Beta Subclass Of Proteobacteria Research Articles

Ammonia-oxidizers’ diversity in wastewater treatment processes

ABSTRACTThe diversity of ammonia-oxidizing bacteria (AOB) within the beta-subclass of Proteobacteria was investigated by genus- and family-specific real-time quantitative PCR (qPCR) assays on samples drawn from wastewater treatment systems. The 16S rRNA gene copy numbers ranged from 7.0 × 103 to 6.8 × 106, 1.1 × 107 to 1.8 × 107, and 2.9 × 105 to 1.5 × 107 copies/mL, respectively. Volumetric ammonium load (VAL) in the wastewater treatment systems calculated using the AOB numbers was in the range of 2.1–12.6 mM/d. Distribution patterns of eutrophic (i.e. Nitrosomonas europaea and Nitrosomonas nitrosa clusters) and oligotrophic (i.e. Nitrosomonas cryotolerans cluster) AOB groups were correlated with the VAL values. A high possibility of potential false-positive detection by family-specific qPCR assays was established by evaluating theoretical specificity in in silico and experimental investigations. The specificities of genus-specific qPCR assays were confirmed by amoA PCR, followed by cloning and sequencing. VAL must be the factor influencing the inclusion of AOB species. However, there was no significant correlation between the volatile suspended solid concentration representing chemical oxygen demand and N. europaea’s community population, indicating that the degree of ammonia oxidation influenced the community cluster of Nitrosomonas relatively more.

Effect of nitrogen deficiency during SBR operation on PHA storage and microbial diversity

In this study, changes in microbial diversity and polyhydroxyalkanoate storage ability of activated sludge under aerobic dynamic feeding conditions were investigated. Two sequencing batch reactors were operated with and without nitrogen limitation, by applying a moderate sludge retention time. Polymer storage abilities of the biomasses were improved significantly under dynamic conditions, in terms of specific polymer storage rate, polymer storage yield and polymer content of activated sludge. Moreover, aerobic dynamic feeding conditions together with nutrient limitation further improved the storage ability of the mixed population. Polymer storage yields of the biomass enriched under nitrogen-sufficient and nitrogen-deficient conditions were 0.43 and 0.61 Cmmol PHA/Cmmol substrate, respectively. This study also contributes to the knowledge of activated sludge microbiology, providing detailed information about temporal changes in community structure under dynamic conditions. Microbial community structure was determined by 16S rDNA clone library construction. Also changes in communities under different operating conditions were monitored by DGGE analysis based on bacterial 16S rDNA. The beta subclass of Proteobacteria was the most abundant phylum in both reactors during the operation periods. Changes in the community structure occurred in terms of relative abundance of the operational taxonomic units (OTUs) rather than the OTU types present in the system.

Gammaproteobacteria occurrence and microdiversity in Tyrrhenian Sea sediments as revealed by cultivation-dependent and -independent approaches

Bacterial diversity in Tyrrhenian Sea sediments was assessed using cultivation-dependent and -independent approaches. Samples collected from the different sediment layers (up to 30 cm) relative to four seamount and non-seamount stations, at depths from 3425 to 3580 m, were subjected to DNA extraction and 16S rRNA amplification targeting the V3 region. Denaturing gradient gel electrophoresis (DGGE) showed several heterogeneous profiles and 27 single bands were excised and sequenced. Sequence analysis revealed the presence of Firmicutes, Actinobacteria and Chloroflexi in 26% of the DGGE bands and a predominance of sequences affiliated to cultivable and uncultivable clones of Gammaproteobacteria (55%). To corroborate these findings, cultivation attempts were performed that allowed the isolation of 87 strains assigned to the proteobacterial classes. Identification was achieved by means of automated ribosomal intergenic spacer analysis (ARISA) and by 16S rDNA sequencing. The isolates were related to the gamma, alpha and beta subclasses of Proteobacteria with respective percentages of 77, 17 and 6%. The most predominant Gammaproteobacteria isolates, assigned to the Psychrobacter marincola and P. submarinus clade ( n = 53) and to Halomonas aquamarina ( n = 14), showed a huge intraspecific diversity with 29 distinct ARISA haplotypes. The detection by both approaches of these psychrophilic and moderately halophilic species and their extensive microdiversity indicated their predominance in Tyrrhenian Sea sediments where they constituted the indigenous microflora.

