Characterization of the bacterial population structure in an anaerobic-aerobic activated sludge system on the basis of respiratory quinone profiles

Bacterial respiratory quinones were used as biomarkers for studying the bacterial population structure, especially the content of Acinetobacter species, in a laboratory-scale anaerobic-aerobic activated sludge system and in the standard aerobic system. All tested sludges contained both ubiquinone and menaquinone, with a molar ratio of about 1:0.5. High-performance liquid chromatography showed that ubiquinone with eight isoprene units (Q-8) was present as the predominant ubiquinone, Q-10 was the second most common type, and Q-9 and other homologs were minor components in the anaerobic-aerobic sludge and the standard aerobic sludge. Bacteriological examination indicated that, in both sludge systems, Q-8-containing bacteria constituted a large proportion of the aerobic heterotrophic bacterial flora, but only a few strains with Q-9 were found. These findings demonstrate that the population of Acinetobacter species, which contain Q-9 as the major quinone, is negligible in those environments. The present results suggest that the introduction of anaerobic conditions into the aerobic batch process has little influence on the bacterial community structure.     

Ubiquinone system of the form-genusChrysosporium

The ubiquinone (coenzyme Q: Q) system of 17 strains of the form-genusChrysosporium was analyzed by high performance liquid chromatography (HPLC) and found to show a heterogeneous distribution of the major ubiquinone. Q-9, Q-10 or Q-10(H₂) was found to be the major ubiquinone in 3, 9 and 5 strains, respectively. It was further demonstrated that the teleomorphs of the species characterized by Q-9 and Q-10 could be classified into two separate families, Arthrodermataceae (Q-9) and Onygenaceae (Q-10), which were defined within the revised order Onygenales by Currah. Teleomorphs ofChrysosporium species having Q-10(H₂) have not been found. This paper also includes the ubiquinone system of dermatophytes which relate to the form-genusChrysosporium morphologically.     

The role of ubiquinones in regulating lipid metabolism in rat thymocytes

The effects of ubiquinones Q-1, Q-2, Q-8 and Q-9 on lipid biosynthesis in thymocytes in vitro were studied during incubation of cells with ubiquinones within the concentration range of 1-100 mM. A 2-fold inhibition of cholesterol synthesis in thymocytes by ubiquinone Q-9 occurred, when the exogenous ubiquinone concentration in the medium was no less than 40 mM. Incubation of thymocytes with unrelated ubiquinone (Q-1 and Q-2-40 and 100 mM, respectively) resulted in the inhibition of cholesterol synthesis. The inhibiting effect was of the same order of magnitude as that during incubation with ubiquinone Q-9. Ubiquinone Q-8 showed a tendency to inhibit cholesterol synthesis in rat thymocytes. The inhibiting effect of ubiquinone on cholesterol metabolism in thymocytes is specific and is not coupled with fatty acid metabolism.     

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.     

Bulking sludge in biological nutrient removal systems

Bulking sludge problems are commonly reported in biological nutrient removal (BNR) systems. This has led to the general belief that intrinsic BNR conditions favor the growth of undesirable and excessive filamentous bacteria. The present study shows that other factors have a major role in bulking, and not the BNR conditions. These factors have been verified in well-controlled, strictly anoxic-aerobic and strictly anaerobic-aerobic sequencing batch reactor systems. The experimental results show that conditions known to be responsible for bulking sludge in aerobic systems (i.e., low concentration of electron donor and/or electron acceptor) did not lead to bulking. Even when acetate was present at very low concentrations in the aerobic stage of an anaerobic-aerobic bio-P system, the sludge settleability remained very good. This clearly demonstrates that good bio-P activity can stabilize and improve sludge settleability. The presence of microaerophilic conditions in the anoxic stage of the anoxic-aerobic system was the only factor leading to worsening sludge settling characteristics. The results are discussed in light of our previous hypothesis about the importance of diffusion-limited substrate uptake for the development of filamentous structures in biological flocs. The hypothesis is extended to anaerobic-aerobic and anoxic-aerobic conditions, typical of BNR-activated sludge systems. Taking into account the effect of feeding patterns on biochemical rates and on the development of filamentous bacterial structures, we recommend the adoption of plug-flow selector configurations, with strictly anaerobic and/or strictly anoxic conditions, wherein microaerophilic conditions are excluded, in order to maintain reliable and robust BNR performance.     

