Anaerobic biodegradability of phthalic acid isomers and related compounds

All three phthalic acid isomers ( ortho, meta and para benzene dicarboxylic acid) are produced in massive amounts, and used in the chemical industry as plasticizers or for the production of polyester. Wastestreams generated during the production of phthalate isomers generally contain high concentrations of aromatic acids. To study the potential biodegradability of these primarily anthropogenic compounds in anaerobic bioreactors, biodegradability studies were performed. Compounds tested were benzoate, ortho-phthalate, isophthalate, terephthalate, dimethyl phthalate, dimethyl terephthalate, para-toluate and para-xylene. Seed materials tested were two types of granular sludge and digested sewage sludge. It was found that all phthalate isomers and their corresponding dimethyl-esters, could be completely mineralized by all seed materials studied. Lag phases required for 50% degradation of these compounds, ranged from 17 to 156 days. The observed degradation curves could be explained by growth of an initially small amount of organisms in the inoculum with the specific ability to degrade one phthalate isomer. The observed order in the length of the lag phases for the phthalate isomers is: phthalate < terephthalate < isophthalate. This order appears to be related to the environmental abundancy of the different phthalate isomers. The initial step in the degradation pathway of both dimethyl phthalate esters was hydrolysis of the ester sidechain, resulting in the formation of the corresponding mono-methyl-phthalate isomer and phthalate isomer. The rate limiting step in mineralization of both dimethyl phthalate and dimethyl terephthalate was found to be fermentation of the phthalate isomer. Para-toluate was degraded only by digested sewage sludge after a lag phase of 425 days. The observed degradation rates of this compound were very low. No mineralization of para-xylene was observed. In general, the differences in the lag phases between different seed materials were relatively small. These results indicate that the time needed for the start-up of anaerobic bioreactors treating wastewaters containing phthalic acid isomers, depends little on the microbial composition of the seed material applied, but may take several months.     

Identification and cultivation of anaerobic, syntrophic long-chain fatty acid-degrading microbes from mesophilic and thermophilic methanogenic sludges

We investigated long-chain fatty acid (LCFA)-degrading anaerobic microbes by enrichment, isolation, and RNA-based stable isotope probing (SIP). Primary enrichment cultures were made with each of four LCFA substrates (palmitate, stearate, oleate, or linoleate, as the sole energy source) at 55 degrees C or 37 degrees C with two sources of anaerobic granular sludge as the inoculum. After several transfers, we obtained seven stable enrichment cultures in which LCFAs were converted to methane. The bacterial populations in these cultures were then subjected to 16S rRNA gene-based cloning, in situ hybridization, and RNA-SIP. In five of seven enrichment cultures, the predominant bacteria were affiliated with the family Syntrophomonadaceae. The other two enrichment cultures contained different bacterial populations in which the majority of members belonged to the phylum Firmicutes and the class Deltaproteobacteria. After several attempts to isolate these dominant bacteria, strain MPA, belonging to the family Syntrophomonadaceae, and strain TOL, affiliated with the phylum Firmicutes, were successfully isolated. Strain MPA converts palmitate to acetate and methane in syntrophic association with Methanospirillum hungatei. Even though strain TOL assimilated [(13)C]palmitate in the original enrichment culture, strain TOL has not shown the ability to degrade LCFAs after isolation. These results suggest that microbes involved in the degradation of LCFAs under methanogenic conditions might not belong only to the family Syntrophomonadaceae, as most anaerobic LCFA-degrading microbes do, but may also be found in phylogenetically diverse bacterial groups.     

Non-sulfate-reducing, syntrophic bacteria affiliated with desulfotomaculum cluster I are widely distributed in methanogenic environments