Nitrogen removal in a Sequencing Batch Biofilm Reactor: effect of carbon availability and intermittent aeration

This study aimed to investigate the effects of carbon availability and intermittent aeration on nitrogen removal in a Sequencing Batch Biofilm Reactor (SBBR). The percentage of nitrogen removal in the SBBRs operating with dump fill and slow fill with optimum intermittent aeration was quite similar, 75.7% and 69.2%, respectively, indicating that intermittent aeration allowed a considerable energy saving without compromising significantly nitrogen removal. Accumulation of storage polymers by heterotrophic bacteria was only observed in the dump fill mode of operation. FISH analyses of the biofilm indicated that ammonia-oxidisers belonged to the beta-subclass Proteobacteria and nitrite-oxidisers were affiliated with the genus Nitrospira.

Changes of catabolic genes and microbial community structures during biodegradation of nonylphenol ethoxylates and nonylphenol in natural water microcosms

Changes of possible key catabolic genes and microbial community structures during the degradation of NPEOs and NP in natural water microcosms were investigated using the most-probable-number-polymerase chain reaction (MPN-PCR) and terminal restriction fragment length polymorphism (T-RFLP). The copy number of catechol 2,3-dioxygenase ( C23O) DNA increased significantly during NPEO and NP degradation, suggesting that meta-cleavage of the aromatic rings of NPEOs and NP might have happened. Catechol 1,2-dioxygenase ( C12O) DNA, alkane-catabolic genes ( alk), and 16S rDNA, on the other hand, did not change notably, suggesting that the two genes might not be the relevant genes for NPEOs and NP degradation. The 16S rRNA gene-based T-RFLP analysis results indicated that specific and different dominant (or degrading) bacteria should be selected, depending on the substances. A strain with a DNA length of 78 bp, which might be affiliated with the beta subclass of Proteobacteria, became the dominant species for NPEO degradation, while strains at 88 and 198 bp were dominant in the NP microcosm. Diversity of microbial community structure tended to be simplified after NPEO degradation, while that in the NP microcosm remained relatively stable. Five clusters were obtained according to the similarity in community structures of different microcosms by cluster analysis, which were consistent with the biodegradation behaviors of different microcosms. This is the first report on genetic evidence of a possible aromatic ring meta-cleaving pathway of NPEOs and NP in an aquatic environment.

Comparative analysis of ammonia-oxidizing bacterial communities in two lakes in North Germany and the Baltic Sea

One important pathway of the nitrogen cycle in aquatic environments is the oxidation of ammonia to nitrite by ammonia-oxidizing bacteria (AOB). In this study the composition of AOB communities was compared between fresh (lakes Plusssee and Schohsee) and brackish (Baltic Sea) water at two different levels: i) between environments and ii) within different depths in each environment. Changes in the community structure were studied by denaturing gradient gel electrophoresis (DGGE) and clone libraries of PCR products of 16S rRNA genes (rDNA) from AOB of the beta subclass of proteobacteria. Each environment displayed a particular DGGE band pattern. In Plusssee and the Baltic Sea, the differentiation of communities in epi- and metalimnion from those in hypolimnion coincided with a distinct stratification of the water column. In Schohsee with an aerobic hypolimnion, the communities at all depths were similar. AOB communities in sediments were different from those in the water column. The composition of clone libraries showed the presence of specific Nitrosomonas and Nitrosospira-like sequences in each environment and habitat.

Isolation and characterization of Dechlorospirillum anomalous strain JB116 from a sewage treatment plant

The dissimilatory perchlorate reducers mainly belong to two monophyletic groups, viz. Dechloromonas and Azospira in the beta subclass of Proteobacteria. The present study describes isolation and genetic characterization of Dechlorospirillum anomalous strain JB116 that belongs to alpha subclass of Proteobacteria. The strain JB116 was isolated under facultative anaerobic conditions on a growth medium containing sodium perchlorate and sodium acetate as electron (e(-)) acceptor and e(-) donor, respectively. The strain is a spirillum shaped, dissimilatory perchlorate and nitrate reducer that prefers nitrate to perchlorate. It grows heterotrophically with acetate at temperatures between 25-35 degrees C, NaCl concentrations between 0-0.5% and pH of 7-7.8. The strain JB116 is the second only representative strain within D. anomalous that shares 99% 16S rDNA sequence similarity with the type strain D. anomalous strain WD.

