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.     

Microbial population dynamics during sludge granulation in an anaerobic-aerobic biological phosphorus removal system

The evolution of a microbial community was investigated during sludge granulation using a wide range of micro-scale and molecular biology techniques. Experimental results demonstrate that polyphosphate-accumulating granules were successfully cultured during the anaerobic/aerobic cycle. Improvement in sludge sedimentation performance occurred prior to the formation of granular sludge and was not affected by change in granule size. Rod-shaped and filamentous bacteria appeared to initiate granule formation and generate the structures that supported further granule growth. It was observed that mature granules supported microbial populations that differed from nascent granules and were predominantly packed with coccoid bacteria. It was further observed that the diversity of the granular microbial community increased as the granules grew. Accumulibacter, Nitrosospira and Thauera were mainly responsible for nutrient removal while microorganisms such as Rhodocyclus and Hyphomicrobiaceae appeared to be primarily responsible for forming and maintaining the granule structure.     

Ecophysiology of Defluviicoccus-related tetrad-forming organisms in an anaerobic-aerobic activated sludge process

A group of uncultured tetrad-forming organisms (TFOs) was enriched in an acetate-fed anaerobic-aerobic sequencing membrane bioreactor showing deteriorated enhanced biological phosphorus removal capacity. Based on 16S rRNA gene clone library and fluorescence in situ hybridization (FISH) analyses, these TFOs were identified as novel members of the Defluviicoccus cluster in the Alphaproteobacteria, accounting for 90 +/- 5% of the EUBmix FISH-detectable bacterial cell area in the reactor biomass. Microautoradiography in combination with FISH and polyhydroxyalkanoate (PHA) staining revealed that these Defluviicoccus-related TFOs could take up and transform acetate, lactate, propionate and pyruvate, but not aspartic acid and glucose, into PHA under anaerobic conditions. In contrast, under continuous anaerobic-aerobic cultivation, Defluviicoccus vanus, the only cultured strain from the cluster, was able to take up glucose with concurrent glycogen consumption and PHA production under anaerobic conditions. Under subsequent aerobic conditions, the accumulated PHA was utilized and the biomass glycogen levels were restored. These findings not only re-confirmed these Defluviicoccus-related TFOs as glycogen-accumulating organisms, but also revealed unexpected levels of physiological, phylogenetic and morphological diversity among members of the Defluviicoccus cluster.     

Changes in respiratory quinone profiles of enhanced biological phosphorus removal activated sludge under different influent phosphorus/carbon ratio conditions

Changes in the microbial community of an enhanced biological phosphorus removal (EBPR) activated sludge system under different influent phosphorus/carbon (P/C) ratio conditions were investigated through evaluation of population respiratory quinone profiles. A total of 13 types of respiratory quinone homologs consisting of 3 types of ubiquinones (UQ) and 10 types of menaquinones (MK) were identified in this study. The dominant quinones were UQ-8 and MK-7 throughout the operational period. A higher P/C ratio (0.1) in the influent stimulated an increase in the mole fractions of UQ-8, MK-7, MK-8(H₄), MK-9(H₄) and MK-8(H₈), suggesting that actinobacterial polyphosphate-accumulating organisms (PAO) containing partially hydrogenated MK, mainly MK-8(H₄), were contributing to EBPR. However, when the P/C ratio gradually decreased from 0.1 to 0.01, the mole fractions of UQ-8 increased from 0.46 to 0.58, while MK-7, MK-8(H₂), MK-8(H₄), MK-9(H₄), MK-8(H₈) and MK-9(H₆) markedly decreased. These changes in the respiratory quinone profiles suggest that glycogen-accumulating organisms corresponding to some Gammaproteobacteria had become dominant populations with a decrease in actinobacterial PAO. On the other hand, increasing abruptly the P/C ratio to 0.1 further caused an increase in the mole fraction of UQ-8, indicating that Rhodocyclus-related organisms were important PAO.     

