Biocatalytic chlorination of aromatic hydrocarbons by chloroperoxidase of Caldariomyces fumago

Chloroperoxidase from Caldariomyces fumago was able to chlorinate 17 of 20 aromatic hydrocarbons assayed in the presence of hydrogen peroxide and chloride ions. Reaction rates varied from 0.6 min(-1) for naphthalene to 758 min(-1) for 9-methylanthracene. Mono-, di- and tri-chlorinated compounds were obtained from the chloroperoxidase-mediated reaction on aromatic compounds. Dichloroacenaphthene, trichloroacenaphthene, 9,10-dichloroanthracene, chloropyrene, dichloropyrene, dichlorobiphenylene and trichlorobiphenylene were identified by mass spectral analyses as products from acenaphthene, anthracene, pyrene and biophenylene respectively. Polycyclic aromatic hydrocarbons with 5 and 6 aromatic rings were also substrates for the chloroperoxidase reaction. The importance of the microbial chlorination of aromatic pollutants and its potential environmental impact are discussed.     

Carboxylation of phenylphosphate by phenol carboxylase, an enzyme system of anaerobic phenol metabolism.

Several lines of evidence indicate that the first step in the anaerobic metabolism of phenol is phenol carboxylation to 4-hydroxybenzoate; this reaction is considered a biological Kolbe-Schmitt carboxylation. A phenol carboxylase system was characterized by using a denitrifying Pseudomonas strain, K 172, which catalyzes an isotope exchange between 14CO2 and the carboxyl group of 4-hydroxybenzoate. The enzymatic isotope exchange activity (100 nmol min-1 mg-1 of protein) requires Mn2+ and K+. We show that this system also catalyzes the carboxylation of phenylphosphate (the phosphoric acid monophenyl ester) to 4-hydroxybenzoate and phosphate. The specific activity of phenylphosphate carboxylation at the optimal pH of 6.5 is 12 nmol of CO2 fixed min-1 mg-1 of protein. Phenylphosphate cannot be replaced by Mg(2+)-ATP and phenol. The carboxylase activity requires Mn2+ but, in contrast to the isotope exchange activity, does not require K+. The apparent Km values are 1.5 mM dissolved CO2 and 0.2 mM phenylphosphate. Several convenient assays for phenylophosphate carboxylation are described. The isotope exchange reaction and the net carboxylation reaction are catalyzed by the same oxygen-sensitive enzyme, which has a half-life in an air-saturated solution of less than 1 min. Both activities cochromatographed with a protein with a Mr of 280,000, and both activities were induced only after anaerobic growth on phenol. The carboxylation of phenylphosphate suggests that phenylphosphate itself is the physiological CO2 acceptor molecular of this novel CO2 fixation reaction. Alternatively, phenylphosphate could simulate the unknown natural precursor. It is suggested that the formation of an enzyme-bound phenolate anion from the activated phenolic compound is the rate-determining step in the carboxylation reaction.     

Evidence for aromatic ring reduction in the biodegradation pathwayof carboxylated naphthalene by a sulfate reducing consortium

Naphthalene was used as a model compound in order to study the anaerobic pathway of polycyclic aromatic hydrocarbon degradation. Previously we had determined that carboxylation is an initial step for anaerobic metabolism of naphthalene, but no other intermediate metabolites were identified (Zhang & Young 1997). In the present study we further elucidate the pathway with the identification of six novel naphthalene metabolites detected when cultures were fed naphthalene in the presence of its analog 1-fluoronaphthalene. Results from cultures supplemented with either deuterated naphthalene or non-deuterated naphthalene plus [¹³C]bicarbonate confirm that the metabolites originated from naphthalene. Three of these metabolites were identified by comparison with the following standards: 2-naphthoic acid (2-NA), 5,6,7,8-tetrahydro-2-naphthoic acid, and decahydro-2-naphthoic acid. The presence of 5,6,7,8-tetrahydro-2-NA as a metabolite of naphthalene degradation indicates that the first reduction reaction occurs at the unsubstituted ring, rather than the carboxylated ring. The overall results suggest that after the initial carboxylation of naphthalene, 2-NA is sequentially reduced to decahydro-2-naphthoic acid through 5 hydrogenation reactions, each of which eliminated one double bond. Incorporation of deuterium atoms from D₂O into 5,6,7,8-tetrahydro-2-naphthoic acid suggests that water is the proton source for hydrogenation.     