Fluorescent In Situ Hybridization (FISH) for the Detection of Bacterial Community in Activated Sludge from Textile Factories

Conventional methods used to study the bacterial community structure in activated sludge are not sufficient enough to determine the compositions of the bacterial populations responsible for biodegradation. Activated sludge samples from 3 textile factories were analyzed by fluorescent in situ hybridization (FISH) using rRNA probes and by phase-contrast microscopy. In Factory-I, the predominant groups were the beta-subclass of Proteobacteria and the cytophaga-flavobacterium (CF) cluster (33.3% and 31.0%) followed by gamma-subclass (17.1%), high G+C DNA (HGC) gram-positive (15.4%) and alpha-subclass (3.2%). Factory-II showed a similar pattern (32.7%, 31.8%, 17.5%, 16.4%, 1.6%) but with lower concentrations, while Factory-III showed predominant alpha- and beta-subclasses (25.2%, 25.0%) and CF cluster (24.8%) followed by the gamma-subclass (13.6%) and the HGC (11.4%) at much lower concentrations. The floc characteristic for factory-I and -II was normal, however factory-III had diffuse and atypical flocs. In conclusion, the FISH technique provided comprehensive information on the bacterial consortia of activated sludge samples. The compositions of the bacterial community and their concentrations together with the floc characteristics might be some of the reasons that affect the operational efficiencies among the 3 textile factories.

The Phylogenetic Diversity of Aerobic Organotrophic Bacteria from the Dagang High-Temperature Oil Field

The distribution and species diversity of aerobic organotrophic bacteria in the Dagan high-temperature oil field (China), which is exploited via flooding, have been studied. Twenty-two strains of the most characteristic thermophilic and mesophilic aerobic organotrophic bacteria have been isolated from the oil stratum. It has been found that, in a laboratory, the mesophilic and thermophilic isolates grow in the temperature, pH, and salinity ranges characteristic of the injection well near-bottom zones or of the oil stratum, respectively, and assimilate a wide range of hydrocarbons, fatty acids, lower alcohols, and crude oil, thus exhibiting adaptation to the environment. Using comparative phylogenetic 16S rRNA analysis, the taxonomic affiliation of the isolates has been established. The aerobic microbial community includes gram-positive bacteria with a high and low G+C content of DNA, and gamma and beta subclasses of Proteobacteria. The thermophilic bacteria belong to the genera Geobacillus and Thermoactinomyces, and the mesophilic strains belong to the genera Bacillus, Micrococcus, Cellulomonas, Pseudomonas, and Acinetobacter. The microbial community of the oil stratum is dominated by known species of the genus Geobacillus (G. subterraneus, G. stearothermophilus, and G. thermoglucosidasius) and a novel species "Geobacillus jurassicus." A number of novel thermophilic oil-oxidizing bacilli have been isolated.

Comparative Analysis of Nitrifying Bacteria in Full–Scale Oxidation Ditch and Aerated Nitrification Biofilter by Using Fluorescent In Situ Hybridization (FISH) and Denaturing Gradient Gel Electrophoresis (DGGE)

In this study, nitrification performances and composition of nitrifying populations in a full–scale oxidation ditch and a high–rate submerged media nitrification biofilter were comparatively analyzed. In addition to different reactor configurations, effects of differing operational conditions on the nitrification efficiency and bacterial diversity were also explored and evaluated thoroughly. In microbial analysis of sludge samples fluorescent in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE) techniques were used complementary to each other. The extended aeration oxidation ditch subjected to the study is operated as a nitrogen and phosphorus removal system consisting of anaerobic, anoxic, and aerobic zones. The high–rate submerged media aerated filter is operated as nitrification step following the conventional activated sludge unit and the nitrified wastewater is discharged to the sea without complete nitrogen removal. In situ hybridization results have indicated that Nitrosomonas–like ammonia oxidizing and Nitrospira–related nitrite oxidizing bacteria were intensively present in vigorous flocs in nitrification biofilter while carbonaceous bacteria belong to beta subclass of Proteobacteria were considerably dominant in oxidation ditch. Low quantities of nitrifiers in oxidation ditch were also confirmed by the dissimilarity in intensive bands between two systems obtained with DGGE analysis.

