Recent developments in the biochemistry and ecology of enhanced biological phosphorus removal

Most of the genes encoding the enzymes involved in polyP synthesis and degradation and in phosphate transport have been studied in various Gram-negative bacteria. Progress has also been made in studying the biochemical mechanisms underlying the process of enhanced biological phosphorus removal (EBPR), in particular in lab-scale systems fed with acetate or acetate plus glucose as the sole carbon and energy sources. By applying 13C-NMR, previous models concerning anaerobic carbon metabolism have been advanced and the role of glycogen in providing reducing equivalents in EBPR is definitely demonstrated. The role of the citric acid cycle in supplying reducing equivalents for the conversion of acetyl-CoA into poly-beta-hydroxybutyrate and poly-beta-hydroxyvalerate has been discussed. An incomplete citric acid cycle has been proposed to provide a small part of the reducing equivalents. Polyphosphate:AMP phosphotransferase and polyphosphatase were readily detectable in EBPR sludge fed with acetate plus glucose, but polyphosphate kinase remained undetected. In a lab-scale EBPR system, fed for several months with only acetate as carbon source, a Rhodocyclus-like bacterium (R6) was highly enriched and is therefore probably responsible for EBPR in systems fed with acetate only. This R6-type bacterium was however also present in other EBPR sludges (but to a lesser extent), and may therefore play an important role in EBPR in general. This organism accumulates polyhydroxyalkanoates anaerobically and polyP under aerobic conditions. Unlike members of the genus Rhodocyclus, bacterium R6 cannot grow phototrophically. Therefore a provisional new genus Candidatus and species Accumulibacter phosphatis was proposed.     

Phylogeny and in situ identification of a morphologically conspicuous bacterium, Candidatus Magnospira bakii, present at very low frequency in activated sludge

A morphologically conspicuous bacterium that constituted a very small fraction (< 0.01%) of the total microbial community of activated sludge was enriched and analysed phylogenetically by a combination of cultivation-independent molecular and physical methods. The large, corkscrew-shaped, filamentous bacteria were first detected in municipal activated sludge by light microscopy owing to their unusual rotating gliding motility. Various attempts at microbiological enrichment and pure culture isolation with traditional techniques failed, as did attempts to retrieve the morphotype of interest by micromanipulation. In situ hybridization with the group-specific, rRNA-targeted oligonucleotide probe CF319a indicated a phylogenetic affiliation to the Cytophaga-Flexibacter group of the Cytophaga-Flavobacterium-Bacteroides phylum. Based on strong morphological resemblance to members of the genus Saprospira, additional 16S rRNA-targeted oligonucleotides with more narrow specificity were designed and evaluated for in situ hybridization to the morphotype of interest. Flow cytometric cell sorting based on the fluorescence conferred by probe SGR1425 and forward scatter enabled a physical enrichment of the helical coiled cells. Subsequent polymerase chain reaction (PCR) amplification of 16S rDNA fragments from whole fixed sorted cells with a primer pair based on probes CF319a and SGR1425 resulted in the retrieval of 12 almost identical partial 16S rDNA fragments with sequence similarities among each other of more than 99.2%. In situ hybridizations proved that the sequences that showed the highest similarity (88.4%) to the 16S rRNA of Saprospira grandis were indeed retrieved from the corkscrew-shaped filaments. The bacterium is likely to be a member of a genus of which no species has been cultured hitherto. It was consequently tentatively named 'Magnospira bakii' and has the taxonomic rank of Candidatus Magnospira bakii, as the ultimate taxonomic placement has to await its cultivation. In this study, it was demonstrated that even bacteria occurring at very low frequencies in highly complex environmental samples can be retrieved selectively without cultivation for further molecular analysis.     