Anaerobic naphthalene degradation by Gram-positive, iron-reducing bacteria

An anaerobic naphthalene-degrading culture (N49) was enriched with ferric iron as electron acceptor. A closed electron balance indicated the total oxidation of naphthalene to CO(2). In all growing cultures, the concentration of the presumed central metabolite of naphthalene degradation, 2-naphthoic acid, increased concomitantly with growth. The first metabolite of anaerobic methylnaphthalene degradation, naphthyl-2-methyl-succinic acid, was not identified in culture supernatants, which does not support a methylation to methylnaphthalene as the initial activation reaction of naphthalene, but rather a carboxylation, as proposed for other naphthalene-degrading cultures. Substrate utilization tests revealed that the culture was able to grow on 1-methyl-naphthalene, 2-methyl-naphthalene, 1-naphthoic acid or 2-naphthoic acid, whereas it did not grow on 1-naphthol, 2-naphthol, anthracene, phenanthrene, indane and indene. Terminal restriction fragment length polymorphism and 16S rRNA gene sequence analyses revealed that the microbial community of the culture was dominated by one bacterial microorganism, which was closely related (99% 16S sequence similarity) to the major organism in the iron-reducing, benzene-degrading enrichment culture BF [ISME J (2007) 1: 643; Int J Syst Evol Microbiol (2010) 60: 686]. The phylogenetic classification supports a new candidate species and genus of Gram-positive spore-forming iron-reducers that can degrade non-substituted aromatic hydrocarbons. It furthermore indicates that Gram-positive microorganisms might also play an important role in anaerobic polycyclic aromatic hydrocarbon-degradation.     

红树林厌氧环境对多环芳烃类有毒物的降解预测

红树林是连接陆地和海洋的重要生态系统,由于潮汐活动,氧化还原条件表现出明显的昼夜间的交替,这一生态体系中不但有大量的动植物种类,同时还有数量极高的不同种类的细菌,包括好氧和厌氧类型,厌养的硫酸(盐)还原菌已证实在降解多环芳烃有机物方面有其独特的生化优势,但从红树林中分离出的此类纯细菌还很少,在降解方面,已初步确定萘的厌氧降解途径异于好氧细菌,厌氧降解时的一系列代谢中间产物也有明显的专一性,羰基化反应是开始的一个重要步骤,而后的每步生化反应还有待进一步验证。从现有的结果可以看出,红树林中厌养的硫酸还原菌应在降解多环芳烃有机物中起到非常重要的作用。     

Identical ring cleavage products during anaerobic degradation of naphthalene, 2-methylnaphthalene, and tetralin indicate a new metabolic pathway