Analysis of microbial community structure in a biofilm on membrane surface in the submerged membrane bioreactor treating domestic wastewater on the basis of respiratory quinone profiles

The objective of this study was to investigate the microbial community structure of the biofouling film formed on hollow-fiber membrane surfaces in the submerged membrane bioreactor (SMBR) with a nitrification-denitrification process. In this experiment, aeration was conducted intermittently (60 min off, 90 min on) cyclic anoxic and oxic conditions in the SMBR. The dominant quinone types of biofilm on the membrane surface in an intermittently aerated SMBR were ubiquinone (UQs)-8, -10, followed by menaquinones (MKs)-8(H4), -8(H2) and -7, but those of suspended microorganisms were UQ-8, UQ-10 followed by MKs-8, -9(H4) and -6. The change in quinone profiles of biofilm on the membrane surface suggested that UQ-9, MK-7, MK-8(H2) and MK-8(H4) contributed to microbiological fouling in the intermittently aerated SMBR treating domestic wastewater. The microbial diversities of suspended microorganisms and biofilm, calculated based on the composition of all quinones, were 9.5 and 10.9, respectively.     

Characterization of virucidal agents in activated sludge.

A comprehensive study was carried out to determine the properties of agents responsible for loss of virus infectivity in mixed-liquor suspended solids (MLSS) of activated sludge. Initial experiments revealed that model enteric viruses (poliovirus-1 and rotavirus SA-11) were irreversibly inactivated in MLSS and released their RNA genomes. Enteric viruses belonging to other genera (echovirus-12, coxsackievirus A13, reovirus-3) were also shown to lose infectivity in MLSS. Although the virucidal activity decreased at reduced temperatures, MLSS still retained significant activity at 4 degrees C. The virucidal agents in MLSS were stable for months at 4 degrees C, but their activity decreased approximately 50% during 4 days of aeration at 26 degrees C. Primary effluent, the nutrient source for activated sludge, also contained virucidal activity. After centrifugation of MLSS, almost all virucidal activity was found in the particulate fraction because of inhibitory substances retained in the supernatant fraction. Decreasing or increasing the solids concentration of the particulate fraction did not increase the virucidal activity of the fraction. The effects of heat and antibiotics on the virucidal activity of MLSS, coupled with the finding that the activity can be produced in autoclaved primary effluent seeded with MLSS, strongly support the conclusion that microorganisms are responsible for this activity. Attempts to characterize the virucidal microbial components of MLSS indicated that treatments that resulted in the inactivation or removal of microorganisms also caused a loss of virucidal activity. Thus, it appears that the virucidal components of microorganisms are either short-lived or active only while bound to the organisms themselves.     

Effect of shock-loading of heavy metals on total organic carbon and phosphate removal in an anaerobic-aerobic activated sludge process

The effect of shock-loading of zinc, copper and cadmium ions on the removal of total organic carbon (TOC) and phosphate in an anaerobic-aerobic activated sludge process was investigated. TOC removal was not sensitive to shock-loading of Zn²⁺ and Cd²⁺ ions, and complete removal was achieved even at 20 mg Zn²⁺/l and 20 mg Cd²⁺/l. However, with over 1 mg Cu²⁺/1 TOC removal efficiency decreased. PO _inf4 ^sup3- removal, in contrast, was extremely sensitive to these metal ions, with the threshold being 1 mg Zn²⁺/l and 1 mg Cd²⁺/l. Higher concentrations adversely affected PO _inf4 ^sup3- removal. Copper again proved detrimental; no PO _inf4 ^sup3- removal was achieved even at 1 mg Cu/l. These results highlight the sensitivity of the removal efficiencies of TOC and PO _inf4 ^sup3- to shock loadings of these heavy metals.Y.P. Ting is with the Department of Chemical Engineering, National University of Singapore, Kent Ridge, 0511, Singapore; H. Imai and S. Kinoshita are with the Department of Chemical Process Engineering, Hokkaido University, Sapporo 060, Japan.     

Nitrification in activated sludge batch reactors is linked to protozoan grazing of the bacterial population

Protozoa feed upon free-swimming bacteria and suspended particles inducing flocculation and increasing the turnover rate of nutrients in complex mixed communities. In this study, the effect of protozoan grazing on nitrification was examined in activated sludge in batch cultures maintained over a 14-day period. A reduction in the protozoan grazing pressure was accomplished by using either a dilution series or the protozoan inhibitor cycloheximide. As the dilutions increased, the nitrification rate showed a decline, suggesting that a reduction in protozoan or bacterial concentration may cause a decrease in nitrification potential. In the presence of cycloheximide, where the bacterial concentration was not altered, the rates of production of ammonia, nitrite, and nitrate all were significantly lower in the absence of active protozoans. These results suggest that a reduction in the number or activity of the protozoans reduces nitrification, possibly by limiting the availability of nutrients for slow-growing ammonia and nitrite oxidizers through excretion products. Furthermore, the ability of protozoans to groom the heterotrophic bacterial population in such systems may also play a role in reducing interspecies competition for nitrification substrates and thereby augment nitrification rates.     