The classical perception of members of the gram-positive Desulfotomaculum cluster I as sulfate-reducing bacteria was recently challenged by the isolation of new representatives lacking the ability for anaerobic sulfate respiration. For example, the two described syntrophic propionate-oxidizing species of the genus Pelotomaculum form the novel Desulfotomaculum subcluster Ih. In the present study, we applied a polyphasic approach by using cultivation-independent and culturing techniques in order to further characterize the occurrence, abundance, and physiological properties of subcluster Ih bacteria in low-sulfate, methanogenic environments. 16S rRNA (gene)-based cloning, quantitative fluorescence in situ hybridization, and real-time PCR analyses showed that the subcluster Ih population composed a considerable part of the Desulfotomaculum cluster I community in almost all samples examined. Additionally, five propionate-degrading syntrophic enrichments of subcluster Ih bacteria were successfully established, from one of which the new strain MGP was isolated in coculture with a hydrogenotrophic methanogen. None of the cultures analyzed, including previously described Pelotomaculum species and strain MGP, consumed sulfite, sulfate, or organosulfonates. In accordance with these phenotypic observations, a PCR-based screening for dsrAB (key genes of the sulfate respiration pathway encoding the alpha and beta subunits of the dissimilatory sulfite reductase) of all enrichments/(co)cultures was negative with one exception. Surprisingly, strain MGP contained dsrAB, which were transcribed in the presence and absence of sulfate. Based on these and previous findings, we hypothesize that members of Desulfotomaculum subcluster Ih have recently adopted a syntrophic lifestyle to thrive in low-sulfate, methanogenic environments and thus have lost their ancestral ability for dissimilatory sulfate/sulfite reduction.     

High rate treatment of terephthalic acid production wastewater in a two-stage anaerobic bioreactor

The feasibility was studied of anaerobic treatment of wastewater generated during purified terephthalic acid (PTA) production in two-stage upflow anaerobic sludge blanket (UASB) reactor system. The artificial influent of the system contained the main organic substrates of PTA-wastewater: acetate, benzoate, and terephthalate. Three parallel operated reactors were used for the second stage, and seeded with a suspended terephthalate degrading culture, with and without additional methanogenic granular sludge (two different types). The first stage UASB-reactor was seeded with methanogenic granular sludge. Reactors were operated at 37 degrees C and pH 7. During the first 300 days of operation a clear distinction between the biomass grown in both reactor stages was obtained. In the first stage, acetate and benzoate were degraded at a volumetric loading rate of 40 g-COD/L . day at a COD-removal efficiency of 95% within the first 25 days of operation. No degradation of terephthalate was obtained in the first stage during the first 300 days of operation despite operation of the reactor at a decreased volumetric loading rate with acetate and benzoate of 9 g-COD/L . day from day 150. Batch incubation of biomass from the reactor with terephthalate showed that the lag-phase prior to terephthalate degradation remained largely unchanged, indicating that no net growth of terephthalate degrading biomass occurred in the first stage reactor. From day 300, however, terephthalate degradation was observed in the first stage, and the biomass in this reactor could successfully be enriched with terephthalate degrading biomass, resulting in terephthalate removal capacities of 15 g-COD/L . day. Even though no single reason could be identified why (suddenly) terephthalate degradation was obtained after such a long period of operation, it is suggested that the solid retention time as well the prevailing reactor concentrations acetate and benzoate may have played an important role. From day 1 of operation, terephthalate was degraded in the second stage. In presence of methanogenic granular biomass, high terephthalate removal capacities were obtained in these reactors (15 g-COD/L . day) after approximately 125 days of operation. From the results obtained it is concluded that terephthalate degradation is the bottleneck during anaerobic treatment of PTA-wastewater. Pre-removal of acetate and benzoate in staged bioreactor reduces the lag-phase prior to terephthalate degradation in latter stages, and enables high rate treatment of PTA-wastewater.     

Pelotomaculum terephthalicum sp. nov. and Pelotomaculum isophthalicum sp. nov.: two anaerobic bacteria that degrade phthalate isomers in syntrophic association with hydrogenotrophic methanogens