Phosphate removal under denitrifying conditions by Brachymonas sp. strain P12 and Paracoccus denitrificans PP15

In this study, we used the denitrifying phosphorus-removing bacterium Brachymonas sp. strain P12 to investigate the enhanced biologic phosphorus-removal (EBPR) mechanism involved with polyhydroxybutyrate (PHB), glycogen, and phosphorus uptake in the presence of acetate under anoxic or aerobic conditions. The results showed that excess acetate concentration and aerobic cultivation can enhance PHB formation efficiency and that PHB formation might be stimulated by glycogenolysis of the cellular glycogen. The efficiency of the uptake of anoxic phosphorus was greater when PHB production was lower. The EBPR mechanism of Brachymonas sp. strain P12 for PHB, phosphorus, and glycogen was similar to the conventional anaerobic-aerobic (or anaerobic-anoxic) EBPR models, but these models were developed under anoxic or aerobic conditions only, without an anaerobic stage. The anoxic or aerobic log phase of growth is divided into two main phases: the early log phase, in which acetate and glycogen are consumed to supply enough energy and reducing power for PHB formation and cell growth (phosphorus assimilation), and the late log phase, which ends the simultaneous degradation of PHB and remaining acetate for polyphosphate accumulation. Glycogenolysis plays a significant role in the alternate responses between PHB formation and phosphorus uptake under anoxic or aerobic conditions. After the application of the denitrifying phosphorus-removing bacterium Brachymonas sp. strain P12, aerobic cultivation increases the level of PHB production, and anoxic cultivation further increases phosphorus uptake.     

Fine-scale population structure of Accumulibacter phosphatis in enhanced biological phosphorus removal sludge

To investigate the diversities of Accumulibacter phosphatis and its polyhydroxyalkanoate (PHA) synthase gene (phaC) in enhanced biological phosphorus removal (EBPR) sludge, an acetate-fed sequencing batch reactor was operated. Analysis of microbial communities using fluorescence in situ hybridization and 16S rRNA gene clone libraries showed that the population of Accumulibacter phosphatis in the EBPR sludge comprised more than 50% of total bacteria, and was clearly divided into two subgroups with about 97.5% sequence identity of the 16S rRNA genes. PAO phaC primers targeting the phaC genes of Accumulibacter phosphatis were designed and applied to retrieve fragments of putative phaC homologs of Accumulibacter phosphatis from EBPR sludge. PAO phaC primers targeting G1PAO, G2PAO, and G3PAO groups produced PCR amplicons successfully; the resulting sequences of the phaC gene homologs were diverse, and were distantly related to metagenomic phaC sequences of Accumulibacter phosphatis with 75-98% DNA sequence identities. Degenerate NPAO (non-PAO) phaC primers targeting phaC genes of non- Accumulibacter phosphatis bacteria were also designed and applied to the EBPR sludge. Twenty-four phaC homologs retrieved from NPAO phaC primers were different from the phaC gene homologs derived from Accumulibacter phosphatis, which suggests that the PAO phaC primers were specific for the amplification of phaC gene homologs of Accumulibacter phosphatis, and the putative phaC gene homologs by PAO phaC primers were derived from Accumulibacter phosphatis in the EBPR sludge. Among 24 phaC homologs, a phaC homolog (G1NPAO-2), which was dominant in the NPAO phaC clone library, showed the strongest signal in slot hybridization and shared approximately 60% nucleotide identity with the G4PAO group of Accumulibacter phosphatis, which suggests that G1NPAO-2 might be derived from Accumulibacter phosphatis. In conclusion, analyses of the 16S rRNA and phaC genes showed that Accumulibacter phosphatis might be phylogenetically and metabolically diverse.     

Phylogenetic characterization and in situ detection of a Cytophaga-Flexibacter-Bacteroides phylogroup bacterium in Tuber borchii vittad. Ectomycorrhizal mycelium

Mycorrhizal ascomycetous fungi are obligate ectosymbionts that colonize the roots of gymnosperms and angiosperms. In this paper we describe a straightforward approach in which a combination of morphological and molecular methods was used to survey the presence of potentially endo- and epiphytic bacteria associated with the ascomycetous ectomycorrhizal fungus Tuber borchii Vittad. Universal eubacterial primers specific for the 5' and 3' ends of the 16S rRNA gene (16S rDNA) were used for PCR amplification, direct sequencing, and phylogenetic analyses. The 16S rDNA was amplified directly from four pure cultures of T. borchii Vittad. mycelium. A nearly full-length sequence of the gene coding for the prokaryotic small-subunit rRNA was obtained from each T. borchii mycelium studied. The 16S rDNA sequences were almost identical (98 to 99% similarity), and phylogenetic analysis placed them in a single unique rRNA branch belonging to the Cytophaga-Flexibacter-Bacteroides (CFB) phylogroup which had not been described previously. In situ detection of the CFB bacterium in the hyphal tissue of the fungus T. borchii was carried out by using 16S rRNA-targeted oligonucleotide probes for the eubacterial domain and the Cytophaga-Flexibacter phylum, as well as a probe specifically designed for the detection of this mycelium-associated bacterium. Fluorescent in situ hybridization showed that all three of the probes used bound to the mycelium tissue. This study provides the first direct visual evidence of a not-yet-cultured CFB bacterium associated with a mycorrhizal fungus of the genus Tuber.     

