Bacteriophage-based biocontrol of biological sludge bulking in wastewater

In a previous paper, the first ever application of lytic bacteriophage (virus)-mediated biocontrol of biomass bulking in the activated sludge process using Haliscomenobacter hydrossis as a model filamentous bacterium was demonstrated. In this work we extended the biocontrol application to another predominant filamentous bacterium, Sphaerotilus natans, notoriously known to cause filamentous bulking in wastewater treatment systems. Very similar to previous study, one lytic bacteriophage was isolated from wastewater that could infect S. natans and cause lysis. Significant reduction in sludge volume index and turbidity of the supernatant was observed in batches containing S. natans biomass following addition of lytic phages. Microscopic examination confirmed that the isolated lytic phage can trigger the bacteriolysis of S. natans. This extended finding further strengthens our hypothesis of bacteriophage-based biocontrol of overgrowth of filamentous bacteria and the possibility of phage application in activated sludge processes, the world's widely used wastewater treatment processes.     

Studies on the survival of enterohemorrhagic and environmental Escherichia coli strains in wastewater and in activated sludges from dairy sewage treatment plants

Survival of Escherichia coli O157:H7 strain isolated from milk in Poland and an environmental E. coli strain in wastewater from Garwolin and ?owicz dairies and in activated sludges from dairy sewage treatment plants as well as in dairy wastewater with activated sludges was examined. Environmental materials were contaminated with about 10(8) of target bacteria/ml of sample. The experiments were performed under temperature conditions typical of autumn-winter (6 degrees) and spring-summer (24 degrees C) seasons. It was found that the non-pathogenic E. coli strain survived longer in all media than the enterohemorrhagic serotype. E. coli O157:H7 bacteria were not detected (in direct plating method) in activated sludges after 21-28 days; in dairy wastewater as well as in wastewater with activated sludges after 21-25 days. These periods for environmental E. coli strain were 35-42 days (activated sludges), 25-28 days (wastewater with activated sludges). At higher temperature environmental E. coli were not detected in wastewater from ?owicz dairy sewage treatment plant after 25 days, but the bacteria were still present in wastewater from Garwolin dairy sewage tratment plant after 34 days. The obtained results show that the lack of environmental E. coli bacteria (as a indicator bacteria of fecal contamination) in dairy wastewater and in dairy wastewater with activated sludges could indicate the absence of pathogenic E. coli bacteria. Prolonged existence of the enterohemorrhagic serotype in activated sludges shows the need to treat activated sludges prior to the utilization of these materials as fertilizer.     

A <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strain HPC248 from an effluent treatment plant with antimicrobial activity

This study reports the identification and demonstration of an organism with antimicrobial activity isolated from activated biomass of an effluent treatment plant (ETP) treating wastewater containing pesticides. While assessing the heterotrophic diversity of biomass collected from ETP, clear zones were observed on Luria Bertani plates. The bacterial isolate producing the zone as well as the bacterial cells surrounding the zone were isolated and purified by sub-culturing. Both isolates were identified by partial sequencing of the 16S rDNA clone. Presence of antimicrobial activity was demonstrated against various laboratory strains, isolated from different treatment plants and also against waterborne pathogens. The isolate that produced antimicrobial activity was identified as Bacillus subtilis strain HPC248 and the sensitive strain was identified as Bacillus sphaericus strain HPC249.     

Detection of polyhydroxyalkanoate-accumulating bacteria from domestic wastewater treatment plant using highly sensitive PCR primers

Polyhydroxyalkanoate (PHA) is a class of biodegradable plastics that have great potential applications in the near future. In this study, the micro-biodiversity and productivity of PHA-accumulating bacteria in activated sludge from a domestic wastewater treatment plant were investigated. A previously reported primer set and a selfdesigned primer set (phaCF1BO/phaCR2BO) were both used to amplify the PHA synthase (phaC) gene of isolated colonies. The new primers demonstrated higher sensitivity for phaC, and combining the PCR results of the two primer sets was able to widen the range of detected genera and raise the sensitivity to nearly 90%. Results showed that 85.3% of the identified bacteria were Gram-negative, with Ralstonia as the dominant genus, and 14.7% were Gram-positive. In addition, Zoogloea and Rhizobium contained the highest amounts of intracellular PHA. It is apparent that glucose was a better carbon source than pentone or tryptone for promoting PHA production in Micrococcus. Two different classes, class I and class II, of phaC were detected from alphaproteobacteria, betaproteobacteria, and gammaproteobacteria, indicating the wide diversity of PHA-accumulating bacteria in this particular sampling site. Simultaneous wastewater treatment and PHA production is promising by adopting the high PHAaccumulating bacteria isolated from activated sludge.     