The role of ubiquinones in the regulation of lipid metabolism in rat thymocytes

The effect of ubiquinones Q-1, Q-2, Q-8 and Q-9 on lipid metabolism in rat thymocytes in vitro was studied. The cells were incubated in a medium containing ubiquinones within the concentration range from 1 to 100 microM. A 2-fold decreased cholesterol synthesis was observed in thymocytes incubated with ubiquinone Q-9 at a concentration of exogenous ubiquinone of no less than 40 microM. Incubation of thymocytes with ubiquinones UQ-1 and UQ-2 that are characteristic of rats (40 microM and 100 microM) resulted in a decrease of cholesterol synthesis. Ubiquinone-8 had a tendency to inhibit the cholesterogenesis in rat thymocytes.     

Ubiquinone systems of Coccidioides immitis, the causative agent of coccidioidomycosis

Abstract The ubiquinone (coenzyme Q) systems of eleven strains of Coccidioides immitis were determined by high performance liquid chromatography (HPLC). The ubiquinone profile of the fungi was shown to be homogeneous: in all of the strains, ubiquinone-10 (Q-10) was demonstrated to be the major component, with Q-9 as a minor component. The results imply that the ubiquinone system may serve as an additional phenotypic criterion for identifying the fungus.     

Quinone profiling of bacterial communities in natural and synthetic sewage activated sludge for enhanced phosphate removal

Respiratory quinones were used as biomarkers to study bacterial community structures in activated sludge reactors used for enhanced biological phosphate removal (EBPR). We compared the quinone profiles of EBPR sludges and standard sludges, of natural sewage and synthetic sewage, and of plant scale and laboratory scale systems. Ubiquinone (Q) and menaquinone (MK) components were detected in all sludges tested at molar MK/Q ratios of 0.455 to 0.981. The differences in MK/Q ratios were much larger when we compared different wastewater sludges (i.e., raw sewage and synthetic sewage) than when we compared sludges from the EBPR and standard processes or plant scale and laboratory scale systems. In all sludges tested a Q with eight isoprene units (Q-8) was the most abundant quinone. In the MK fraction, either tetrahydrogenated MK-8 or MK-7 was the predominant type, and there was also a significant proportion of MK-6 to MK-8 in most cases. A numerical cluster analysis of the profiles showed that the sludges tested fell into two major clusters; one included all raw sewage sludges, and the other consisted of all synthetic sewage sludges, independent of the operational mode and scale of the reactors and the phosphate accumulation. These data suggested that Q-8-containing species belonging to the class Proteobacteria (i.e., species belonging to the beta subclass) were the major constituents of the bacterial populations in the EBPR sludge, as well as in standard activated sludge. Members of the class Actinobacteria (gram-positive bacteria with high DNA G+C contents) were the second most abundant group in both types of sludge. The bacterial community structures in activated sludge processes may be affected more by the nature of the influent wastewater than by the introduction of an anaerobic stage into the process or by the scale of the reactors.     

A phylogenetic study of ubiquinone Q-8 species of the genera Candida,Pichia, and Citeromyces based on 18S ribosomal DNA sequence divergence

To clarify phylogenetic relationships among species of the anamorphic ascomycetous genus Candida with ubiquinone Q-8, we determined complete sequences of 18S ribosomal RNA genes (18S rDNAs) from the type strains of 20 species of the genus Candida and 7 of the teleomorphic ascomycetous genera Pichia and Citeromyces, which have Q-8 as the major ubiquinone. Q-8-forming Candida species were divided into six clusters and were phylogenetically distant from a group of Candida species that included the type species of the genus. One Q-8-forming species from each of the genera Pichia, Citeromyces, or Clavispora was included in five of six clusters. Cluster 1 comprised C. ishiwadae, C. ernobii, C. karawaiewii, C. anatomiae, C. populi, and Pichia holstii. Cluster 2 comprised C. globosa and its teleomorph, Citeromyces matritensis. Cluster 3 comprised C. molischiana and Pichia capsulata. Cluster 4 comprised C. silvanorum, C. sequanensis, C. fennica, C. entomophila, C. homilentoma, C. rhagii, C. gotoi, and Pichia burtonii. Cluster 5 comprised C. fructus, C. musae, and C. lusitaniae (anamorph of Clavispora lusitaniae). Cluster 6 comprised C. stellata, C. lactiscondensi, C. galacta, and C. incommunis and was a heterogeneous group with large interspecific divergence. Pichia pastoris was quite divergent and phylogenetically distant from other Pichia species examined. Pichia methanolica and its synonym, P. cellobiosa, which have both Q-7 and Q-8 as major ubiquinones, were closely associated with Q-7-forming Williopsis salicorniae. Based on this comparative analysis of 18S rDNA sequences, it is evident that Q-8 Candida species and Q-8 Pichia species are polyphyletic.     