Rubrobacter-related bacteria associated with rosy discolouration of masonry and lime wall paintings

A molecular approach was chosen to analyse the correlation between bacterial colonisation and rosy discolouration of masonry and lime wall paintings of two historically important buildings in Austria and Germany. The applied molecular method included PCR amplification of genes encoding the small subunit rRNA of bacteria (16S rDNA), genetic fingerprinting by denaturing gradient gel electrophoresis (DGGE), construction of 16S rDNA clone libraries, and comparative phylogenetic sequence analyses. The bacterial community of one red-pigmented biofilm sampled in Herberstein (Austria) contained bacteria phylogenetically related to the genera Saccharopolyspora, Nocardioides, Pseudonocardia, Rubrobacter, and to a Kineococcus-like bacterium. The bacterial community of the second red-pigmented biofilm sampled in Herberstein contained bacteria related to Arthrobacter, Comamonas, and to Rubrobacter. Rubrobacter-related 16S rDNA sequences were the most abundant. In the red-pigmented biofilm sampled in Burggen (Germany), only Rubrobacter-related bacteria were identified. No Rubrobacter-related bacteria were detected in non-rosy biofilms. The majority of sequences (70%) obtained from the bacterial communities of the three investigated rosy biofilms were related to sequences of the genus Rubrobacter (red-pigmented bacteria), demonstrating a correlation between Rubrobacter-related bacteria and the phenomenon of rosy discolouration of masonry and lime wall paintings.     

Microbial Composition and Structure of a Rotating Biological Contactor Biofilm Treating Ammonium-Rich Wastewater without Organic Carbon

High nitrogen losses were observed in a rotating biological contactor (RBC) treating ammonium-rich (up to 500 mg NH4(+)-N/L) but organic-carbon-poor leachate from a hazardous waste landfill in K?lliken, Switzerland. The composition and spatial structure of the microbial community in the biofilm on the RBC was analyzed with specific attention for the presence of aerobic ammonium and nitrite oxidizing bacteria and anaerobic ammonium oxidizers. Anaerobic ammonium oxidation (anammox) involves the oxidation of ammonium with nitrite to N2. First the diversity of the biofilm community was determined from sequencing cloned PCR-amplified 16S rDNA fragments. This revealed the presence of a number of very unusual 16S rDNA sequences, but very few sequences related to known ammonium or nitrite oxidizing bacteria. From analysis of biofilm samples by fluorescence in situ hybridization with known phylogenetic probes and by dot-blot hybridization of the same probes to total RNA purified from biofilm samples, the main groups of microorganisms constituting the biofilm were found to be ammonium-oxidizing bacteria from the Nitrosomonas europaea/eutropha group, anaerobic ammonium-oxidizing bacteria of the "Candidatus Kuenenia stuttgartiensis" type, filamentous bacteria from the phylum Bacteroidetes, and nitrite-oxidizing bacteria from the genus Nitrospira. Aerobic and anaerobic ammonium-oxidizing bacteria were present in similar amounts of around 20 to 30% of the biomass, whereas members of the CFB phylum were present at around 7%. Nitrite oxidizing bacteria were only present in relatively low amounts (less than 5% determined with fluorescence in situ hybridization). Data from 16S rRNA dot-blot and in situ hybridization were not in all cases congruent. FISH analysis of thin-sliced and fixed biofilm samples clearly showed that the aerobic nitrifiers were located at the top of the biofilm in an extremely high density and in alternating clusters. Anammox bacteria were exclusively present in the lower half of the biofilm, whereas CFB-type filamentous bacteria were present throughout the biofilm. The structure and composition of these biofilms correlated very nicely with the proposed physiological functional separations in ammonium conversion.     