An anaerobic phthalate isomer-degrading strain (JT^T) that we previously isolated was characterized. In addition, a strictly anaerobic, mesophilic, syntrophic phthalate isomer-degrading bacterium, designated strain JI^T, was isolated and characterized in this study. Both were non-motile rods that formed spores. In both strains, the optimal growth was observed at temperatures around 37°C and neutral pH. In syntrophic co-culture with the hydrogenotrophic methanogen Methanospirillum hungatei, both strains could utilize two or three phthalate isomers for growth, and produce acetate and methane as end products. Strain JT^T was able to grow on isophthalate, terephthalate, and a number of low-molecular weight aromatic compounds, such as benzoate, hydroquinone, 2-hydroxybenzoate, 3-hydroxybenzoate, 2,5-dihydroxybenzoate, 3-phenylpropionate in co-culture with M. hungatei. It could also grow on crotonate, hydroquinone and 2,5-dihydroxybenzoate in pure culture. Strain JI^T utilized all of the three phthalate isomers as well as benzoate and 3-hydroxybenzoate for growth in co-culture with M. hungatei. No substrates were, however, found to support the axenic growth of strain JI^T. Neither strain JT^T nor strain JI^T could utilize sulfate, sulfite, thiosulfate, nitrate, fumarate, Fe (III) or 4-hydroxybenzoate as electron acceptor. Phylogenetically, strains JT^T and JI^T were relatively close to the members of the genera Pelotomaculum and Cryptanaerobacter in ‘Desulfotomaculum lineage I’. Physiological and chemotaxonomic characteristics indicated that the two isolates should be classified into the genus Pelotomaculum, creating two novel species for them. Here, we propose Pelotomaculum terephthalicum sp. nov. and Pelotomaculum isophthalicum sp. nov. for strain JT^T and strain JI^T, respectively. The type strains are strains JT^T (= DSM 16121^T = JCM 11824^T = NBRC 100523^T) and JI^T (= JCM 12282^T = BAA-1053^T) for P. terephthalicum and P. isophthalicum, respectively.Nucleotide sequence accession number: The GenBank/EMBL/DDBJ accession numbers of the 16S rRNA gene sequences of strains JT^T and JI^T are AB091323 and AB232785, respectively     

Cultivation of methanogens from shallow marine sediments at Hydrate Ridge, Oregon

Little is known about the methanogenic degradation of acetate, the fate of molecular hydrogen and formate or the ability of methanogens to grow and produce methane in cold, anoxic marine sediments. The microbes that produce methane were examined in permanently cold, anoxic marine sediments at Hydrate Ridge (44 degrees 35' N, 125 degrees 10' W, depth 800 m). Sediment samples (15 to 35 cm deep) were collected from areas of active methane ebullition or areas where methane hydrates occurred. The samples were diluted into enrichment medium with formate, acetate or trimethylamine as catabolic substrate. After 2 years of incubation at 4 degrees C to 15 degrees C, enrichment cultures produced methane. PCR amplification and sequencing of the rRNA genes from the highest dilutions with growth suggested that each enrichment culture contained a single strain of methanogen. The level of sequence similarity (91 to 98%) to previously characterized prokaryotes suggested that these methanogens belonged to novel genera or species within the orders Methanomicrobiales and Methanosarcinales. Analysis of the 16S rRNA gene libraries from DNA extracted directly from the sediment samples revealed phylotypes that were either distantly related to cultivated methanogens or possible anaerobic methane oxidizers related to the ANME-1 and ANME-2 groups of the Archaea. However, no methanogenic sequences were detected, suggesting that methanogens represented only a small proportion of the archaeal community.     

The role of benzoate in anaerobic degradation of terephthalate

The effects of acetate, benzoate, and periods without substrate on the anaerobic degradation of terephthalate (1, 4-benzene-dicarboxylate) by a syntrophic methanogenic culture were studied. The culture had been enriched on terephthalate and was capable of benzoate degradation without a lag phase. When incubated with a mixture of benzoate and terephthalate, subsequent degradation with preference for benzoate was observed. Both benzoate and acetate inhibited the anaerobic degradation of terephthalate. The observed inhibition is partially irreversible, resulting in a decrease (or even a complete loss) of the terephthalate-degrading activity after complete degradation of benzoate or acetate. Irreversible inhibition was characteristic for terephthalate degradation only because the inhibition of benzoate degradation by acetate could well be described by reversible noncompetitive product inhibition. Terephthalate degradation was furthermore irreversibly inhibited by periods without substrate of only a few hours. The inhibition of terephthalate degradation due to periods without substrate could be overcome through incubation of the culture with a mixture of benzoate and terephthalate. In this case no influence of a period without substrate was observed. Based on these observations it is postulated that decarboxylation of terephthalate, resulting in the formation of benzoate, is strictly dependent on the concomitant fermentation of benzoate. In the presence of higher concentrations of benzoate, however, benzoate is the favored substrate over terephthalate, and the culture loses its ability to degrade terephthalate. In order to overcome the inhibition of terephthalate degradation by benzoate and acetate, a two-stage reactor system is suggested for the treatment of wastewater generated during terephthalic acid production.     

