction at a distance.     

Characterization of Protein-Protein Interactions Involved in Iron Reduction by Shewanella oneidensis MR-1

The interaction of proteins implicated in dissimilatory metal reduction by Shewanella oneidensis MR-1 (outer membrane [OM] proteins OmcA, MtrB, and MtrC; OM-associated protein MtrA; periplasmic protein CctA; and cytoplasmic membrane protein CymA) were characterized by protein purification, analytical ultracentrifugation, and cross-linking methods. Five of these proteins are heme proteins, OmcA (83 kDa), MtrC (75 kDa), MtrA (32 kDa), CctA (19 kDa), and CymA (21 kDa), and can be visualized after sodium dodecyl sulfate-polyacrylamide gel electrophoresis by heme staining. We show for the first time that MtrC, MtrA, and MtrB form a 198-kDa complex with a 1:1:1 stoichiometry. These proteins copurify through anion-exchange chromatography, and the purified complex has the ability to reduce multiple forms of Fe(III) and Mn(IV). Additionally, MtrA fractionates with the OM through sucrose density gradient ultracentrifugation, and MtrA comigrates with MtrB in native gels. Protein cross-linking of whole cells with 1% formaldehyde show new heme bands of 160, 151, 136, and 59 kDa. Using antibodies to detect each protein separately, heme proteins OmcA and MtrC were shown to cross-link, yielding the 160-kDa band. Consistent with copurification results, MtrB cross-links with MtrA, forming high-molecular-mass bands of approximately 151 and 136 kDa.     

Promotion of Iron Oxide Reduction and Extracellular Electron Transfer in Shewanella oneidensis by DMSO

The dissimilatory metal reducing bacterium Shewanella oneidensis MR-1, known for its capacity of reducing iron and manganese oxides, has great environmental impacts. The iron oxides reducing process is affected by the coexistence of alternative electron acceptors in the environment, while investigation into it is limited so far. In this work, the impact of dimethyl sulphoxide (DMSO), a ubiquitous chemical in marine environment, on the reduction of hydrous ferric oxide (HFO) by S. oneidensis MR-1 was investigated. Results show that DMSO promoted HFO reduction by both wild type and ΔdmsE, but had no effect on the HFO reduction by ΔdmsB, indicating that such a promotion was dependent on the DMSO respiration. With the DMSO dosing, the levels of extracellular flavins and omcA expression were significantly increased in WT and further increased in ΔdmsE. Bioelectrochemical analysis show that DMSO also promoted the extracellular electron transfer of WT and ΔdmsE. These results demonstrate that DMSO could stimulate the HFO reduction through metabolic and genetic regulation in S. oneidensis MR-1, rather than compete for electrons with HFO. This may provide a potential respiratory pathway to enhance the microbial electron flows for environmental and engineering applications.     

Microbial Reduction and Precipitation of Vanadium by Shewanella oneidensis

Shewanella oneidensis couples anaerobic oxidation of lactate, formate, and pyruvate to the reduction of vanadium pentoxide (V^V). The bacterium reduces V^V (vanadate ion) to V^IV (vanadyl ion) in an anaerobic atmosphere. The resulting vanadyl ion precipitates as a V^IV-containing solid.     

Anaerobic biodecolorization mechanism of methyl orange by Shewanella oneidensis MR-1

In this work, we investigated the anaerobic decolorization of methyl orange (MO), a typical azo dye, by Shewanella oneidensis MR-1, which can use various organic and inorganic substances as its electron acceptor in natural and engineered environments. S. oneidensis MR-1 was found to be able to obtain energy for growth through anaerobic respiration accompanied with dissimilatory azo-reduction of MO. Chemical analysis shows that MO reduction occurred via the cleavage of azo bond. Block of Mtr respiratory pathway, a transmembrane electron transport chain, resulted in a reduction of decolorization rate by 80%, compared to the wild type. Knockout of cymA resulted in a substantial loss of its azo-reduction ability, indicating that CymA is a key c-type cytochrome in the electron transfer chain to MO. Thus, the MtrA-MtrB-MtrC respiratory pathway is proposed to be mainly responsible for the anaerobic decolorization of azo dyes such as MO by S. oneidensis.     