Structure and functional analysis of the microbial community in an aerobic: anaerobic sequencing batch reactor (SBR) with no phosphorus removal

The bacterial community of an aerobic:anaerobic non-P removing SBR biomass fed a mixture of acetate and glucose was analysed using several 16S rRNA based methods. Populations responsible for anaerobic glucose and acetate assimilation were determined with fluorescent in situ hybridization (FISH) in combination with microautoradiography (FISH/MAR). At 'steady state' this community consisted of alpha-Proteobacteria (26%) and gamma-Proteobacteria (14%), mainly appearing as large cocci in tetrads (i.e. typical 'G-Bacteria'). Large numbers of low G+C bacteria (22%), and high G+C Gram-positive bacteria (29%) seen as small cocci in clusters or in sheets were also detected after FISH. DGGE fingerprinting of PCR amplified 16S rDNA fragments and subsequent cloning and sequencing of several of the major bands led to the identification of some of these populations. They included an organism 98% similar in its 16S rRNA sequence to Micropruina glycogenica, and ca. 76% of the high G+C bacteria responded to a probe MIC 184, designed against it. The rest responded to the KSB 531 probe designed against a high G+C clone sequence, sbr-gs28 reported in other similar systems. FISH analyses showed that both these high G+C populations were almost totally dominated by small clustered cocci. Only ca. 2% of cells were beta-Proteobacteria. None of the alpha- and gamma-Proteobacterial 'G-bacteria' responded to FISH probes designed for the 'G-Bacteria' Amaricoccus spp. or Defluvicoccus vanus. FISH/MAR revealed that not all the alpha-Proteobacterial 'G-Bacteria' could take up acetate or glucose anaerobically. Almost all of the gamma-Proteobacterial 'G-Bacteria' assimilated acetate anaerobically but not glucose, the low G+C clustered cocci only took up glucose, whereas the high G+C bacteria including M. glycogenica and the sbr-gs28 clone assimilated both acetate and glucose. All bacteria other than the low G+C small cocci and a few of the alpha-Proteobacteria accumulated PHB. The low G+C bacteria showing anaerobic glucose assimilation ability were considered responsible for the lactic acid produced anaerobically by this SBR biomass, and M. glycogenica for its high glycogen content.     

Microbial selection of polyphosphate-accumulating bacteria in activated sludge wastewater treatment processes for enhanced biological phosphate removal

Activated sludge processes with alternating anaerobic and aerobic conditions (the anaerobic-aerobic process) have been successfully used for enhanced biological phosphate removal (EBPR) from wastewater. It is known that polyphosphate-accumulating bacteria (PAB) play an essential role for EBPR in the anaerobic-aerobic process. The present paper reviews limited information available on the metabolism and the microbial community structure of EBPR, highlighting the microbial ecological selection of PAB in EBPR processes. Exposure of microorganisms to alternate carbon-rich anaerobic environments and carbon-poor aerobic environments in the anaerobic-aerobic process induces the key metabolic characteristics of PAB, which include organic substrate uptake followed by its conversion to stored polyhydroxyalkanoate (PHA) and hydrolysis of intracellular polyphosphate accompanied by subsequent Pi release under anaerobic conditions. Intracellular glycogen is assumed to function as a regulator of the redox balance in the cell. Storage of glycogen is a key strategy for PAB to maintain the redox balance in the anaerobic uptake of various organic substrates, and hence to win in the microbial selection. Acinetobacter spp., Microlunatus phosphovorus, Lampropedia spp., and the Rhodocyclus group have been reported as candidates of PAB. PAB may not be composed of a few limited genospecies, but involve phylogenetically and taxonomically diverse groups of bacteria. To define microbial community structure of EBPR processes, it is needed to look more closely into the occurrence and behavior of each species of PAB in various EBPR processes mainly by molecular methods because many of PAB seem to be impossible to culture.     