Anaerobic degradation of naphthalene, 2-methylnaphthalene, and tetralin (1,2,3,4-tetrahydronaphthalene) was investigated with a sulfate-reducing enrichment culture obtained from a contaminated aquifer. Degradation studies with tetralin revealed 5,6,7,8-tetrahydro-2-naphthoic acid as a major metabolite indicating activation by addition of a C(1) unit to tetralin, comparable to the formation of 2-naphthoic acid in anaerobic naphthalene degradation. The activation reaction was specific for the aromatic ring of tetralin; 1,2,3,4-tetrahydro-2-naphthoic acid was not detected. The reduced 2-naphthoic acid derivatives tetrahydro-, octahydro-, and decahydro-2-naphthoic acid were identified consistently in supernatants of cultures grown with either naphthalene, 2-methylnaphthalene, or tetralin. In addition, two common ring cleavage products were identified. Gas chromatography-mass spectrometry (GC-MS) and high-resolution GC-MS analyses revealed a compound with a cyclohexane ring and two carboxylic acid side chains as one of the first ring cleavage products. The elemental composition was C(11)H(16)O(4) (C(11)H(16)O(4)-diacid), indicating that all carbon atoms of the precursor 2-naphthoic acid structure were preserved in this ring cleavage product. According to the mass spectrum, the side chains could be either an acetic acid and a propenic acid, or a carboxy group and a butenic acid side chain. A further ring cleavage product was identified as 2-carboxycyclohexylacetic acid and was assumed to be formed by beta-oxidation of one of the side chains of the C(11)H(16)O(4)-diacid. Stable isotope-labeling growth experiments with either (13)C-labeled naphthalene, per-deuterated naphthalene-d(8), or a (13)C-bicarbonate-buffered medium showed that the ring cleavage products derived from the introduced carbon source naphthalene. The series of identified metabolites suggests that anaerobic degradation of naphthalenes proceeds via reduction of the aromatic ring system of 2-naphthoic acid to initiate ring cleavage in analogy to the benzoyl-coenzyme A pathway for monoaromatic hydrocarbons. Our findings provide strong indications that further degradation goes through saturated compounds with a cyclohexane ring structure and not through monoaromatic compounds. A metabolic pathway for anaerobic degradation of bicyclic aromatic hydrocarbons with 2-naphthoic acid as the central intermediate is proposed.     

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.     

ATP‐dependent/‐independent enzymatic ring reductions involved in the anaerobic catabolism of naphthalene

Polycyclic aromatic hydrocarbons are among the most hazardous environmental pollutants. However, in contrast to aerobic degradation, the respective degradation pathways in anaerobes are greatly unknown which has so far prohibited many environmental investigations. In this work, we studied the enzymatic dearomatization reactions involved in the degradation of the PAH model compounds naphthalene and 2‐methylnaphthalene in the sulfate‐reducing enrichment culture N47. Cell extracts of N47 grown on naphthalene catalysed the sodium dithionite‐dependent four‐electron reduction of the key intermediate 2‐naphthoyl‐coenzyme A (NCoA) to 5,6,7,8‐tetrahydro‐2‐naphthoyl‐CoA (THNCoA). The NCoA reductase activity was independent of ATP and was, surprisingly, not sensitive to oxygen. In cell extracts in the presence of various electron donors the product THNCoA was further reduced by a two‐electron reaction to most likely a conjugated hexahydro‐2‐naphthoyl‐CoA isomer (HHNCoA). The reaction assigned to THNCoA reductase strictly depended on ATP and was oxygen‐sensitive with a half‐life time between 30 s and 1 min when exposed to air. The rate was highest with NADH as electron donor. The results indicate that two novel and completely different dearomatizing ring reductases are involved in anaerobic naphthalene degradation. While the THNCoA reducing activity shows some properties of ATP‐dependent class I benzoyl‐CoA reductases, NCoA reduction appears to be catalysed by a previously unknown class of dearomatizing aryl‐carboxyl‐CoA reductases.     

Identical Ring Cleavage Products during Anaerobic Degradation of Naphthalene, 2-Methylnaphthalene, and Tetralin Indicate a New Metabolic Pathway