Bioaugmentation treatment for coking wastewater containing pyridine and quinoline in a sequencing batch reactor

Two pyridine-degrading bacteria and two quinoline-degrading bacteria were introduced for bioaugmentation to treat the coking wastewater. Sequencing batch reactors (SBRs) were used for a comparative study on the treatment efficiency of pyridine, quinoline, and chemical oxygen demand. Results showed that the treatment efficiency with coking-activated sludge plus a mixture of the four degrading bacteria was much better than that ones with coking-activated sludge only or mixed degrading bacteria only. Moreover, a 52-day continuous operation of the bioaugmented and general SBRs was investigated. The bioaugmented SBR showed better treatment efficiency and stronger capacity to treat high pyridine and quinoline shock loading. The general SBR failed to cope with the shock loading, and the biomass of the activated sludge decreased significantly. In order to monitor the microbial ecological variation during the long-term treatment, the bacterial community in both reactors was monitored by the amplicon length heterogeneity polymerase chain reaction technique. The diversity of the bacterial community decreased in both reactors, but the introduced highly efficient bacteria were dominant in the bioaugmented SBR. Our experiment showed clearly that the use of highly efficient bacteria in SBR process could be a feasible method to treat wastewater containing pyridine or/and quinoline.     

造纸废水活性污泥的微生物群落结构及多样性分析

为探究造纸废水活性污泥中微生物群落结构多样性以及对造纸废水处理效果的影响，利用Illumina MiSeq 高通量测序方法，分析在处理造纸废水过程中，同一运行阶段两个并联氧化沟内活性污泥的微生物群落与多样性组成。结果表明，系统中处理造纸废水的活性污泥在同一废水条件下微生物群落结构总体稳定，优势细菌为绿弯菌门(Chloroflexi)、拟杆菌门（Bacteroidota）、变形菌门（Proteobacteria）、Myxococcota、放线菌门(Actinobacteria）、厚壁菌门(Firmicutes）等。最重要的优势细菌类群为Chloroflexi，相对丰度占比为47.67%~48.22%，远远高于其他废水中Chloroflexi的占比，其中厌氧绳菌纲（Anaerolineae）是其主要成员，占比84.39%~88.34%，可针对性地去除造纸废水中的污染物。造纸废水活性污泥样品中存在大量特殊功能菌群，其在废水中污染物尤其是木质素的去除中发挥着重要作用。     

Configuration of biological wastewater treatment line and influent composition as the main factors driving bacterial community structure of activated sludge

The structure of microbial consortia in wastewater treatment facilities is a resultant of environmental conditions created by the operational parameters of the purification process. In the research, activated sludge from nine Polish wastewater treatment plants (WWTPs) was investigated at a molecular level to determine the impact of the complexity of biological treatment line and the influent composition on the species structure and the diversity of bacterial consortia. The community fingerprints and technological data were subjected to the canonical correspondence and correlation analyses. The number of separated biological processes realized in the treatment line and the presence of industrial wastewater in the influent were the key factors determining the species structure of total and ammonia-oxidizing bacteria in biomass. The N₂O-reducers community composition depended significantly on the design of the facility; the highest species richness of denitrifiers was noted in the WWTPs with separated denitrification tanks. The contribution of industrial streams to the inflow affected the diversity of total and denitrifying bacterial consortia and diminished the diversity of ammonia oxidizers. The obtained data are valuable for engineers since they revealed the main factors, including the design of wastewater treatment plant, influencing the microbial groups critical for the stability of purification processes.     