Anaerobic degradation of benzoate: batch studies

Response of benzoate along with phenol to different anaerobic inocula has been investigated in batch reactors. In Phase I, the anaerobic biodegradability of benzoate and phenol were evaluated using (a) washed acclimatized granular sludge (WAGS) collected from a passive phenol fed bench-scale up-flow anaerobic sludge blanket reactor (UASBR) and (b) unacclimatized flocculent sludge (UFS) from a UASB based sewage treatment plant (STP). The effect of varying concentrations of benzoate has been investigated in Phase II using acclimatized granular sludge (AGS) from a bench-scale UASBR. Extent of degradation of benzoate was more than the phenol. Increasing benzoate COD from 2500 to 11,700mg/L, resulted in decrease in (i) rate constant, k from 0.79 to 0.11/d and (ii) ultimate biochemical methane potential (microb, g CH4-COD formed/g benzoate COD) from 84% to 60%. Temporal trend conforming to logistic S-curve indicated stressed conditions at higher benzoate concentration. Benzoate degradation was found to be sensitive to nature as well as quantity i.e. food to microorganism (F/M) of inocula used.     

<Emphasis Type="Italic">Sporotomaculum syntrophicum</Emphasis> sp. nov., a novel anaerobic,syntrophic benzoate-degrading bacterium isolated from methanogenic sludge treating wastewater from terephthalate manufacturing

An anaerobic, mesophilic, syntrophic benzoate-degrading bacterium, designated strain FB(T), was isolated from methanogenic sludge which had been used to treat wastewater from the manufacture of terephthalic acid. Cells were non-motile gram-positive rods that formed spores. The optimum temperature for growth was 35-40 degrees C, and the optimum pH was 7.0-7.2. A co-culture with the hydrogenotrophic methanogen Methanospirillum hungatei converted benzoate to acetate, carbon dioxide, and methane. Butyrate transiently accumulated at a high concentration of 2.5 mM during degradation. Besides benzoate, no other compound tested supported growth of the co-culture. Crotonate supported growth of strain FB(T) in pure culture. Furthermore, the strain degraded benzoate in pure culture with crotonate as co-substrate to produce acetate and butyrate. The strain was not able to utilize sulfate, sulfite, thiosulfate, nitrate, fumarate, or Fe(III) as electron acceptor. The G+C content of the DNA was 46.8 mol%. Strain FB(T) contained MK-7 as the major quinone and C(16:1) as the major fatty acid. 16S rDNA sequence analysis revealed that the strain was a member of the genus Sporotomaculum, even though it exhibited significant differences, such as the capacity for syntrophic growth, to the known member of the genus. Hence, we propose the name Sporotomaculum syntrophicum sp. nov. for strain FB(T). The type strain is strain FB(T) (DSM 14795, JCM 11475).     

木质纤维素燃料乙醇蒸馏废水的高温厌氧处理及菌群结构分析

【目的】为开发高效的高浓度木质纤维素燃料乙醇蒸馏废水厌氧处理及资源化利用工艺,以活性炭为载体,在实验室规模上对高温厌氧流化床反应器处理木质纤维素燃料乙醇蒸馏废水进行研究。【方法】反应器经65 d梯度驯化后启动,对工艺参数进行一系列优化,并通过基于16S rRNA基因的分子生态学技术分析厌氧污泥中的优势菌群。【结果】实验获得了最优的反应条件和处理效果:厌氧流化床反应器(Anaerobic fluidized bed reactor,AFBR)在温度55±1°C、有机负荷率(OLR)13.8 g COD/(L·d)及水力停留时间(HRT)48 h操作时,COD去除率达到90%以上,同时甲烷产率达到290 mL/g COD;菌群鉴定分析结果显示高温厌氧活性污泥中Clostridia所占比例最大,产甲烷菌属以Methanoculleus和Methanosarcina为主,其它功能菌群主要为Alphaproteobacteria等。【结论】AFBR反应器可高效降解木质纤维素燃料乙醇蒸馏废水并产生生物能源甲烷,其反应体系内微生物种类丰富。     

Anaerobic degradation of xenobiotics by organisms from municipal solid waste under landfilling conditions