Molecular evidence for genus level diversity of bacteria capable of catalyzing anaerobic ammonium oxidation

Recently, a bacterium capable to oxidize ammonium anaerobically at a high rate was identified as novel member of the Planctomycetales (Strous, M., Fuersi, J. A., Kramer, E. H. M., Logemann, S., Muyzer, G., van de Pas-Schoonen, K. T., Webb, R. I., Kufnen, J. G., and Jetten, M. S. M.: Nature 400, 446-449, 1999). Here we investigated the microbial community structure of a trickling filter biofilm with a high anaerobic ammonium oxidation activity. Fluorescence in situ hybridization (FISH) with a set of nine probes designed for specific identification of the recently described anaerobic ammonium oxidizer demonstrated that only one probe hybridized to bacteria within the biofilm. For phylogenetic characterization of putative biofilm anaerobic ammonium oxidizers a full-cycle 16S rDNA approach was performed by using a Planctomycetales-specific forward primer for PCR amplification. Of the twenty-five 16S rDNA fragments (1364 bp in length) amplified from the biofilm, nine were affiliated to the Planctomycetales. Comparative analysis showed that these sequences were more than 98.9% similar to each other but only distantly related to the previously recognized anaerobic ammonium oxidizer (below 91% similarity) and all other organisms represented in public 16S rRNA databases (similarities of below 79%). The retrieved sequences and the previously recognized anaerobic ammonium oxidizer represent two well-separated groups of a deep-branching lineage within the Planctomycetales. Quantitative FISH analysis with a newly designed specific probe showed that the novel bacterium, provisionally classified as "Candidatus Kuenenia stuttgartiensis" constituted the dominant fraction of the biofilm bacteria. In situ probing revealed that ammonia-oxidizing bacteria of the beta-subclass of Proteobacteria were also present, albeit in significant smaller amounts, within the anoxic biofilm. Comparative sequence analysis of a stretch of the gene encoding ammonia-monooxygenase (amoA) demonstrated the occurrence of the DNA of at least three different populations of beta-subclass ammonia oxidizers within the biofilm.     

Polynucleotide probes that target a hypervariable region of 16S rRNA genes to identify bacterial isolates corresponding to bands of community fingerprints

Temperature gradient gel electrophoresis (TGGE) is well suited for fingerprinting bacterial communities by separating PCR-amplified fragments of 16S rRNA genes (16S ribosomal DNA [rDNA]). A strategy was developed and was generally applicable for linking 16S rDNA from community fingerprints to pure culture isolates from the same habitat. For this, digoxigenin-labeled polynucleotide probes were generated by PCR, using bands excised from TGGE community fingerprints as a template, and applied in hybridizations with dot blotted 16S rDNA amplified from bacterial isolates. Within 16S rDNA, the hypervariable V6 region, corresponding to positions 984 to 1047 (Escherichia coli 16S rDNA sequence), which is a subset of the region used for TGGE (positions 968 to 1401), best met the criteria of high phylogenetic variability, required for sufficient probe specificity, and closely flanking conserved priming sites for amplification. Removal of flanking conserved bases was necessary to enable the differentiation of closely related species. This was achieved by 5' exonuclease digestion, terminated by phosphorothioate bonds which were synthesized into the primers. The remaining complementary strand was removed by single-strand-specific digestion. Standard hybridization with truncated probes allowed differentiation of bacteria which differed by only two bases within the probe target site and 1.2% within the complete 16S rDNA. However, a truncated probe, derived from an excised TGGE band of a rhizosphere community, hybridized with three phylogenetically related isolates with identical V6 sequences. Only one of the isolates comigrated with the excised band in TGGE, which was shown to be due to identical sequences, demonstrating the utility of a combined TGGE and V6 probe approach.     