分子检测技术对活性污泥中氨氧化细菌的比较研究

采用PCR扩增、随机克隆测序等技术,分析处理含高浓度氨氮的废水处理系统不同驯化时期的4个活性污泥样品,对样品中氨氧化细菌（AOB）的种类和氨单加氧酶（AMO）的活性进行分析比较,并在国内首次采用PCR变性梯度凝胶电泳（DGGE）相结合的技术对样品中总的细菌类群的差异进行研究。结果表明所检测到的氨氧化细菌优势菌群均属于变形细菌的β亚类,与Nitrosomonas sp.具有较高的相似性。活性污泥驯化成熟后,废水处理系统中AMO的活性有明显提高,活性污泥中的细菌类群更加集中,优势菌群相对稳定,系统对废水的处理效率也相应提高。结果表明采用分子检测技术有利于更全面地了解AOB的类群和功能,进而改善废水处理系统的处理效果。     

活性污泥中菌群多样性及其功能调控研究进展

活性污泥是污水处理厂生物处理工艺的功能主体,活性污泥中菌群的种类、数量及活性是提高污水处理能力与效果的重要基础。本文综述了活性污泥处理工艺中的主要功能细菌(絮凝菌、脱氮菌、除磷菌等)生物群落的多样性与生态特征,并对目前主流的菌群鉴定方式进行总结,最后从运行条件、定向驯化及生物强化3个方面对菌群调控进行论述,以期为活性污泥法污水处理工艺提供一些理论指导。     

The structure of the planktic microbial community in the lower reaches of the Ob River near Salekhard

We studied the distribution of the main components of planktic food web: bacteria, heterotrophic flagellates, and ciliates in the lower reaches of the Ob River near the town of Salekhard. In the early summer, the abundance, biomass, and diversity of protozoans were low; the plankton community was dominated by heterotrophic bacteria whose biomass was higher than that of phyto- and zooplankton. Downstream of Salekhard, biomass and abundance of bacterioplankton were increased due to household and industrial wastewater. The data obtained showed the importance of heterotrophic organisms in trophic dynamics and self-purification of the lower reaches of the Ob River.     

Effect of a water extract of Moringa oleifera seeds on the hydrolytic microbial species diversity of a UASB reactor treating domestic wastewater

The effect of a continuous supply of a water extract of Moringa oleifera seeds (WEMOS) on the hydrolytic microbial population of biomass grown in mesophilic upflow anaerobic sludge blanket reactors treating domestic wastewater was investigated. The WEMOS-treated sludge had seemingly a wider diversity, with enterobacter and klebsiella as dominant hydrolytic bacteria, compared with the control sludge. Additional tests indicated that various hydrolytic bacteria could degrade WEMOS. It appeared that a continuous supply of WEMOS to an anaerobic digester, treating domestic wastewater, increased the diversity of hydrolytic bacteria and therefore enhanced the biological start-up of the reactor.     

解烃细菌处理油田污水的研究

从油田污水中分离到一组可高效降解石油烃的混合菌群F6。室内模拟实验证明,该菌群适于油田含油污水的净化处理。采用粒状活性炭为载体的生物流化床系统处理辽河油田“兴一联”（辽河油田兴隆台第一联合站）污水,可使含油量由45mg/L降至4.1mg/L、CODcr由 470mg/L降至42mg/L。处理后的石油污水达到国家石油污水一级排放标准。     

Viral composition and context in metagenomes from biofilm and suspended growth municipal wastewater treatment plants

Wastewater treatment plants (WWTPs) contain high density and diversity of viruses which can significantly impact microbial communities in aquatic systems. While previous studies have investigated viruses in WWTP samples that have been specifically concentrated for viruses and filtered to exclude bacteria, little is known about viral communities associated with bacterial communities throughout wastewater treatment systems. Additionally, differences in viral composition between attached and suspended growth wastewater treatment bioprocesses are not well characterized. Here, shotgun metagenomics was used to analyse wastewater and biomass from transects through two full-scale WWTPs for viral composition and associations with bacterial hosts. One WWTP used a suspended growth activated sludge bioreactor and the other used a biofilm reactor (trickling filter). Myoviridae, Podoviridae and Siphoviridae were the dominant viral families throughout both WWTPs, which are all from the order Caudovirales. Beta diversity analysis of viral sequences showed that samples clustered significantly both by plant and by specific sampling location. For each WWTP, the overall bacterial community structure was significantly different than community structure of bacterial taxa associated with viral sequences. These findings highlight viral community composition in transects through different WWTPs and provide context for dsDNA viral sequences in bacterial communities from these systems.     