Assessment of denitrifying bacterial composition in activated sludge

The abundance and structure of denitrifying bacterial community in different activated sludge samples were assessed, where the abundance of denitrifying functional genes showed nirS in the range of 10(4)-10(5), nosZ with 10(4)-10(6) and 16S rRNA gene in the range 10(9)-10(10) copy number per ml of sludge. The culturable approach revealed Pseudomonas sp. and Alcaligenes sp. to be numerically high, whereas culture independent method showed betaproteobacteria to dominate the sludge samples. Comamonas sp. and Pseudomonas fluorescens isolates showed efficient denitrification, while Pseudomonas mendocina, Pseudomonas stutzeri and Brevundimonas diminuta accumulated nitrite during denitrification. Numerically dominant RFLP OTUs of the nosZ gene from the fertilizer factory sludge samples clustered with the known isolates of betaproteobacteria. The data also suggests the presence of different truncated denitrifiers with high numbers in sludge habitat.     

Characterization of virucidal agents in activated sludge

A comprehensive study was carried out to determine the properties of agents responsible for loss of virus infectivity in mixed-liquor suspended solids (MLSS) of activated sludge. Initial experiments revealed that model enteric viruses (poliovirus-1 and rotavirus SA-11) were irreversibly inactivated in MLSS and released their RNA genomes. Enteric viruses belonging to other genera (echovirus-12, coxsackievirus A13, reovirus-3) were also shown to lose infectivity in MLSS. Although the virucidal activity decreased at reduced temperatures, MLSS still retained significant activity at 4 degrees C. The virucidal agents in MLSS were stable for months at 4 degrees C, but their activity decreased approximately 50% during 4 days of aeration at 26 degrees C. Primary effluent, the nutrient source for activated sludge, also contained virucidal activity. After centrifugation of MLSS, almost all virucidal activity was found in the particulate fraction because of inhibitory substances retained in the supernatant fraction. Decreasing or increasing the solids concentration of the particulate fraction did not increase the virucidal activity of the fraction. The effects of heat and antibiotics on the virucidal activity of MLSS, coupled with the finding that the activity can be produced in autoclaved primary effluent seeded with MLSS, strongly support the conclusion that microorganisms are responsible for this activity. Attempts to characterize the virucidal microbial components of MLSS indicated that treatments that resulted in the inactivation or removal of microorganisms also caused a loss of virucidal activity. Thus, it appears that the virucidal components of microorganisms are either short-lived or active only while bound to the organisms themselves.     

Pyrosequencing reveals the inhibitory impact of chronic exposure to erythromycin on activated sludge bacterial community structure

The study investigated changes in the microbial population structure sustained at two different sludge ages of 10 d and 2 d under chronic impact of erythromycin. It intended to observe the experimental correlation between variable process kinetics and changes in the composition of the microbial community induced by erythromycin. Samples from two fill/draw reactors operated with continuous erythromycin dosing of 50 mg/L were collected for the analysis of microbial population structure using high-throughput sequencing of 16SrRNA gene. Significant changes were observed in the composition of microbial community during the exposure period. Richness analysis for slower growing culture indicated that most microbial fractions were inactivated and eliminated in favor of fewer more resistant species in different phyla. Sludge age appeared to control the impact of erythromycin on microbial composition. At a sludge age of 2 d, erythromycin appeared to generate richer community with faster growing and more compatible species. For slower growing culture, elimination of vulnerable species was supported by decrease in the number of shared level OTUs. For faster growing culture, shared species level OTUs also decreased significantly upon exposure to erythromycin, suggesting rapid washout and replacement by more resistant species. Resistance gene analysis yielded positive results for mph(A) gene indicating presence of erythromycin-resistant components in the microbial community.     

Identification of a functionally important population in phenol-digesting activated sludge with antisera raised against isolated bacterial strains.