The potential for biological transformation of 23 xenobiotic compounds by microorganisms in municipal solid waste (MSW) samples from a laboratory scale landfill reactor was studied. In addition the influence of these xenobiotic compounds on methanogenesis was investigated. All R11, 1,1 dichloroethylene, 2,4,6 trichlorophenol, dimethyl phthalate, phenol, benzoate and phthalic acid added were completely transformed during the period of incubation (> 100 days). Parts of the initially added perchloroethylene, trichloroethylene, R12, R114, diethyl phthalate, dibutyl phthalate and benzylbutyl phthalate were transformed. Methanogenesis from acetate was completely inhibited in the presence of 2,5 dichlorophenol, whereas 2,4,6 trichlorophenol and R11 showed an initial inhibition, whenafter methane formation recovered. No transformation or effect on the anaerobic microflora occurred for R13, R22, R114, 3 chlorobenzoate, 2,4,6 trichlorobenzoate, bis(2 ethyl)hexyl phthalate, diisodecyl phthalate and dinonyl phthalate. The results indicate a limited potential for degradation, of the compounds tested, by microorganisms developing in a methanogenic landfill environment as compared with other anaerobic habitats such as sewage digestor sludge and sediments.Abbreviations BBP benzylbutylphthalate - DEHP bis(2 ethylhexyl) phthalate - 3 CB 3 chlorobenzoate - R22 chlorodifluoromethane - CFC chlorofluorocarbon - R13 chlorotrifluoromethane - cis1,2 DCE cis 1,2 dichloroethylene - DBP dibutyl phthalate - R12 dichlorodifluoromethane - 1,1 DCE 1,1 dichloroethylenel - R114 dichlorotetrafluoroethane - 2,5 DCP 2,5 dichlorophenol - DEP diethyl phthalate - DiDP diisodecyl phthalate - DMP Dimethyl phthalate - DNP dinonyl phthalate - MSW dunicipal solid waste - PCE perchloroethylene - PA phthalic acid - PAE phthalic acid esters - R11 trichlorofluoromethane - 2,4,6 TCB 2,4,6 trichlorobenzoate - 2,4,6 TCP 2,4,6 trichlorophenol - VC vinylchloride     

Anaerobic metabolism of phthalate and other aromatic compounds by a denitrifying bacterium.

The anaerobic metabolism of phthalate and other aromatic compounds by the denitrifying bacterium Pseudomonas sp. strain P136 was studied. Benzoate, cyclohex-1-ene-carboxylate, 2-hydroxycyclohexanecarboxylate, and pimelate were detected as predominant metabolic intermediates during the metabolism of three isomers of phthalate, m-hydroxybenzoate, p-hydroxybenzoate, and cyclohex-3-ene-carboxylate. Inducible acyl-coenzyme A synthetase activities for phthalates, benzoate, cyclohex-1-ene-carboxylate, and cyclohex-3-ene-carboxylate were detected in the cells grown on aromatic compounds. Simultaneous adaptation to these aromatic compounds also occurred. A similar phenomenon was observed in the aerobic metabolism of aromatic compounds by this strain. A new pathway for the anaerobic metabolism of phthalate and a series of other aromatic compounds by this strain was proposed. Some properties of the regulation of this pathway were also discussed.     

Long-Term Enrichment on Cellulose or Xylan Causes Functional and Taxonomic Convergence of Microbial Communities from Anaerobic Digesters

Cellulose and xylan are two major components of lignocellulosic biomass, which represents a potentially important energy source, as it is abundant and can be converted to methane by microbial action. However, it is recalcitrant to hydrolysis, and the establishment of a complete anaerobic digestion system requires a specific repertoire of microbial functions. In this study, we maintained 2-year enrichment cultures of anaerobic digestion sludge amended with cellulose or xylan to investigate whether a cellulose- or xylan-digesting microbial system could be assembled from sludge previously used to treat neither of them. While efficient methane-producing communities developed under mesophilic (35°C) incubation, they did not under thermophilic (55°C) conditions. Illumina amplicon sequencing results of the archaeal and bacterial 16S rRNA genes revealed that the mature cultures were much lower in richness than the inocula and were dominated by single archaeal (genus Methanobacterium) and bacterial (order Clostridiales) groups, although at finer taxonomic levels the bacteria were differentiated by substrates. Methanogenesis was primarily via the hydrogenotrophic pathway under all conditions, although the identity and growth requirements of syntrophic acetate-oxidizing bacteria were unclear. Incubation conditions (substrate and temperature) had a much greater effect than inoculum source in shaping the mature microbial community, although analysis based on unweighted UniFrac distance found that the inoculum still determined the pool from which microbes could be enriched. Overall, this study confirmed that anaerobic digestion sludge treating nonlignocellulosic material is a potential source of microbial cellulose- and xylan-digesting functions given appropriate enrichment conditions.     