Anaerobic degradation of naphthalene, 2-methylnaphthalene, and tetralin (1,2,3,4-tetrahydronaphthalene) was investigated with a sulfate-reducing enrichment culture obtained from a contaminated aquifer. Degradation studies with tetralin revealed 5,6,7,8-tetrahydro-2-naphthoic acid as a major metabolite indicating activation by addition of a C₁ unit to tetralin, comparable to the formation of 2-naphthoic acid in anaerobic naphthalene degradation. The activation reaction was specific for the aromatic ring of tetralin; 1,2,3,4-tetrahydro-2-naphthoic acid was not detected. The reduced 2-naphthoic acid derivatives tetrahydro-, octahydro-, and decahydro-2-naphthoic acid were identified consistently in supernatants of cultures grown with either naphthalene, 2-methylnaphthalene, or tetralin. In addition, two common ring cleavage products were identified. Gas chromatography-mass spectrometry (GC-MS) and high-resolution GC-MS analyses revealed a compound with a cyclohexane ring and two carboxylic acid side chains as one of the first ring cleavage products. The elemental composition was C₁₁H₁₆O₄ (C₁₁H₁₆O₄-diacid), indicating that all carbon atoms of the precursor 2-naphthoic acid structure were preserved in this ring cleavage product. According to the mass spectrum, the side chains could be either an acetic acid and a propenic acid, or a carboxy group and a butenic acid side chain. A further ring cleavage product was identified as 2-carboxycyclohexylacetic acid and was assumed to be formed by β-oxidation of one of the side chains of the C₁₁H₁₆O₄-diacid. Stable isotope-labeling growth experiments with either ¹³C-labeled naphthalene, per-deuterated naphthalene-d₈, or a ¹³C-bicarbonate-buffered medium showed that the ring cleavage products derived from the introduced carbon source naphthalene. The series of identified metabolites suggests that anaerobic degradation of naphthalenes proceeds via reduction of the aromatic ring system of 2-naphthoic acid to initiate ring cleavage in analogy to the benzoyl-coenzyme A pathway for monoaromatic hydrocarbons. Our findings provide strong indications that further degradation goes through saturated compounds with a cyclohexane ring structure and not through monoaromatic compounds. A metabolic pathway for anaerobic degradation of bicyclic aromatic hydrocarbons with 2-naphthoic acid as the central intermediate is proposed.     

Carboxylation as an initial reaction in the anaerobic metabolism of naphthalene and phenanthrene by sulfidogenic consortia.

The anaerobic biodegradation of naphthalene (NAP) and phenanthrene (PHE) was investigated by using sediment collected from the Arthur Kill in New York/New Jersey harbor. The initial cultures were composed of 10% sediment and 90% mineral medium containing 20 mM sulfate. Complete loss of NAP and PHE (150 to 200 muM) was observed after 150 days of incubation. Upon refeeding, NAP and PHE were utilized within 14 days. The utilization of both compounds was inhibited in the presence of 20 mM molybdate. [14C]NAP and [14C]PHE were mineralized to 14CO2. The activities could be maintained and propagated by subculturing in mineral medium. In the presence of halogenated analogs, 2-naphthoate was detected in NAP-utilizing enrichments. The mass spectrum of the derivatized 2-napththoate from the enrichment supplemented with both [13C]bicarbonate and NAP indicates the incorporation of 13CO2 into NAP. In the PHE-utilizing enrichment, a metabolite was detected by both high-pressure liquid chromatography and gas chromatography-mass spectrometry analyses. The molecular ion and fragmentation pattern of its mass spectrum indicate that it was phenanthrenecarboxylic acid. The results obtained with [13C] bicarbonate indicate that 13CO2 was incorporated into PHE. It appears, therefore, that carboxylation is an initial key reaction for the anaerobic metabolism and NAP and PHE. To our knowledge, this is the first report providing evidence for intermediates of PAH degradation under anaerobic conditions.     

厌氧微生物降解多环芳烃研究进展

多环芳烃(PAHs)是一类普遍存在于环境介质中的难降解有机污染物,相对于好氧微生物降解PAHs的研究,厌氧微生物降解PAHs的研究则相对较少.本文从厌氧微生物降解PAHs的研究背景,厌氧降解微生物的特点和不同厌氧降解还原反应体系的角度综述了厌氧微生物降解PAHs的概况;结合厌氧微生物降解萘和菲转化途径的介绍,推断了其降解机制的内在原因;同时通过总结影响厌氧微生物降解PAHs的主要因素(包括:PAHs的生物可利用性、外源营养物质的添加、外源电子受体的添加、特定厌氧降解菌的筛选强化和部分环境因素等),指出了制约降解进程的潜在限制因子;并对厌氧微生物降解PAHs研究目前存在的问题和未来的发展方向作了简述与展望.     