Antisera were raised against nine strains which had been isolated from phenol-acclimated oil refinery activated sludge. Although several antisera reacted significantly with the activated sludge during a period of adaptation to phenol, only an antiserum against one of the isolates, Alcaligenes sp. E2, reacted with the activated sludge after the adaptation period. A kinetic pattern of phenol-oxygenating activity of the activated sludge after the adaptation period was similar to that of strain E2. These results suggest that a functionally important population in the phenol-digesting activated sludge was serologically identified.     

Diversity and distribution of a deeply branched novel proteobacterial group found in anaerobic-aerobic activated sludge processes

A novel coccobacilli group found previously in enhanced biological phosphorus removal (EBPR) systems was further revealed to have a high degree of diversity and distribution in various activated sludge systems. Phylogenetic analysis based on 14 existing and 18 newly retrieved 16S rRNA sequences revealed that these sequences formed a novel cohesive cluster with seven subgroups in the gamma-Proteobacteria. Fluorescence in situ hybridization with a set of probes designed specifically targeting the novel group at different hierarchical levels showed that the novel group with a coccoid (2-4 micro m) to occasionally long-rod (up to 20 micro m) shape widely distributed and in some cases predominated in sludge samples taken from nine lab- and full-scale EBPR systems (10-50% of total cells) and four conventional activated sludge systems (1-10%). Variation of predominance was also observed among those subgroups in systems showing deteriorated or effective EBPR activity.     

活性污泥微生物菌群研究方法进展

活性污泥是活性污泥法处理污水系统的功能主体。人类对活性污泥微生物菌群的认识随着其研究方法的发展而逐步深入。传统培养方法只能检测到活性污泥中1％～15％的微生物。随着一系列基于免培养的分子生物学技术的出现,活性污泥中菌群的复杂性和多样性以惊人的速度被人们认识,大量依靠传统检测方法未能发现却在活性污泥中起关键作用的微生物逐渐被发现。许多模拟活性污泥菌群生存环境条件的现代培养技术开始发展,且已成功培养了一部分传统培养方法不能培养的细菌类群,这为研究基于免培养方法发现的大量新的微生物菌群的生理特性和作用机制提供了可能,也无疑将把人们对活性污泥菌群的认识推向一个新的层次．主要介绍活性污泥微生物菌群研究的一系列方法,从传统培养方法到基于免培养的现代分子生物学技术,再到现代培养技术,着重论述了现代分子生物学技术及其在活性污泥微生物菌群研究中的进展。     

Role of glycogen in acetate uptake and polyhydroxyalkanoate synthesis in anaerobic-aerobic activated sludge with a minimized polyphosphate content

The role of glycogen in the uptake of acetate in anaerobic-aerobic activated sludge without enhanced biological phosphorus removal were investigated. Although the polyphosphate content of the sludge was minimized by lowering the phosphorus feeding concentration, significant acetate uptake and accumulation of polyhydroxyalkanoates (PHAs) were observed in proportion to glycogen consumption under anaerobic conditions. The results of anaerobic inhibition studies, which showed suppressive effects on acetate uptake by a glycolysis inhibitor (iodoacetate) but not by a membrane ATPase inhibitor (N,N′-dicyclohexyl carbodiimide), supported an assumption that glycogen degradation through glycolysis supplies the required ATP and reducing power for PHA synthesis from acetate and consumed glycogen. Under subsequent aerobic conditions, the accumulated PHAs were depleted and the consumed glycogen recovered to the same level as that at the start of the anaerobic phase. Iodoacetate also inhibited the recovery of glycogen under aerobic conditions, suggesting that nearly 50% of the PHAs depleted was used for glycogen synthesis through reversed glycolysis.     