Diversity, localization, and physiological properties of filamentous microbes belonging to Chloroflexi subphylum I in mesophilic and thermophilic methanogenic sludge granules

We previously reported that the thermophilic filamentous anaerobe Anaerolinea thermophila, which is the first cultured representative of subphylum I of the bacterial phylum Chloroflexi, not only was one of the predominant constituents of thermophilic sludge granules but also was a causative agent of filamentous sludge bulking in a thermophilic (55 degrees C) upflow anaerobic sludge blanket (UASB) reactor in which high-strength organic wastewater was treated (Y. Sekiguchi, H. Takahashi, Y. Kamagata, A. Ohashi, and H. Harada, Appl. Environ. Microbiol. 67:5740-5749, 2001). To further elucidate the ecology and function of Anaerolinea-type filamentous microbes in UASB sludge granules, we surveyed the diversity, distribution, and physiological properties of Chloroflexi subphylum I microbes residing in UASB granules. Five different types of mesophilic and thermophilic UASB sludge were used to analyze the Chloroflexi subphylum I populations. 16S rRNA gene cloning-based analyses using a 16S rRNA gene-targeted Chloroflexi-specific PCR primer set revealed that all clonal sequences were affiliated with the Chloroflexi subphylum I group and that a number of different phylotypes were present in each clone library, suggesting the ubiquity and vast genetic diversity of these populations in UASB sludge granules. Subsequent fluorescence in situ hybridization (FISH) of the three different types of mesophilic sludge granules using a Chloroflexi-specific probe suggested that all probe-reactive cells had a filamentous morphology and were widely distributed within the sludge granules. The FISH observations also indicated that the Chloroflexi subphylum I bacteria were not always the predominant populations within mesophilic sludge granules, in contrast to thermophilic sludge granules. We isolated two mesophilic strains and one thermophilic strain belonging to the Chloroflexi subphylum I group. The physiological properties of these isolates suggested that these populations may contribute to the degradation of carbohydrates and other cellular components, such as amino acids, in the bioreactors.     

Cultivation and in situ detection of a thermophilic bacterium capable of oxidizing propionate in syntrophic association with hydrogenotrophic methanogens in a thermophilic methanogenic granular sludge

The thermophilic, anaerobic, propionate-oxidizing bacterial populations present in the methanogenic granular sludge in a thermophilic (55 degrees C) upflow anaerobic sludge blanket reactor were studied by cultivation and in situ hybridization analysis. For isolation of propionate-degrading microbes, primary enrichment was made with propionate as the sole energy source at 55 degrees C. After several attempts to purify the microbes, a thermophilic, syntrophic, propionate-oxidizing bacterium, designated strain SI, was isolated in both pure culture and coculture with Methanobacterium thermoautotrophicum. Under thermophilic (55 degrees C) conditions, strain SI oxidized propionate, ethanol, and lactate in coculture with M. thermoautotrophicum. In pure culture, the isolate was found to ferment pyruvate. 16S ribosomal DNA sequence analysis revealed that the strain was relatively close to members of the genus Desulfotomaculum, but it was only distantly related to any known species. To elucidate the abundance and spatial distribution of organisms of the strain SI type within the sludge granules, a 16S rRNA-targeted oligonucleotide probe specific for strain SI was developed and applied to thin sections of the granules. Fluorescence in situ hybridization combined with confocal laser scanning microscopy revealed that a number of rod-shaped cells were present in the middle and inner layers of the thermophilic granule sections and that they formed close associations with hydrogenotrophic methanogens. They accounted for approximately 1.1% of the total cells in the sludge. These results demonstrated that strain SI was one of the significant populations in the granular sludge and that it was responsible for propionate oxidation in the methanogenic granular sludge in the reactor.     