Bacterial community shifts in nonylphenol polyethoxylates-enriched activated sludge

A molecular approach was used to evaluate the effect of nonylphenol ethoxylate surfactants on the bacterial diversity in lab-scale activated sludge reactors. Separate bench-scale units were fed synthetic wastewater with and without addition of branched nonylphenol ethoxylates (NP nEO). The performance of the reactors, in terms of carbonaceous removal was largely unaffected by the presence of NP10EO in the feeding solution. However, addition of NP10EO exerted a pronounced shift in bacterial community composition. In situ hybridization analyzing larger phylogenetic groups of bacteria with ribosomal RNA-targeted oligonucleotide probes revealed the dominance of clusters composed of Betaproteobacteria, accounting for up to one-third of 4′,6-diamidino-2-phenylindol-dihydrochloride (DAPI)-stained cells in NP10EO amended reactors and only 5% of DAPI-stained cells in the controls. These shifts in populations of larger phylogenetic groups were confirmed by dot-blot analysis of rRNA. Members of gamma subclass of Proteobacteria were present in low numbers in all activated sludge samples examined, suggesting that only bacteria affiliated with the beta subclass of Proteobacteria may have a specific role in NP10EO degradation.

Chitinimonas taiwanensis gen. nov., sp. nov., a Novel Chitinolytic Bacterium Isolated from a Freshwater Pond for Shrimp Culture

A bacterial strain, designated cfT was isolated from surface water of a freshwater pond for shrimp (Macrobrachium rosenbergii) culture at Ping-Tung (Southern Taiwan). Cells of this organism were Gram-negative, slightly curved rods which were motile by means of a single polar flagellum. Strain cfT utilized chitin as the exclusive carbon, nitrogen, and energy source for growth, both under aerobic and anaerobic conditions. Optimum conditions for growth were between 25 and 37 degrees C, 0 and 1% NaCl and pH 6 to 8. Strain cfT secreted two chitinolytic enzymes with approximate molecular weight 52 and 64 kDa, which hydrolyzed chitin to produce chitotriose as major product. Sequence comparison of an almost complete 16S rDNA gene showed less than 92% sequence similarity with known bacterial species. Phylogenetic analysis based on the neighbour-joining and other methods indicated that the organism formed a distinct lineage within the beta-subclass of Proteobacteria. The predominant cellular fatty acids of strain cfT were hexadecanoic acid (about 29%), octadecenoic acid (about 12%) and summed feature 3 (16:1 omega7c or 15 iso 2-OH or both [about 49%]). Its DNA base ratio was 62.8 mol% G+C. We propose to classify strain cfT (= CCRC 17210T = LMG 22011T) as Chitinimonas taiwanensis gen. nov., sp. nov.

Isolation and characterization of bacteria capable of degrading phenol and reducing nitrate under low-oxygen conditions.

Nitrate-reducing bacteria capable of degrading phenol were isolated from natural and contaminated environments under low-oxygen conditions with nitrate-containing media, using phenol as a sole carbon and energy source. A total of 27 bacteria able to degrade phenol and reduce nitrate under low-oxygen conditions were isolated from all of the inoculum samples, regardless of previous phenol contamination. For all of these bacteria, oxygen was an essential requirement for phenol degradation. Nitrate reduction by 19 of the strains was insensitive to 10 mM sodium azide, and these strains were placed into the alpha- and beta-subclasses of Proteobacteria and two were Gram-positive bacteria. To date, the order of Rhizobiales has hardly been reported to have an ability to degrade aromatic compounds. Interestingly, our study showed that all isolates that were placed into the alpha-subclass of Proteobacteria are in the order of Rhizobiales. Furthermore, the genus Agrobacterium was isolated from most inoculum samples and one genus of Gram-positive bacteria, Staphylococcus, was also isolated. In the case of the remaining eight strains, nitrate reduction was inhibited by 10 mM sodium azide. Of these strains, seven were placed into the gamma-subclass of Proteobacteria.