Microbial diversity differences within aerobic granular sludge and activated sludge flocs

In this study, we investigated during 400 days the microbial community variations as observed from 16S DNA gene DGGE banding patterns from an aerobic granular sludge pilot plant as well as the from a full-scale activated sludge treatment plant in Epe, the Netherlands. Both plants obtained the same wastewater and had the same relative hydraulic variations and run stable over time. For the total bacterial population, a similarity analysis was conducted showing that the community composition of both sludge types was very dissimilar. Despite this difference, general bacterial population of both systems had on average comparable species richness, entropy, and evenness, suggesting that different bacteria were sharing the same functionality. Moreover, multi-dimensional scaling analysis revealed that the microbial populations of the flocculent sludge system moved closely around the initial population, whereas the bacterial population in the aerobic granular sludge moved away from its initial population representing a permanent change. In addition, the ammonium-oxidizing community of both sludge systems was studied in detail showing more unevenness than the general bacterial community. Nitrosomonas was the dominant AOB in flocculent sludge, whereas in granular sludge, Nitrosomonas and Nitrosospira were present in equal amounts. A correlation analysis of process data and microbial data from DGGE gels showed that the microbial diversity shift in ammonium-oxidizing bacteria clearly correlated with fluctuations in temperature.     

Isolation and structural elucidation of a dihydroubiquinone-9 from the fungus Aureobasidium pullulans

Abstract A naturally occurring member of ubiquinone (Q) group, a dihydroubiquinone-9 (Q-9 (H₂)), has been isolated as a minor ubiquinone component from the fungus Aureobasidium pullulans . By ultraviolet absorption, mass and nuclear magnetic resonance spectrometric studies, the structure of Q-9 (H₂) was found to be 2,3-dimethoxy-5-methyl-6-IX-dihydromultiprenyl⁹-1,4-benzoquinone (I).     

Lysobacter ginsengisoli sp. nov., a novel species isolated from soil in Pocheon Province, South Korea

A Gram-negative, aerobic, rod-shaped, nonspore-forming bacterial strain, designated Gsoil 357T, was isolated from soil sample of a ginseng field in Pocheon Province (South Korea). The isolate contained Q-8 as the predominant ubiquinone and iso-C16:0, iso-C17:1 9c, and iso-C15:0 as the major fatty acids. The G+C content of the genomic DNA was 69.3mol%. A phylogenetic analysis based on 16S rRNA gene sequences revealed that strain Gsoil 357T was most closely related to Lysobacter gummosus (97.6%) and Lysobacter antibioticus (97.6%). However, the DNA-DNA relatedness value between strain Gsoil 357T and its phylogenetically closest neighbors was less than 17%. On the basis of its phenotypic properties and phylogenetic distinctiveness, strain Gsoil 357T should be classified as representing a novel species in the genus Lysobacter, for which the name Lysobacter ginsengisoli sp. nov. is proposed. The type strain is Gsoil 357T (=KCTC 12602T=DSM 18420T).     

Evolutionary relationships within Aspergillus section Flavi based on sequences of the intergenic transcribed spacer regions and the 5.8S rRNA gene

Sequences of the intergenic transcribed spacer regions and the 5.8S rRNA gene (455 nucleotides) of type strains or representative isolates of 23 species and subspecies either currently assigned to Aspergillus subgenus Circumdati section Flavi or other closely related sections, were analyzed. Parsimony analysis of sequence data indicated that species of Aspergillus section Flavi form distinct clades. The three main clades identified based on sequence data could also be distinguished based on colony color, and their ubiquinone systems. The 'A. flavus' clade includes species characterized with Q-10(H(2)) as their main ubiquinone, conidial colors in shades of green, and dark sclerotia. The 'A. tamarii' clade involves species with ubiquinone system Q-10(H(2)), and conidia in shades of olive to brown, while the 'A. alliaceus' clade consists of species with Q-10 ubiquinone system, and conidia in shades of ocher. The synnematous species A. coremiiformis was found to be closely related to species in the 'A. tamarii' clade. A. thomii and A. terricola var. americana were found to be related to the 'A. flavus' clade in spite of producing brownish colonies. Three species, A. nomius, A. avenaceus, and A. leporis were found to form separate lineages not closely related to any of the main clades identified. It is suggested that A. clavatoflavus and A. zonatus be excluded from Aspergillus section Flavi. Phylogenetic analysis of partial 26S rRNA gene sequences (564 nucleotides) supported our findings.     