Aerobic and anaerobic catabolism of phthalic acid by a nitrate-respiring bacterium

A Bacillus sp., isolated by anaerobic enrichment on a o-phthalic acid-nitrate medium, grew either aerobically or anaerobically on phthalic acid. Cells grown anaerobically on phthalate immediately oxidized phthalate and benzoate with nitrate, whereas aerobic oxidation only occurred after a lag period and was inhibited by chloramphenicol. 2-Fluoro-and 3-fluorobenzoate were formed from 3-fluorophthalate by cells grown anaerobically on phthalate. Aerobically grown cells immediately oxidized phthalate, benzoate, 3-hydroxybenzoate and gentisate with oxygen. The aerobic and anaerobic route of catabolism of phthalate may thus share an initial decarboxylation to benzoate. This is the first report of the anaerobic dissimilation of phthalic acid by a pure bacterial culture.     

Detection of anaerobic processes and microorganisms in immobilized activated sludge of a wastewater treatment plant with intense aeration

Attached activated sludge from the Krasnaya Polyana (Sochi) wastewater treatment plant was studied after the reconstruction by increased aeration and water recycle, as well as by the installation of a bristle carrier for activated sludge immobilization. The activated sludge biofilms developing under conditions of intense aeration were shown to contain both aerobic and anaerobic microorganisms. Activity of a strictly anaerobic methanogenic community was revealed, which degraded organic compounds to methane, further oxidized by aerobic methanotrophs. Volatile fatty acids, the intermediates of anaerobic degradation of complex organic compounds, were used by both aerobic and anaerobic microorganisms. Anaerobic oxidation of ammonium with nitrite (anammox) and the presence of obligate anammox bacteria were revealed in attached activated sludge biofilms. Simultaneous aerobic and anaerobic degradation of organic contaminants by attached activated sludge provides for high rates of water treatment, stability of the activated sludge under variable environmental conditions, and decreased excess sludge formation.     

Syntrophorhabdus aromaticivorans gen. nov., sp. nov., the first cultured anaerobe capable of degrading phenol to acetate in obligate syntrophic associations with a hydrogenotrophic methanogen

Phenol degradation under methanogenic conditions has long been studied, but the anaerobes responsible for the degradation reaction are still largely unknown. An anaerobe, designated strain UI(T), was isolated in a pure syntrophic culture. This isolate is the first tangible, obligately anaerobic, syntrophic substrate-degrading organism capable of oxidizing phenol in association with an H(2)-scavenging methanogen partner. Besides phenol, it could metabolize p-cresol, 4-hydroxybenzoate, isophthalate, and benzoate. During the degradation of phenol, a small amount of 4-hydroxybenzoate (a maximum of 4 microM) and benzoate (a maximum of 11 microM) were formed as transient intermediates. When 4-hydroxybenzoate was used as the substrate, phenol (maximum, 20 microM) and benzoate (maximum, 92 microM) were detected as intermediates, which were then further degraded to acetate and methane by the coculture. No substrates were found to support the fermentative growth of strain UI(T) in pure culture, although 88 different substrates were tested for growth. 16S rRNA gene sequence analysis indicated that strain UI(T) belongs to an uncultured clone cluster (group TA) at the family (or order) level in the class Deltaproteobacteria. Syntrophorhabdus aromaticivorans gen. nov., sp. nov., is proposed for strain UI(T), and the novel family Syntrophorhabdaceae fam. nov. is described. Peripheral 16S rRNA gene sequences in the databases indicated that the proposed new family Syntrophorhabdaceae is largely represented by abundant bacteria within anaerobic ecosystems mainly decomposing aromatic compounds.     

Aerobic degradation of diethyl phthalate by Sphingomonas sp

An aerobic diethyl phthalate (DEP) degrading bacterium, DEP-AD1, was isolated from activated sludge. Based on its 16S rDNA sequence, this isolate was identified belonging to Sphingomonas genus with 99% similarity to Sphingomonas sp. strain C28242 and 98% similarity to S. capsulate. The specific degradation rate of DEP was concentration dependent with a maximum of 14 mg-DEP/(Lh). Results of degradation tests showed that DEP-AD1 could also degrade monoethyl phthalate (MEP), dimethyl phthalate (DMP), dibutyl phthalate (DBP), and diethylhexyl phthalate (DEHP), but not phthalate and benzoate.