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-(13)C]naphthalene or deuterated D(8)-naphthalene, all metabolites mentioned derived from the introduced labeled naphthalene. When a [(13)C]bicarbonate-buffered growth medium was used in conjunction with unlabeled naphthalene, (13)C 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.     

Identification of new enzymes potentially involved in anaerobic naphthalene degradation by the sulfate-reducing enrichment culture N47

The sulfate-reducing highly enriched culture N47 is capable to anaerobically degrade naphthalene, 2-methylnaphthalene, and 2-naphthoic acid. A proteogenomic investigation was performed to elucidate the initial activation reaction of anaerobic naphthalene degradation. This lead to the identification of an alpha-subunit of a carboxylase protein that was two-fold up-regulated in naphthalene-grown cells compared to 2-methylnaphthalene-grown cells. The putative naphthalene carboxylase subunit showed 48% similarity to the anaerobic benzene carboxylase from an iron-reducing, benzene-degrading culture and 45% to alpha-subunit of phenylphosphate carboxylase of Aromatoleum aromaticum EbN1. A gene for the beta-subunit of putative naphthalene carboxylase was located nearby on the genome and was expressed with naphthalene. Similar to anaerobic benzene carboxylase, there were no genes for gamma- and delta-subunits of a putative carboxylase protein located on the genome which excludes participation in degradation of phenolic compounds. The genes identified for putative naphthalene carboxylase subunits showed only weak similarity to 4-hydroxybenzoate decarboxylase excluding ATP-independent carboxylation. Several ORFs were identified that possibly encode a 2-naphthoate-CoA ligase, which is obligate for activation before the subsequent ring reduction by naphthoyl-CoA reductase. One of these ligases was exclusively expressed on naphthalene and 2-naphthoic acid and might be the responsible naphthoate-CoA-ligase.     

Continuous removal of aromatic hydrocarbons by an AF reactor under denitrifying conditions

Removal of three typical aromatic hydrocarbons, benzene, biphenyl and naphthalene by an anaerobic filter (AF) reactor under continuous mode and denitrifying conditions was studied. Results showed that the AF reactor could degrade these aromatic hydrocarbons effectively under above-mentioned conditions. When influent wastewater contained 900 mg COD/l and about 60 mg (total aromatic hydrocarbons)/l, 90% and 84% removal efficiency could be achieved for them respectively. When COD/NO₃ ⁻-N ratio (C/N) was in the range 5–30, the removal of benzene was slightly influenced by C/N and it remained stable at about 90%. However, degradation of naphthalene, biphenyl and total COD was greatly influenced by C/N, and highest removal was achieved at C/N = 15, it was 90%, 85% and 82% for COD, naphthalene and biphenyl, respectively. Degradation of these three aromatic hydrocarbons followed the order: benzene > naphthalene > biphenyl.     

Bicarbonate exchange kinetics at equilibrium across the erythrocyte membrane by 13C NMR

The rate of exchange of 13C-labelled bicarbonate across the membranes of human erythrocytes in suspension, at thermal and chemical equilibrium, was measured using 13C NMR spectroscopy: the permeability coefficient (3.34 X 10(-4) cm s-1) agrees well with previous values obtained with other methods. Data analysis was complicated by the need to consider the Donnan ratio of the charged species inside and outside the cells. This work appears to be the first, involving the present NMR procedure, for studying fast membrane transport of a molecule other than water.     