Comparison of PCR-DGGE and selective plating methods for monitoring the dynamics of a mixed culture population in synthetic brewery wastewater

Enrichment of an activated sludge inoculum in synthetic brewery wastewater, which included glucose, maltose, and ethanol, was conducted in batch experiments to identify the dominant microbes present, to determine methodologies capable of monitoring the mixed culture population dynamics, and to determine the consortium's substrate degradation behavior. These results and methodologies were subsequently used in the determination of the population dynamics of suspended and attached microorganisms in a sequencing batch system in the second part of this research work. The three-membered microbial community comprised two bacterial and one fungal species that were identified as Acinetobacter sp., Enterobacter sp., and Candida sp. PCR-DGGE and plating on selective media were used to track the population dynamics of the consortium during the degradation of different substrates in synthetic wastewater containing glucose, maltose, and ethanol. Enterobacter sp. could degrade glucose and maltose but not ethanol, whereas Acinetobacter and Candida could degrade all three carbon sources. In buffered batch mixed culture experiments, Enterobacter was the predominant bacterium until the sugar concentrations decreased to levels that enabled Acinetobacter and Candida to degrade ethanol. PCR-DGGE was effective for detecting the dominant species, but culture-based methods were more accurate for monitoring the population dynamics of these microorganisms during growth in the wastewater medium.     

Analysis of the polyphosphate-accumulating microflora in phosphorus-eliminating, anaerobic-aerobic activated sludge systems by using diaminopropane as a biomarker for rapid estimation of Acinetobacter spp.

Polyphosphate-accumulating gram-negative bacteria were isolated from different anaerobic-aerobic activated sludge systems with diverse processes for enhanced biological phosphorus (P) elimination. Of 22 isolates, 10 were allocated to the genus Acinetobacter by using multiple-test systems and soluble protein and polyamine patterns. As diaminopropane (DAP) appears to be the characteristic main polyamine compound produced by Acinetobacter spp., it was used as a biomarker for the genus. The high DAP contents of representative samples from municipal wastes with enhanced biological P elimination indicated that Acinetobacter spp. can be dominant organisms in sewage treatment plants with low organic loading and nitrification and denitrification steps. Contrary to accepted opinion, sludge from treatment plants with efficient P removal and high organic loading had a low DAP content, indicating that bacteria other than Acinetobacter spp. are responsible for enhanced biological P elimination in these plants.     

Analysis of the polyphosphate-accumulating microflora in phosphorus-eliminating, anaerobic-aerobic activated sludge systems by using diaminopropane as a biomarker for rapid estimation of Acinetobacter spp.

Polyphosphate-accumulating gram-negative bacteria were isolated from different anaerobic-aerobic activated sludge systems with diverse processes for enhanced biological phosphorus (P) elimination. Of 22 isolates, 10 were allocated to the genus Acinetobacter by using multiple-test systems and soluble protein and polyamine patterns. As diaminopropane (DAP) appears to be the characteristic main polyamine compound produced by Acinetobacter spp., it was used as a biomarker for the genus. The high DAP contents of representative samples from municipal wastes with enhanced biological P elimination indicated that Acinetobacter spp. can be dominant organisms in sewage treatment plants with low organic loading and nitrification and denitrification steps. Contrary to accepted opinion, sludge from treatment plants with efficient P removal and high organic loading had a low DAP content, indicating that bacteria other than Acinetobacter spp. are responsible for enhanced biological P elimination in these plants.     

Genomic fingerprints, ARDRA profiles and quinone systems for classification of Pasteurella sensu stricto