In situ characterization of nitrifiers in an activated sludge plant: detection of Nitrobacter Spp

Aims: The purpose of this work was to investigate microbial ecology of nitrifiers at the genus level in a typical full-scale activated sludge plant. Methods and Results: Grab samples of mixed liquor were collected from a plug-flow reactor receiving domestic wastewater. Fluorescent in situ hybridization technique (FISH) was used to characterize both ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) in combination with Confocal Scanning Laser Microscope (CSLM). Fluorescently labelled, 16S rRNA-targeted oligonucleotide probes were used in this study. Both Nitrosomonas and Nitrosospira genera as AOB and Nitrobacter and Nitrospira genera as NOB were sought with genus specific probes Nsm156, Nsv443 and NIT3 and NSR1156, respectively. Conclusions: It was shown that Nitrosospira genus was dominant in the activated sludge system studied, although Nitrosomonas is usually assumed to be the dominant genus. At the same time, Nitrobacter genus was detected in activated sludge samples. Significance and Impact of the Study: Previous studies based on laboratory scale pilot plants employing synthetic wastewater suggested that only Nitrospira are found in wastewater treatment plants. We have shown that Nitrobacter genus might also be present. We think that these kinds of studies may not give a valid indication of the microbial diversity of the real full-scale plants fed with domestic wastewater.     

Monitoring filamentous bulking in activated sludge systems fed by synthetic or municipal wastewater

The stability with respect to filamentous bulking of two activated sludge fully-aerobic systems, one with a completely mixed tank and one with a channel reactor, fed either by a synthetic wastewater or by a primary-settled municipal wastewater, of variable composition and flow rate, has been investigated. The morphological characteristics of the biomass in terms of floc size and roughness and of filamentous bacteria abundance have been monitored by image analysis. Severe bulking was only observed in the well-mixed tank fed at a constant flow rate by synthetic substrate of constant concentration, when the channel reactor fed in a similar manner was fully stable. Variations of biomass characteristics as well as of settling properties were observed on both systems fed with the real wastewater, but these events were related to the characteristics of the wastewater, as similar changes were observed on the full-scale plant fed with the same substrate. In any case, automated image analysis was an efficient way to monitor in detail the fate of the activated sludge at pilot and full scale.     

Cultivation of denitrifying bacteria: optimization of isolation conditions and diversity study

An evolutionary algorithm was applied to study the complex interactions between medium parameters and their effects on the isolation of denitrifying bacteria, both in number and in diversity. Growth media with a pH of 7 and a nitrogen concentration of 3 mM, supplemented with 1 ml of vitamin solution but not with sodium chloride or riboflavin, were the most successful for the isolation of denitrifiers from activated sludge. The use of ethanol or succinate as a carbon source and a molar C/N ratio of 2.5, 20, or 25 were also favorable. After testing of 60 different medium parameter combinations and comparison with each other as well as with the standard medium Trypticase soy agar supplemented with nitrate, three growth media were highly suitable for the cultivation of denitrifying bacteria. All evaluated isolation conditions were used to study the cultivable denitrifier diversity of activated sludge from a municipal wastewater treatment plant. One hundred ninety-nine denitrifiers were isolated, the majority of which belonged to the Betaproteobacteria (50.4%) and the Alphaproteobacteria (36.8%). Representatives of Gammaproteobacteria (5.6%), Epsilonproteobacteria (2%), and Firmicutes (4%) and one isolate of the Bacteroidetes were also found. This study revealed a much more diverse denitrifying community than that previously described in cultivation-dependent research on activated sludge.     

Comparing the dehalogenase gene pool in cultivated alpha-halocarboxylic acid-degrading bacteria with the environmental metagene pool

Culture-dependent and culture-independent approaches were used to determine the relationship between the dehalogenase gene pool in bacteria enriched and isolated on 2,2-dichloropropionic acid (22DCPA) and the environmental metagene pool (the collective gene pool of both the culturable and uncultured microbes) from which they were isolated. The dehalogenases in the pure-cultures isolates, which were able to degrade 22DCPA, were similar to previously described group I and II dehalogenases. Significantly, the majority of the dehalogenases isolated from activated sludge by degenerate PCR with primers specific for alpha-halocarboxylic acid dehalogenases were not closely related to the dehalogenases in any isolate. Furthermore, the dehalogenases found in the pure cultures predominated in the enrichments but were a minor component of the community used to inoculate the batch cultures. Phylogenetic analysis of the dehalogenase sequences isolated by degenerate PCR showed that the diversity of the group II deh gene was greater than that of the group I deh gene. Direct plating of the activated sludge onto minimal media supplemented with 22DCPA resulted in biomass and DNA from which dehalogenases were amplified. Analysis of the sequences revealed that they were much more closely related to the sequences found in the community used to start the enrichments. However, no pure cultures were obtained with this isolation method, and thus no pure cultures were available for identification. In this study we examined the link between genes found in pure cultures with the metagene pool from which they were isolated. The results show that there is a large bias introduced by culturing, not just in the bacteria isolated but also the degradative genes that they contain. Moreover, our findings serve as a caveat for studies involving the culturing of pure cultures of bacteria and conclusions which are drawn from analysis of these organisms.     