Biodegradation of soluble aromatic compounds of jet fuel under anaerobic conditions: laboratory batch experiments

Laboratory batch experiments were performed with contaminated aquifer sediments and four soluble aromatic components of jet fuel to assess their biodegradation under anaerobic conditions. The biodegradation of four aromatic compounds, toluene, o-xylene, 1,2,4-trimethylbenzene (TMB), and naphthalene, separately or together, was investigated under strictly anaerobic conditions in the dark for a period of 160 days. Of the aromatic compounds, toluene and o-xylene were degraded both as a single substrate and in a mixture with the other aromatic compounds, while TMB was not biodegraded as a single substrate, but was biodegraded in the presence of the other aromatic hydrocarbons. Substrate interaction is thus significant in the biodegradation of TMB. Biodegradation of naphthalene was not observed, either as a single substrate or in a mixture of other aromatic hydrocarbons. Although redox conditions were dominated by iron reduction, a clear relation between degradation and sulfate reduction was observed. Methanogenesis took place during the later stages of incubation. However, the large background of Fe(II) masked the increase of Fe(II) concentration due to iron reduction. Thus, although microbial reduction of Fe(III) is an important process, the evidence is not conclusive. Our results have shown that a better understanding of the degradation of complex mixtures of hydrocarbons under anaerobic conditions is important in the application of natural attenuation as a remedial method for soil and groundwater contamination.     

Anaerobic degradation of naphthalene and 2-methylnaphthalene by strains of marine sulfate-reducing bacteria

The anaerobic biodegradation of naphthalene, an aromatic hydrocarbon in tar and petroleum, has been repeatedly observed in environments but scarcely in pure cultures. To further explore the relationships and physiology of anaerobic naphthalene-degrading microorganisms, sulfate-reducing bacteria (SRB) were enriched from a Mediterranean sediment with added naphthalene. Two strains (NaphS3, NaphS6) with oval cells were isolated which showed naphthalene-dependent sulfate reduction. According to 16S rRNA gene sequences, both strains were Deltaproteobacteria and closely related to each other and to a previously described naphthalene-degrading sulfate-reducing strain (NaphS2) from a North Sea habitat. Other close relatives were SRB able to degrade alkylbenzenes, and phylotypes enriched anaerobically with benzene. If in adaptation experiments the three naphthalene-grown strains were exposed to 2-methylnaphthalene, this compound was utilized after a pronounced lag phase, indicating that naphthalene did not induce the capacity for 2-methylnaphthalene degradation. Comparative denaturing gel electrophoresis of cells grown with naphthalene or 2-methylnaphthalene revealed a striking protein band which was only present upon growth with the latter substrate. Peptide sequences from this band perfectly matched those of a protein predicted from genomic libraries of the strains. Sequence similarity (50% identity) of the predicted protein to the large subunit of the toluene-activating enzyme (benzylsuccinate synthase) from other anaerobic bacteria indicated that the detected protein is part of an analogous 2-methylnaphthalene-activating enzyme. The absence of this protein in naphthalene-grown cells together with the adaptation experiments as well as isotopic metabolite differentiation upon growth with a mixture of d(8)-naphthalene and unlabelled 2-methylnaphthalene suggest that the marine strains do not metabolize naphthalene by initial methylation via 2-methylnaphthalene, a previously suggested mechanism. The inability to utilize 1-naphthol or 2-naphthol also excludes these compounds as free intermediates. Results leave open the possibility of naphthalene carboxylation, another previously suggested activation mechanism.     

Methylation is the initial reaction in anaerobic naphthalene degradation by a sulfate-reducing enrichment culture

The sulfate-reducing culture N47 can utilize naphthalene or 2-methylnaphthalene as the sole carbon source and electron donor. Here we show that the initial reaction in the naphthalene degradation pathway is a methylation to 2-methylnaphthalene which then undergoes the subsequent oxidation to the central metabolite 2-naphthoic acid, ring reduction and cleavage. Specific metabolites occurring exclusively during anaerobic degradation of 2-methylnaphthalene were detected during growth on naphthalene, i.e. naphthyl-2-methyl-succinate and naphthyl-2-methylene-succinate. Additionally, all three enzymes involved in anaerobic degradation of 2-methylnaphthalene to 2-naphthoic acid that could be measured in vitro so far, i.e. naphthyl-2-methyl-succinate synthase, succinyl-CoA:naphthyl-2-methyl-succinate CoA-transferase and naphthyl-2-methyl-succinyl-CoA dehydrogenase were also detected in naphthalene-grown cells with similar activities. Induction experiments were performed to study the growth behaviour of the cell when transferred from naphthalene to 2-methylnaphthalene or vice versa. When the cells were transferred from naphthalene to 2-methylnaphthalene they grew immediately, indicating that no new enzymes had to be induced. On the contrary, the transfer of cells from 2-methylnaphthalene to naphthalene caused a lag-phase of almost 100 days demonstrating that an additional catabolic enzyme has to be activated in this case. We propose the methylation as a novel general mechanism of activation reactions in anaerobic degradation of unsubstituted aromatic hydrocarbons.     