In order to investigate the relationships between species of the genus Pasteurella sensu stricto such as Pasteurella multocida, Pasteurella canis, Pasteurella stomatis, Pasteurella dagmatis, Pasteurella avium, Pasteurella volantium, Pasteurella gallinarum, Pasteurella species A, Pasteurella species B and "Pasteurella leonis" MCCM 00659 their genomic fingerprints and ARDRA profiles were compared and their quinone systems were analysed. Visual comparison of band patterns from rep-PCR (ERIC-, REP- and BOX-PCR) and the analyses of the combined band patterns by UPGMA (unweighted pair group method with averages) dendrogram derived from the combined fingerprint profiles demonstrated that each strain displays a distinct genomic fingerprint. In members of the same species several similarities in the band patterns were observed. Combined ARDRA profiles, obtained after digestion of amplified 23S rRNA coding genes with the enzymes DdeI, MseI and RsaI, revealed a dissection of the members of the genus Pasteurella sensu stricto into two groups which was in agreement with the two groups obtained from our analyses of the quinone systems. These two groups corresponded with the two phylogenetically determined subclusters 3A and 3B described previously. The species of subcluster 3A displayed a quinone system with ubiquinone Q-7 (32-56%) and ubiquinone Q-8 (44-63%) as major compounds. Members of subcluster 3B had a quinone system with ubiquinone Q-8 (86-97%) as the major compound. Based on these results it can be suggested that the genus Pasteurella sensu stricto should be restricted to the species of subcluster 3B including the species Pasteurella multocida, Pasteurella canis, Pasteurella stomatis, Pasteurella dagmatis and Pasteurella species B. In addition, evidence was found which would indicate that: 1) Pasteurella canis MCCM 00927 is misnamed and should be reclassified with Pasteurella multocida; 2) Pasteurella multocida subsp. septica may be classified as a separate species; and 3) "Pasteurella leonis" MCCM 00659 represents a separate species within subcluster 3B and thus could be described as a species of Pasteurella sensu stricto (also in a redefined genus) when more strains become available.     

Tahibacter aquaticus gen. nov., sp. nov., a new gammaproteobacterium isolated from the drinking water supply system of Budapest (Hungary)

Three Gram-stain negative, aerobic, non-motile, non-spore-forming, rod-shaped bacterial strains, PYM5-11^T, RaM5-2 and PYM5-8, were isolated from the drinking water supply system of Budapest (Hungary) and their taxonomic positions were investigated by a polyphasic approach. All three strains grew optimally at 20-28 °C and pH 5-7 without NaCl. The G+C content of the DNA of the type strain was 65.4 mol%. On the basis of 16S rRNA gene sequence analysis, the isolates showed 94.5-94.9% sequence similarity to the type strain of Dokdonella koreensis and a similarity of 93.0-94.1% to the species of the genera Aquimonas and Arenimonas. The major isoprenoid quinone of the strains was ubiquinone Q-8. The predominant fatty acids were iso-C_15:0, iso-C_17:1ω9c, C_16:1ω7c, and C_16:0. Diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and phosphatidylmonomethylethanolamine, as well as several unidentified aminolipids and phospholipids were present. The 16S rRNA gene sequence analysis, the predominant fatty acids, the polar lipid composition, RiboPrint patterns, physiological and biochemical characteristics showed that the three strains were related but distinct from the type strains of the four recognized species of the genus Dokdonella, and indicated that the strains represented a new genus within the Gammaproteobacteria. The strain PYM5-11 (=DSM 21667^T=NCAIM B 02337^T) is proposed as the type strain of a new genus and species, designated as Tahibacter aquaticus gen. nov., sp. nov.     

Polyphosphate accumulation among denitrifying bacteria in activated sludge

Bacterial polyphosphate accumulation and denitrification are important processes in biological removal of nutrients from wastewater. It has been suggested that phosphorus accumulators are able to denitrify. However, the bacteria known as the most important phosphorus accumulators, belonging to the genus Acinetobacter are generally not known to denitrify. To clarify how commonly both physiological traits are present in the same organism, we screened 165 isolates from activated sludge and wastewater for their ability to denitrify, and the ability of the denitrifying isolates to accumulate polyphosphate. Of the 165 isolates, 149 were from acetate mineral medium (87 of these identified as Acinetobacter by the API 20 NE identification system) and 16 were from nutrient broth and nitrate medium. Only 15 of 165 isolates tested showed true respiratory denitrification activity. In the presence of acetylene they converted more than 80% of 5mM NO3- to N2O in 6 days. None of the Acinetobacter isolates were among the 15 respiratory denitrifiers. The denitrifying isolates were identified as species of Pseudomonas, Agrobacterium, Pasteurella, Sphingomonas or could not be identified by the API 20 NE identification system. According to the BIOLOG identification system the denitrifiers were species of Pseudomonas, Hydrogenophaga, Citrobacter, Xanthomonas or they could not be identified. The ability of confirmed denitrifiers to accumulate phosphate was measured in experiments where cells pregrown under phosphorus limitation were exposed to phosphate (8 mg P/L) under aerobic conditions. The rates of excess phosphate uptake varied from 0.3 to more than 23 mg P/g dry matter/h. Rates for four isolates were higher than those reported for Acinetobacter strains. These results show that polyphosphate accumulation and denitrification in activated sludge can be carried out by the same organisms.