Siderophore Production by Pseudomonas stutzeri under Aerobic and Anaerobic Conditions

The siderophore production of the facultative anaerobe Pseudomonas stutzeri, strain CCUG 36651, grown under both aerobic and anaerobic conditions, was investigated by liquid chromatography and mass spectrometry. The bacterial strain has been isolated at a 626-m depth at the Äspö Hard Rock Laboratory, where experiments concerning the geological disposal of nuclear waste are performed. In bacterial culture extracts, the iron in the siderophore complexes was replaced by gallium to facilitate siderophore identification by mass spectrometry. P. stutzeri was shown to produce ferrioxamine E (nocardamine) as the main siderophore together with ferrioxamine G and two cyclic ferrioxamines having molecular masses 14 and 28 atomic mass units lower than that of ferrioxamine E, suggested to be ferrioxamine D₂ and ferrioxamine X₁, respectively. In contrast, no siderophores were observed from anaerobically grown P. stutzeri. None of the siderophores produced by aerobically grown P. stutzeri were found in anaerobic natural water samples from the Äspö Hard Rock Laboratory.     

Comparative Studies of Glucose Catabolism by Escherichia coli Grown in a Complex Medium under Aerobic and Anaerobic Conditions

Escherichia coli HB101 was grown in complex medium under anaerobic and aerobic conditions. Cells prepared under these two different conditions were characterized by two-dimensional protein gel electrophoresis, by NMR measurements under identical (anaerobic) conditions, and by measuring the kinetics of glucose uptake and catabolite end-product appearance in the medium under identical anaerobic conditions. Specific rates of glucose uptake and end-product formation were significantly greater for the anaerobically grown cells, which also exhibited lower intracellular concentrations of sugar phosphates, nucleoside di-and triphosphates, UDPG, and NAD(H). Two-dimensional gel electrophoretic analyses reveal changes in the intracellular levels of proteins involved in pyruvate catabolism that have been observed previously for E. coli grown in minimal medium under aerobic and anaerobic conditions. Enzymes involved in the TCA cycle were not detected in cells grown aerobically or anaerobically in complex medium.     

Fe(III) Oxide Reduction by Anaerobic Biofilm Formation-DeficientS-Ribosylhomocysteine Lyase (LuxS) Mutant of Shewanella oneidensis

Shewanella oneidensis respires a variety of terminal electron acceptors, including solid phase Fe(III) oxides. S. oneidensis transfers electrons to Fe(III) oxides via direct (outer membrane- or nanowire-localized c-type cytochromes) and indirect (electron shuttling and Fe(III) solubilization) pathways. In the present study, the influence of anaerobic biofilm formation on Fe(III) oxide reduction by S. oneidensis was determined. The gene encoding the activated methyl cycle (AMC) enzyme S-ribosylhomocysteine lyase (LuxS) was deleted in-frame to generate the corresponding mutant ΔluxS. Conventional biofilm assays and visual inspection via confocal laser scanning microscopy indicated that the wild-type strain formed anaerobic biofilms on Fe(III) oxide-coated silica surfaces, while the ΔluxS mutant was severely impaired in anaerobic biofilm formation on such surfaces. Cell-hematite attachment isotherms demonstrated that the ΔluxS mutant was also severely impaired in attachment to hematite surfaces under anaerobic conditions. The S. oneidensis ΔluxS mutant, however, reduced Fe(III) at wild-type rates during anaerobic incubation with Fe(III) oxide-coated silica surfaces or in batch cultures with Fe(III) oxide or hematite as a terminal electron acceptor. Anaerobic biofilm formation by the ΔluxS mutant was restored to wild-type rates by providing a wild-type copy of luxS in trans or by the addition of AMC or transsulfurylation pathway metabolites involved in organic sulfur metabolism. LuxS is thus required for wild-type anaerobic biofilm formation on Fe(III) oxide surfaces, yet the inability to form wild-type anaerobic biofilms on Fe(III) oxide surfaces does not alter Fe(III) oxide reduction activity.     