Co-metabolic conversion of toluene in anaerobic n-alkane-degrading bacteria

Diverse microorganisms have been described to degrade petroleum hydrocarbons anaerobically. Strains able to utilize n-alkanes do not grow with aromatic hydrocarbons, whereas strains able to utilize aromatic hydrocarbons do not grow with n-alkanes. To investigate this specificity in more detail, three anaerobic n-alkane degraders (two denitrifying, one sulfate-reducing) and eight anaerobic alkylbenzene degraders (five denitrifying, three sulfate-reducing) were incubated with mixtures of n-alkanes and toluene. Whereas the toluene degradationers formed only the characteristic toluene-derived benzylsuccinate and benzoate, but no n-alkane-derived metabolites, the n-alkane degraders formed toluene-derived benzylsuccinate, 4-phenylbutanoate, phenylacetate and benzoate besides the regular n-alkane-derived (1-methylalkyl)succinates and methyl-branched alkanoates. The co-metabolic conversion of toluene by anaerobic n-alkane degraders to the level of benzoate obviously follows the anaerobic n-alkane degradation pathway with C-skeleton rearrangement and decarboxylation rather than the β-oxidation pathway of anaerobic toluene metabolism. Hence, petroleum-derived aromatic metabolites detectable in anoxic environments may not be exclusively formed by genuine alkylbenzene degraders. In addition, the hitherto largely unexplored fate of fumarate hydrogen during the activation reactions was examined with (2,3-(2) H(2) )fumarate as co-substrate. Deuterium was completely exchanged with hydrogen at the substituted carbon atom (C-2) of the succinate adducts of n-alkanes, whereas it is retained in toluene-derived benzylsuccinate, regardless of the type of enzyme catalysing the fumarate addition reaction.     

Genomic insights into the metabolic potential of the polycyclic aromatic hydrocarbon degrading sulfate‐reducing Deltaproteobacterium N47

Anaerobic degradation of polycyclic aromatic hydrocarbons (PAHs) is an important process during natural attenuation of aromatic hydrocarbon spills. However, knowledge about metabolic potential and physiology of organisms involved in anaerobic degradation of PAHs is scarce. Therefore, we introduce the first genome of the sulfate‐reducing Deltaproteobacterium N47 able to catabolize naphthalene, 2‐methylnaphthalene, or 2‐naphthoic acid as sole carbon source. Based on proteomics, we analysed metabolic pathways during growth on PAHs to gain physiological insights on anaerobic PAH degradation. The genomic assembly and taxonomic binning resulted in 17 contigs covering most of the sulfate reducer N47 genome according to general cluster of orthologous groups (COGs) analyses. According to the genes present, the Deltaproteobacterium N47 can potentially grow with the following sugars including d ‐mannose, d ‐fructose, d ‐galactose, α‐d ‐glucose‐1P, starch, glycogen, peptidoglycan and possesses the prerequisites for butanoic acid fermentation. Despite the inability for culture N47 to utilize NO₃^‐ as terminal electron acceptor, genes for nitrate ammonification are present. Furthermore, it is the first sequenced genome containing a complete TCA cycle along with the carbon monoxide dehydrogenase pathway. The genome contained a significant percentage of repetitive sequences and transposase‐related protein domains enhancing the ability of genome evolution. Likewise, the sulfate reducer N47 genome contained many unique putative genes with unknown function, which are candidates for yet‐unknown metabolic pathways.