Characterization of diverse Acinetobacter isolates for utilization of multiple aromatic compounds

This study demonstrates the multiple catabolic capacities of lab isolates belonging to the genus Acinetobacter. Thirty-one Acinetobacter strains were screened initially for their capacity to utilize ten substrates that includes monocyclic, heterocyclic and polycyclic aromatic compounds. These bacteria were isolated from activated biomass of different effluent treatment plants (ETPs) treating wastewater generated at different industries and selected based on partial sequence data of the 16S rRNA gene. Of these 31 isolates, preliminary plate assay results showed eleven isolates that could utilize multiple substrates. Analytical studies demonstrated multiple degradation of hydrocarbons dibenzothiophene, fluorene, dibenzofuran, benzyl sulfide, and sodium benzoate by two isolates, HPC311 and HPC159.     

Comparing the Dehalogenase Gene Pool in Cultivated α-Halocarboxylic Acid-Degrading Bacteria with the Environmental Metagene Pool

Culture-dependent and culture-independent approaches were used to determine the relationship between the dehalogenase gene pool in bacteria enriched and isolated on 2,2-dichloropropionic acid (22DCPA) and the environmental metagene pool (the collective gene pool of both the culturable and uncultured microbes) from which they were isolated. The dehalogenases in the pure-cultures isolates, which were able to degrade 22DCPA, were similar to previously described group I and II dehalogenases. Significantly, the majority of the dehalogenases isolated from activated sludge by degenerate PCR with primers specific for α-halocarboxylic acid dehalogenases were not closely related to the dehalogenases in any isolate. Furthermore, the dehalogenases found in the pure cultures predominated in the enrichments but were a minor component of the community used to inoculate the batch cultures. Phylogenetic analysis of the dehalogenase sequences isolated by degenerate PCR showed that the diversity of the group II deh gene was greater than that of the group I deh gene. Direct plating of the activated sludge onto minimal media supplemented with 22DCPA resulted in biomass and DNA from which dehalogenases were amplified. Analysis of the sequences revealed that they were much more closely related to the sequences found in the community used to start the enrichments. However, no pure cultures were obtained with this isolation method, and thus no pure cultures were available for identification. In this study we examined the link between genes found in pure cultures with the metagene pool from which they were isolated. The results show that there is a large bias introduced by culturing, not just in the bacteria isolated but also the degradative genes that they contain. Moreover, our findings serve as a caveat for studies involving the culturing of pure cultures of bacteria and conclusions which are drawn from analysis of these organisms.     

Bacterial community composition and diversity of a full-scale integrated fixed-film activated sludge system as investigated by pyrosequencing

The integrated fixed-film activated sludge (IFAS) system is a variation of the activated sludge wastewater treatment process, in which hybrid suspended and attached biomass is used to treat wastewater. Although the function and performance of the IFAS system are well studied, little is known about its microbial community structure. In this study, the composition and diversity of the bacterial community of suspended and attached biomass samples were investigated in a full-scale IFAS system using a highthroughput pyrosequencing technology. Distinct bacterial community compositions were examined for each sample and appeared to be important for its features different from conventional activated sludge processes. The abundant bacterial groups were Betaproteobacteria (59.3%), Gammaproteobacteria (8.1%), Bacteroidetes (5.2%), Alphaproteobacteria (3.9%), and Actinobacteria (3.2%) in the suspended sample, whereas Actinobacteria (14.6%), Firmicutes (13.6%), Bacteroidetes (11.6%), Betaproteobacteria (9.9%), Gammaproteobacteria (9.25%), and Alphaproteobacteria (7.4%) were major bacterial groups in the attached sample. Regarding the diversity, totals of 3,034 and 1,451 operational taxonomic units were identified at the 3% cutoff for the suspended and attached samples, respectively. Rank abundance and community analyses demonstrated that most of the diversity was originated from rare species in the samples. Taken together, the information obtained in this study will be a base for further studies relating to the microbial community structure and function of the IFAS system.