Denitrification and bacterial community structure in the cascade of six reservoirs on a tropical river in Brazil

Denitrification activity was measured by the acetylene blockage technique in six reservoirs of the Tiete River, southeastern Brazil, in March 1999 and July 1999. The total number of bacteria was measured by DAPI while bacterial community structures were estimated with fluorescent in situ hybridization (FISH) using oligonucleotide probes specific to ribosomal RNA of the domain Bacteria; the alpha, beta and gamma subclasses of the Class Proteobacteria; the Cytophaga-Flavobacterium group; the Order Planktomycetales; and the Domain Archaea. Denitrification was detected only where anoxia or low concentrations of dissolved oxygen were observed. Maximum in situ denitrification integrated in the water column ranged from 1.36 to 3.79 mmol-N2O m−2 day−1 in the rainy season and in the dry season, respectively, both at Station 1, the upstream portion of Barra Bonita Reservoir influenced by the sewage carried out by the river from Sao Paulo City. Bacterioplankton community analysis by FISH showed that the Alpha subclass of the class Proteobacteria was the main subgroup in the middle and low Tiete River (Fig. 3), represented by 10.3–19.2% of total, followed by the Beta Subclass of Proteobacteria and the Cytophaga/Flavobacterium group, represented by 8.3–13.3% and 3.4–10.9%, respectively). This result differs from the proportion of bacterial subgroups in water bodies of temperate regions, suggesting that bacterial communities of water bodies of the tropical region may differ from those of temperate regions.

Isolation and Molecular Characterization of Thiosulfate-oxidizing Bacteria from an Italian Rice Field Soil

In rice paddy soils an active cycling of sulfur compounds takes place. To elucidate the diversity of thiosulfate-oxidizing bacteria these organisms were enriched from bulk soil and rice roots by the most probable number method in liquid medium. From the MPN enrichment cultures 21 bacterial strains were isolated on solid mineral medium, and could be further shown to produce sulfate from thiosulfate. These strains were characterized by 16S rDNA analyses. The isolates were affiliated to seven different phylogenetic groups within the alpha- and beta-subclass of Proteobacteria. Two of these phylotypes were already described as S-oxidizers in this environment (Xanthobacter sp. and Bosea sp. related strains), but five groups represented new S-oxidizers in rice field soil. These isolates were closely related to Mesorhizobium loti, to Hydrogenophaga sp., to Delftia sp., to Pandoraea sp. or showed sequence similarity to a strain of Achromobacter sp.

Alicycliphilus denitrificans gen. nov., sp. nov., a cyclohexanol-degrading, nitrate-reducing beta-proteobacterium.

A facultatively denitrifying bacterium, strain K601T, was isolated at 30 degrees C from a municipal sewage plant on cyclohexanol as sole carbon source and nitrate as electron acceptor. Under aerobic conditions this strain used acetate, fumarate, lactate, pyruvate, crotonate, indole, glucose, vanillate, 4-hydroxybenzoate, m-cresol, o-cresol and p-cresol. Under denitrifying conditions the strain used cyclohexanol, cyclohexanone, 1,3-cyclohexanedione, 2-cyclohexenone, 1,3-cyclohexanediol (cis and trans), monocarboxylic acids (C2-C7), adipate, pimelate, 5-oxocaproate, citrate, 2-oxoglutarate, succinate, malate, crotonate, lactate, pyruvate and fumarate. Cells were short rods, 0.6 microm wide and 1-2 microm long, motile, non-spore-forming, Gram-negative, and catalase- and oxidase-positive. Strain K601T used nitrate, nitrite and oxygen as electron acceptors, but not sulfate, sulfite or fumarate. The DNA G+C content of strain K601T was 66 mol%. Phylogenetic analysis, based on 16S rDNA sequencing, showed that strain K601T represents a separate lineage of the family Comamonadaceae in the beta-subclass of Proteobacteria. Based on the high 16S rDNA sequence divergence and phenotypic characteristics, the name Alicycliphilus denitrificans gen. nov., sp. nov. is proposed for this strain. The type strain is K60IT (=DSM 14773T =CIP 107495T).

Deammonification in biofilm systems: population structure and function

For the development of alternative concepts for the cost effective treatment of wastewaters with high ammonium content and low C/N-ratio, autotrophic consortia of micro-organisms with the ability to convert ammonium directly into N2 are of particular interest. Several full-scale industrial biofilm plants eliminating nitrogen without carbon source for years in a stable process, are suspected for some time to harbor active anaerobic ammonium oxidizers in deeper, oxygen-limited biofilm layers. In order to identify the processes of the single-stage nitrogen elimination (deammonification) in biofilm systems and to allocate them to the responsible micro-organisms, a deammonifying moving-bed pilot plant was investigated in detail. 15N-labelled tracer compounds were used as well as 16S rDNA libraries and in situ identification of dominant organisms. The usage of rRNA-targeted oligonucleotide probes (FISH) was particularly emphasized on the ammonium oxidizers of the beta-subclass of Proteobacteria and on the members of the order Planctomycetales. The combined application of these methods led to a deeper insight into the population structure and function of a deammonifying biofilm.