高效产氢细菌新种--Ethanologenbacterium sp.X-1的分离鉴定及其产氢效能

为获得高效产氢发酵细菌 ,采用改进的厌氧Hungate培养技术 ,从生物制氢反应器CSTR中分离一株产氢细菌X 1。对该株细菌进行了形态学特征、生理生化指标、16SrDNA和 16S 2 3SrDNA间隔区序列分析等研究。结果表明与最相近的种属Clostridiumcellulosi和Acetanaerobacteriumelongatum等的 16SrRNA基因序列同源性为 94 %以下。16S 2 3SrRNA间隔区基因序列比对分析显示保守区域仅为tRNAAla和tRNAIle序列 ,其它可变部位没有同源性区域 ,鉴定为新属Ethanologenbacteriumsp .。该株细菌为专性厌氧杆菌 ,代谢特征为乙醇发酵 ,葡萄糖发酵产物主要为乙醇、乙酸、H2 和CO2 。在pH4 0和 36℃条件下最大产氢速率是 2 8 3mmolH2 (gdrycell·h)。经鉴定和产氢效能分析表明该菌株是一新属的高效产氢细菌     

Phylogenetic Characterization and In Situ Detection of a Cytophaga-Flexibacter-Bacteroides Phylogroup Bacterium in Tuber borchii Vittad. Ectomycorrhizal Mycelium

Mycorrhizal ascomycetous fungi are obligate ectosymbionts that colonize the roots of gymnosperms and angiosperms. In this paper we describe a straightforward approach in which a combination of morphological and molecular methods was used to survey the presence of potentially endo- and epiphytic bacteria associated with the ascomycetous ectomycorrhizal fungus Tuber borchii Vittad. Universal eubacterial primers specific for the 5′ and 3′ ends of the 16S rRNA gene (16S rDNA) were used for PCR amplification, direct sequencing, and phylogenetic analyses. The 16S rDNA was amplified directly from four pure cultures of T. borchii Vittad. mycelium. A nearly full-length sequence of the gene coding for the prokaryotic small-subunit rRNA was obtained from each T. borchii mycelium studied. The 16S rDNA sequences were almost identical (98 to 99% similarity), and phylogenetic analysis placed them in a single unique rRNA branch belonging to the Cytophaga-Flexibacter-Bacteroides (CFB) phylogroup which had not been described previously. In situ detection of the CFB bacterium in the hyphal tissue of the fungus T. borchii was carried out by using 16S rRNA-targeted oligonucleotide probes for the eubacterial domain and the Cytophaga-Flexibacter phylum, as well as a probe specifically designed for the detection of this mycelium-associated bacterium. Fluorescent in situ hybridization showed that all three of the probes used bound to the mycelium tissue. This study provides the first direct visual evidence of a not-yet-cultured CFB bacterium associated with a mycorrhizal fungus of the genus Tuber.     

Secondary intracellular symbiotic bacteria in aphids of the genus Yamatocallis (Homoptera: Aphididae: Drepanosiphinae).

A novel secondary intracellular symbiotic bacterium from aphids of the genus Yamatocallis (subfamily Drepanosiphinae) was characterized by using molecular phylogenetic analysis, in situ hybridization, and diagnostic PCR detection. In the aphid tissues, this bacterium (tentatively designated YSMS [Yamatocallis secondary mycetocyte symbiont]) was found specifically in large cells surrounded by primary mycetocytes harboring Buchnera cells. Of nine drepanosiphine aphids examined, YSMS was detected in only two species of the same genus, Yamatocallis tokyoensis and Yamatocallis hirayamae. In natural populations of these aphids, YSMS was present in 100% of the individuals. Phylogenetic analysis based on 16S ribosomal DNA (rDNA) sequences demonstrated that YSMS of Y. tokyoensis and Y. hirayamae constitute a distinct and isolated clade in the gamma subdivision of the class Proteobacteria. No 16S rDNA sequences of secondary endosymbionts characterized so far from other aphids showed phylogenetic affinity to YSMS. Based on these results, I suggest that YSMS was acquired by an ancestor of the genus Yamatocallis and has been conserved throughout the evolution of the lineage. By using the nucleotide substitution rate for 16S rDNA of Buchnera spp., the time of acquisition of YSMS was estimated to be about 13 to 26 million years ago, in the Miocene epoch of the Tertiary period.     