Release of Iron from Ferritin by 6-Hydroxydopamine Under Aerobic and Anaerobic Conditions

6-hydroxydopamine (6-OHDA) proved to be a very effective agent for iron release from ferritin. Iron release was enhanced in the presence of SOD, catalase and under anaerobic conditions. Ascorbic acid, a well known agent able to release iron from ferritin, increased the amount of released iron in more than an additive manner when used in combination with 6-OHDA. Similar to 6-OHDA, 6-hydroxydopa (Topa) and 1,2,4-benzenetriol were also able to release iron in large amounts; in contrast, catecholamines and other benzenediols were comparatively ineffective.     

Role of Menaquinones in Fe(III) Reduction by Membrane Fractions of Shewanella putrefaciens

Two Tn5-generated mutants of Shewanella putrefaciens with insertions in menD and menB were isolated and analyzed. Both mutants were deficient in the use of several terminal electron acceptors, including Fe(III). This deficiency was overcome by the addition of menaquinone (vitamin K(2)). Isolated membrane fractions from both mutants were unable to reduce Fe(III) in the absence of added menaquinone when formate was used as the electron donor. These results indicate that menaquinones are essential components for the reduction of Fe(III) by both whole cells and purified membrane fractions when formate or lactate is used as the electron donor.     

Release of Iron from Ferritin by 6-Hydroxydopamine Under Aerobic and Anaerobic Conditions

6-hydroxydopamine (6-OHDA) proved to be a very effective agent for iron release from ferritin. Iron release was enhanced in the presence of SOD, catalase and under anaerobic conditions. Ascorbic acid, a well known agent able to release iron from ferritin, increased the amount of released iron in more than an additive manner when used in combination with 6-OHDA. Similar to 6-OHDA, 6-hydroxydopa (Topa) and 1,2,4-benzenetriol were also able to release iron in large amounts; in contrast, catecholamines and other benzenediols were comparatively ineffective.     

Mechanism and Consequences of Anaerobic Respiration of Cobalt by Shewanella oneidensis Strain MR-1

Bacteria from the genus Shewanella are the most diverse respiratory organisms studied to date and can utilize a variety of metals and metal(loid)s as terminal electron acceptors. These bacteria can potentially be used in bioremediation applications since the redox state of metals often influences both solubility and toxicity. Understanding molecular mechanisms by which metal transformations occur and the consequences of by-products that may be toxic to the organism and thus inhibitory to the overall process is significant to future applications for bioremediation. Here, we examine the ability of Shewanella oneidensis to catalyze the reduction of chelated cobalt. We describe an unexpected ramification of [Co(III)-EDTA]⁻ reduction by S. oneidensis: the formation of a toxic by-product. We found that [Co(II)-EDTA]²⁻, the product of [Co(III)-EDTA]⁻ respiration, inhibited the growth of S. oneidensis strain MR-1 and that this toxicity was partially abolished by the addition of MgSO₄. We demonstrate that [Co(III)-EDTA]⁻ reduction by S. oneidensis requires the Mtr extracellular respiratory pathway and associated pathways required to develop functional Mtr enzymes (the c-type cytochrome maturation pathway) and ensure proper localization (type II secretion). The Mtr pathway is known to be required for a variety of substrates, including some chelated and insoluble metals and organic compounds. Understanding the full substrate range for the Mtr pathway is crucial for developing S. oneidensis strains as a tool for bioremediation.     

Dependency of Nitrate Reduction on Soluble Carbohydrates in Primary Leaves of Barley under Aerobic Conditions

Nitrate reduction was studied as a function of carbohydrate concentration in detached primary leaves of barley (Hordeum vulgare L. cv Numar) seedlings under aerobic conditions in light and darkness. Seedlings were grown either in continuous light for 8 days or under a regimen of 16-hour light and 8-hour dark for 8 to 15 days. Leaves of 8-day-old seedlings grown in continuous light accumulated 4 times more carbohydrates than leaves of plants grown under a light and dark regimen. When detached leaves from these seedlings were supplied with NO₃⁻ in darkness, those with the higher levels of carbohydrates reduced a greater proportion of the NO₃⁻ that was taken up. In darkness, added glucose increased the percentage of NO₃⁻ reduced up to 2.6-fold depending on the endogenous carbohydrate status of the leaves. Both NO₃⁻ reduction and carbohydrate content of the leaves increased with age. Fructose and sucrose also increased NO₃⁻ reduction in darkness to the same extent as glucose. Krebs cycle intermediates, citrate and succinate, did not increase NO₃⁻ reduction, whereas malate slightly stimulated it in darkness.