Phylogenetic analysis of dichloromethane-utilizing aerobic methylotrophic bacteria

The phylogenetic relationships of 12 aerobic dichloromethane-degrading bacteria that implement different C1-assimilation pathways was determined based on 16S ribosomal RNA sequences and DNA–DNA hybridization data. The restricted facultative methylotroph “Methylophilus leisingerii” DM11 with the ribulose monophosphate pathway was found to belong to the genus Methylophilus cluster of the beta subclass of Proteobacteria. The facultative methylotroph Methylorhabdus multivorans DM13 was assigned to a separate branch of the alpha-2 group of Proteobacteria. Paracoccus methylutens DM12, which utilizes C1-compounds via the Calvin cycle, was found to belong to the alpha-3 group of Proteobacteria (more precisely, to the genus Paracoccus cluster). Thus, phylogenetic analysis confirmed the taxonomic status of these recently characterized bacteria. According to the degree of DNA homology, several novel strains of methylotrophic bacteria were divided into three genotypic groups within the alpha-2 group of the Proteobacteria. Genotypic group 1, comprising strains DM1, DM3, and DM5 through DM9, and genotypic group 3, comprising strain DM10, were phylogenetically close to the methylotrophic bacteria of the genus Methylopila, whereas genotypic group 2 (strain DM4) was close to bacteria of the genus Methylobacterium. The genotypic groups obviously represent distinct taxa of methylotrophic bacteria, whose status should be confirmed by phenotypic analysis.

Impact of herbicides on the abundance and structure of indigenous beta-subgroup ammonia-oxidizer communities in soil microcosms.

In this study, mixtures of five herbicide-formulated products (atrazine, dicamba, fluometuron, metolachlor, and sulfentrazone) were applied to soil microcosm columns in increasing concentrations. The toxic impact of herbicides on the indigenous beta-subclass Proteobacteria autotrophic ammonia-oxidizer (beta-AAO) community was assessed. The beta-AAO population abundances were estimated by competitive polymerase chain reaction (PCR) targeting the gene amoA, encoding the alpha-subunit of ammonia monooxygenase. Community structure was examined by PCR and denaturing gradient gel electrophoresis targeting 16S rDNA with band excision and sequence analysis, and by analysis of amoA gene fragment clone libraries. The 16S rDNA analyses showed that a single ribotype of Nitrosospira cluster 3 was the dominant beta-AAO in all treatments. At a finer scale, amoA clone library analysis suggested a shift in community structure corresponding to the 100-ppm application. Competitive PCR indicated significant differences between treatments. The control exhibited relatively stable population abundance over the time period examined. The 10-ppm treatment induced a population increase, but a significant decrease was induced by the 100-ppm application. At 1,000 ppm, the ammonia-oxidizer population dropped below the method detection limit by the first sampling point. An impact on ammonia oxidizers resulting from the application of herbicides was observed, both in abundance and community structure.

Characterization of the microbial diversity in a biotreatment process using non-culture based methods

Molecular techniques were used to compare the microbial community of suspended activated sludge and attached biofilm in a biological nutrient removal process (TNCU-I) operated under various COD/total P (COD/TP) feed ratios. Analysis of restriction fragment length polymorphism of amplified 16S rRNA genes indicated that the microbial population structures were more closely related among those sludge samples taken from aerobic sludge than that taken from biofilm attached on the RBC. The use of different COD/TP feed ratios (300/2.5-300/25) had no significant effect on the change of microbial structures of sludge samples. The 16S rDNA clone library further indicated that at least eight and six different microbial populations were present in activated sludge and RBC biofilm, respectively. The phylogenetic analysis revealed that six of those eight major clones obtained from the suspended activated sludge were from the beta-subclass of the class Proteobacteria. In contrast, only one clone obtained from biofilm belonged to the beta-subclass of the Proteobacteria. This difference in the microbial population structure was possibly attributed to the growth state (suspended or attached) or carbon source (autographs or heterotrophs) of the sludge samples rather than the effect of the COD/TP ratio used.