Polyphasic approaches to the identification of predominant polyphosphate-accumulating organisms in a laboratory-scale anaerobic/aerobic activated sludge system

By combination of denaturing gradient gel electrophoresis of PCR-amplified 16S rDNA (PCR-DGGE), quinone profiling, and 16S rRNA-targeted fluorescence in situ hybridization (FISH), a polyphosphate-accumulating organism (PAO) responsible for phosphate (P)-removal was identified in activated sludge with high P-removal ability from a laboratory-scale anaerobic/aerobic continuous flow reactor. The DNA fragment from the most dense band on the DGGE gel was closely related to that of 'Candidatus Accumulibacter phosphatis' (beta-Proteobacteria). Quinone profiling also suggested the predominance of beta-Proteobacteria. FISH with a specific oligonucleotide probe designed for the sequence showed that the targeted bacterium was dominant in the activated sludge, and the accumulation and consumption of polyphosphate were observed by dual staining with 4',6-diamidino-2-phenylindole. The bacterium was concluded to be the responsible PAO in the reactor. However, when the P-removal ability per cell slightly decreased, the dominance of the PAO greatly diminished in the activated sludge. Such sludge might be dominated by other types of PAOs.     

MOLECULAR IDENTIFICATION OF A BACTERIUM ASSOCIATED WITH GALL FORMATION IN THE MARINE RED ALGA PRIONITIS LANCEOLATA1

The polymerase chain reaction (PCR) was used to amplify eubacterial small-subunit (16S) ribosomal DNA (rDNA) genes from galls of the marine red alga Prionitis lanceolata Harvey (Gigartinales). These tumors consist of hypertrophied algal cells containing large numbers of intercellular bacteria that remain uncultivable. PCR-amplified 16S rDNAs from surface-sterilized gall tissue plugs were cloned, sequenced, and analyzed by alignment to available small-subunit rRNA sequences (University of Illinois Ribosomal Database Project). Variable regions were identified and used to construct a fluorescently labeled, species-specific oligodeoxynucleotide probe for whole cell in situ hybridization to the gall symbiont. Probe 949 (PLANC.949) localized the P. lanceolata bacterial symbiont in preparations from mature gall tissue. This probe did not hybridize to the rDNA of closely related bacteria included as controls in the same hybridization reactions, In situ hybridization revealed the presence of the same bacterium in association with P. lanceolata gall formation from three central California localities. Distance and parsimony analyses suggest that this organism is a member of the Proteobacteria (alpha subdivision; Rhodobacter group) and is most closely related to Roseobacter denitrificans .     

Combined Molecular and Conventional Analyses of Nitrifying Bacterium Diversity in Activated Sludge: Nitrosococcus mobilis and Nitrospira-Like Bacteria as Dominant Populations

The ammonia-oxidizing and nitrite-oxidizing bacterial populations occurring in the nitrifying activated sludge of an industrial wastewater treatment plant receiving sewage with high ammonia concentrations were studied by use of a polyphasic approach. In situ hybridization with a set of hierarchical 16S rRNA-targeted probes for ammonia-oxidizing bacteria revealed the dominance of Nitrosococcus mobilis-like bacteria. The phylogenetic affiliation suggested by fluorescent in situ hybridization (FISH) was confirmed by isolation of N. mobilis as the numerically dominant ammonia oxidizer and subsequent comparative 16S rRNA gene (rDNA) sequence and DNA-DNA hybridization analyses. For molecular fine-scale analysis of the ammonia-oxidizing population, a partial stretch of the gene encoding the active-site polypeptide of ammonia monooxygenase (amoA) was amplified from total DNA extracted from ammonia oxidizer isolates and from activated sludge. However, comparative sequence analysis of 13 amoA clone sequences from activated sludge demonstrated that these sequences were highly similar to each other and to the corresponding amoA gene fragments of Nitrosomonas europaea Nm50 and the N. mobilis isolate. The unexpected high sequence similarity between the amoA gene fragments of the N. mobilis isolate and N. europaea indicates a possible lateral gene transfer event. Although a Nitrobacter strain was isolated, members of the nitrite-oxidizing genus Nitrobacter were not detectable in the activated sludge by in situ hybridization. Therefore, we used the rRNA approach to investigate the abundance of other well-known nitrite-oxidizing bacterial genera. Three different methods were used for DNA extraction from the activated sludge. For each DNA preparation, almost full-length genes encoding small-subunit rRNA were separately amplified and used to generate three 16S rDNA libraries. By comparative sequence analysis, 2 of 60 randomly selected clones could be assigned to the nitrite-oxidizing bacteria of the genus Nitrospira. Based on these clone sequences, a specific 16S rRNA-targeted probe was developed. FISH of the activated sludge with this probe demonstrated that Nitrospira-like bacteria were present in significant numbers (9% of the total bacterial counts) and frequently occurred in coaggregated microcolonies with N. mobilis.     