In light, 73 to 90% of the NO₃⁻ taken up was reduced by the detached leaves; therefore, an exogenous supply of glucose had little additional effect on NO₃⁻ reduction. The results indicate that in darkness the rate of NO₃⁻ reduction in primary leaves of barley depends upon the availability of carbohydrates.

     

Dissimilatory Nitrate and Nitrite Reduction under Aerobic Conditions by an Aerobically and Anaerobically Grown Alcaligenes sp. and by Activated Sludge

The oxygen uptake of an Alcaligenes sp., isolated from activated sludge, was inhibited by small amounts of nitric oxide. The occurrence of this inhibition was dependent on the growth conditions and the pretreatment of the cells. Anaerobically grown cells, which had subsequently been aerated in a nitrogen-free medium, accumulated nitric oxide, after the addition of nitrate or nitrite. When the oxygen uptake was inhibited by nitric oxide, dissimilatory reduction of nitrate and nitrite proceeded under aerobic conditions at the same rate as in the absence of oxygen. Activated sludge removed nitric oxide actively under aerobic conditions and as a consequence the oxygen uptake of the sludge was not inhibited in the presence of nitrite. The rate of nitrate reduction under aerobic conditions was about 20% of that in the absence of oxygen.     

Amylolytic Activities in Cereal Seeds under Aerobic and Anaerobic Conditions

An adequate carbohydrate supply contributes to the survival of seeds under conditions of limited oxygen availability. The amount of soluble, readily fermentable carbohydrates in dry cereal seeds is usually very limited, with starch representing the main storage compound. Starch breakdown during the germination of cereal seeds is the result of the action of hydrolytic enzymes and only through the concerted action of [alpha]-amylase (EC 3.2.1.1), [beta]-amylase (EC 3.2.1.2), debranching enzyme (EC 3.2.1.41), and [alpha]-glucosidase (EC 3.2.1.20) can starch be hydrolyzed completely. We present here data concerning the complete set of starch-degrading enzymes in three cereals, rice (Oryza sativa L.), which is tolerant to anaerobiosis, and wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.), which are unable to germinate under anoxia. Among the cereal seeds tested under anoxia, only rice is able to degrade nonboiled, soluble starch, reflecting the ability to degrade the starch granules in vivo. This is explained by the presence of the complete set of enzymes needed to degrade starch completely either as the result of de novo synthesis ([alpha]-amylase, [beta]-amylase) or activation of preexisting, inactive forms of the enzyme (debranching enzyme, [alpha]-glucosidase). These enzymes are either absent or inactive in wheat and barley seeds kept under anaerobic conditions.     

Reduction of Nitrated Diphenylamine Derivatives under Anaerobic Conditions

2-Nitrodiphenylamine, 4-nitrodiphenylamine, and 2,4-dinitrodiphenylamine were anaerobically metabolized in sediment-water batch enrichments inoculated with mud from the German North Sea coast. The first intermediate in 2,4-dinitrodiphenylamine degradation was 2-amino-4-nitrodiphenylamine, which appeared in large (nearly stoichiometric) amounts before being completely reduced to 2,4-diaminodiphenylamine. Of the second theoretically expected metabolite, 4-amino-2-nitrodiphenylamine, only traces were detected by gas chromatographic-mass spectrometric analysis in highly concentrated extracts. In addition, low levels of 4-nitrodiphenylamine, which may be the product of ortho deamination of intermediately produced 2-amino-4-nitrodiphenylamine, were observed. 2-Nitrodiphenylamine and 4-nitrodiphenylamine were primarily reduced to 2-aminodiphenylamine and 4-aminodiphenylamine, respectively. Diphenylamine was never detected in any experiment as a theoretically possible intermediate. Results from studies with dense cell suspensions of anaerobic, aromatic-compound-mineralizing bacteria confirmed the transformation reactions, which were carried out by microorganisms indigenous to the anaerobic coastal water sediment.