Characterization of Fe-hydrogenase genes diversity and hydrogen-producing population in an acidophilic sludge

According to the DNA sequences of six Fe-hydrogenase genes (FHG) of Clostridium species retrieved from the GenBank, a set of primers specific for Fe-hydrogenase genes were identified from their common conserved regions. The length of DNA fragments amplified using these two primers averaged 313 bps. This primer set was then used to investigate the FHG diversity in an acidophilic rice-degrading sludge by methods based on polymerase chain reaction (PCR). Eight new Fe-hydrogenase gene fragments were identified from the sludge, as a result. Similarity based on amino acids among the 14 hydrogenase genes (8 newly found plus 6 known ones) was 39-97%, which is comparable to the similarity of 41-82% among the 6 known hydrogenase genes alone. The low similarity indicates a great diversity on Fe-hydrogenase among the Clostridium species. The primer set was then used to monitor the change of hydrogen-producing microbial population in a batch reaction using the technique of quantitative real-time polymerase chain reaction (qRT-PCR) with SYBR Green I as the fluorescent reagent. Results showed that the hydrogen producers had an average generation time of 4.2h, and a production rate of 7.0 x 10(16) H2-molecule cell(-1)h(-1).     

Characterization of methanotrophic bacteria on the basis of intact phospholipid profiles

The intact phospholipid profiles (IPPs) of seven species of methanotrophs from all three physiological groups, type I, II and X, were determined using liquid chromatography/electrospray ionization/mass spectrometry. In these methanotrophs, two major classes of phospholipids were found, phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) as well as its derivatives phosphatidylmethylethanolamine (PME) and phosphatidyldimethylethanolamine (PDME). Specifically, the type I methanotrophs, Methylomonas methanica, Methylomonas rubra and Methylomicrobium album BG8 were characterized by PE and PG phospholipids with predominantly C16:1 fatty acids. The type II methanotrophs, Methylosinus trichosporium OB3b and CSC1 were characterized by phospholipids of PG, PME and PDME with predominantly C18:1 fatty acids. Methylococcus capsulatus Bath, a representative of type X methanotrophs, contained mostly PE (89% of the total phospholipids). Finally, the IPPs of a recently isolated acidophilic methanotroph, Methylocella palustris, showed it had a preponderance of PME phospholipids with 18:1 fatty acids (94% of total). Principal component analysis showed these methanotrophs could be clearly distinguished based on phospholipid profiles. Results from this study suggest that IPP can be very useful in bacterial chemotaxonomy.     

Determination of microbial respiratory and redox activity in activated sludge

The tetrazolium salt 5-cyano-2,3-ditolyltetrazolium chloride (CTC) was used for the determination of metabolically active bacteria in active sludge. The method was adapted and optimized to the conditions of activated sludge. The colorless and nonfluorescent tetrazolium salt is readily reduced to a water-insoluble fluorescent formazan product via the microbial electron transport system and indicates mainly dehydrogenase activity. After more than 2 h incubation, no further formation of new formazan crystals was observed, although the existing crystals in active cells continued to grow at the optimal CTC-concentration of 4 mM. The dehydrogenase activity determined by direct epifluorescence microscopic enumeration did not correlate with cumulative measured activity as determined by formazan extraction. The addition of nutrients did not lead to an increase of CTC-active cells. Sample storage conditions such as low temperature or aeration resulted in a significant decrease in dehydrogenase activity within 30 min. The rapid and sensitive method is well suited for the detection and enumeration of metabolically active microorganisms in activated sludge. Extracellular redox activity was measured with the tetrazolium salt 3′-{1-[phenylamino-) carbonyl]-3,4-tetrazolium}-bis(4-methoxy-6-nitro)benzene-sulfonic acid hydrate (XTT), which remains soluble in its reduced state, after extraction of extracellular polymeric substances (EPS) with a cation exchange resin. Received 12 August 1996/ Accepted in revised form 29 May 1997     

Layered structure of bacterial aggregates produced in an upflow anaerobic sludge bed and filter reactor.

The ultrastructure of bacterial granules that were maintained in an upflow anaerobic sludge bed and filter reactor was examined. The reactor was fed a sucrose medium, and it was operated at 35 degrees C. Scanning and transmission electron microscopy revealed that the granular aggregates were three-layered structures. The exterior layer of the granule contained a very heterogeneous population that included rods, cocci, and filaments of various sizes. The middle layer consisted of a slightly less heterogeneous population than the exterior layer. A more ordered arrangement, made up predominantly of bacterial rods, was evident in this second layer. The third layer formed the internal core of the granules. It consisted of large numbers of Methanothrix-like cells. Large cavities, indicative of vigorous gas production, were evident in the third layer. On the basis of these ultrastructural results, a model that presents a possible explanation of granule development is offered.