16S rDNA characterisation of bacterial and archaeal communities during start-up of anaerobic thermophilic digestion of cattle manure

A laboratory-scale continuously stirred anaerobic thermophilic batch digester was inoculated with cattle manure. Bacterial and archaeal communities, as well as digester performances, were analysed during reactor start-up for about 20 days. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) was used for overall detection and for study of the dynamics of microbial populations. Dominant bacteria and archaea 16S rDNAs were sequenced from the sample on day 12. Ten bacteria and 3 archaea OTUs (operational taxonomic units) were identified from the 52 clones sequenced. Sequences corresponding to the dominant bacterial SSCP peak were phylogenetically close to the 16S rDNA sequence of Bacillus thermoterrestris, whereas sequences corresponding to the two dominant archaeal SSCP peaks were phylogenetically close to the 16S rDNA sequence of Methanoculleus thermophilicus and Methanosarcina thermophila.     

Net P-removal deterioration in enriched PAO sludge subjected to permanent aerobic conditions

Recently, some research in the field of enhanced biological phosphorus removal (EBPR) has been focused on studying systems where the electron donor (substrate) and the electron acceptor (nitrate or oxygen) are present simultaneously. This can occur, for example, in a full scale wastewater treatment plant during heavy rainfall periods when the anaerobic hydraulic retention time is temporarily shortened. To study this situation that could induce EBPR failure, the operation of a sequencing batch reactor (SBR) working under alternating anaerobic-aerobic conditions with an enriched EBPR population (50% Candidatus Accumulibacter phosphatis and less than 1% Candidatus Competibacter phosphatis) was shifted to strict aerobic operation. Seven cycle studies were performed during the 11 days of aerobic operation. Net P-removal was observed in this aerobic SBR during the first 4 days of operation but the system could not achieve net-P removal after this period, although the microbial composition, in terms of percentage of Accumulibacter and Competibacter, did not change significantly. The observed changes in the different compounds analysed (phosphorus, acetate, glycogen and PHB) as well as in the OUR profile indicate that metabolic changes are produced for the adaptation of PAO to aerobic conditions.     

Microbial diversity in an in situ reactor system treating monochlorobenzene contaminated groundwater as revealed by 16S ribosomal DNA analysis

A molecular approach based on the construction of 16S ribosomal DNA clone libraries was used to investigate the microbial diversity of an underground in situ reactor system filled with the original aquifer sediments. After chemical steady state was reached in the monochlorobenzene concentration between the original inflowing groundwater and the reactor outflow, samples from different reactor locations and from inflowing and outflowing groundwater were taken for DNA extraction. Small-subunit rRNA genes were PCR-amplified with primers specific for Bacteria, subsequently cloned and screened for variation by restriction fragment length polymorphism (RFLP). A total of 87 bacterial 16S rDNA genes were sequenced and subjected to phylogenetic analysis. The original groundwater was found to be dominated by a bacterial consortium affiliated with various members of the class of Proteobacteria, by phylotypes not affiliated with currently recognized bacterial phyla, and also by sporulating and non-sporulating sulfate-reducing bacteria. The most occurring clone types obtained from the sediment samples of the reactor were related to the beta-Proteobacteria, dominated by sequences almost identical to the widespread bacterium Alcaligenes faecalis, to low G+C gram-positive bacteria and to Acidithiobacillus ferrooxidans (formerly Thiobacillus ferrooxidans) within the gamma subclass of Proteobacteria in the upper reactor sector. Although bacterial phylotypes originating from the groundwater outflow of the reactors also grouped within different subdivisions of Proteobacteria and low G+C gram-positive bacteria, most of the 16S rDNA sequences were not associated with the sequence types observed in the reactor samples. Our results suggest that the different environments were inhabited by distinct microbial communities in respect to their taxonomic diversity, particular pronounced between sediment attached microbial communities from the reactor samples and free-living bacteria from the groundwater in- and outflow.