Motoharu Onuki

The microbiology of biological phosphorus removal in activated sludge systems

Activated sludge systems are designed and operated globally to remove phosphorus microbiologically, a process called enhanced biological phosphorus removal (EBPR). Yet little is still known about the ecology of EBPR processes, the microbes involved, their functions there and the possible reasons why they often perform unreliably. The application of rRNA-based methods to analyze EBPR community structure has changed dramatically our understanding of the microbial populations responsible for EBPR, but many substantial gaps in our knowledge of the population dynamics of EBPR and its underlying mechanisms remain. This review critically examines what we once thought we knew about the microbial ecology of EBPR, what we think we now know, and what still needs to be elucidated before these processes can be operated and controlled more reliably than is currently possible. It looks at the history of EBPR, the currently available biochemical models, the structure of the microbial communities found in EBPR systems, possible identities of the bacteria responsible, and the evidence why these systems might operate suboptimally. The review stresses the need to extend what have been predominantly laboratory-based studies to full-scale operating plants. It aims to encourage microbiologists and process engineers to collaborate more closely and to bring an interdisciplinary approach to bear on this complex ecosystem.

Microbial community structure in activated sludge floc analysed by fluorescence in situ hybridization and its relation to floc stability.

The efficiency of activated sludge treatment plants is dependent on the solid-liquid separation properties of the activated sludge. A critical parameter is the stability of the microbial flocs. Weak flocs deflocculate easily leaving increased concentrations of suspended solids in the effluent. The knowledge about how different bacteria are attached to the flocs and their influence on the bioflocculation is limited. In this study, the deflocculation of different phylogenetic groups of bacteria in activated sludge from a full scale plant was investigated. The experiments were carried out by using a shear method where the sludge flocs are deflocculated under controlled shear conditions. The degree of deflocculation was measured as increase in turbidity of the supernatant. Identification and quantification of the microbial community structure of both total activated sludge and deflocculated bacteria were conducted with group-specific gene probes for broad groups of bacteria (Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria and Actinobacteria) and fluorescence in situ hybridization (FISH). The microbial community structure of the deflocculated bacteria was different compared to the total activated sludge with a higher abundance of Gammaproteobacteria in the supernatant indicating that different groups of bacteria are bound with different strength to the floc surface. The results show that the bacteria in the outer parts of the flocs are relatively loosely bound to the floc matrix and can be easily eroded from the surface when exposed to shear.

Microorganisms involved in anaerobic phenol degradation in the treatment of synthetic coke‐oven wastewater detected by RNA stable‐isotope probing

An RNA-based stable-isotope probing method was used to identify anaerobic phenol-assimilating bacteria present in activated sludge and used to treat synthetic coke-oven wastewater. Activated sludge was fed with nitrate and (13)C-labeled or unlabeled phenol under anaerobic conditions. After the incubation period, RNA was extracted from the activated sludge and separated by isopycnic centrifugation. Bacterial rRNA in each density fraction was analyzed by reverse transcription-PCR-mediated terminal restriction fragment length polymorphism and cloning followed by sequencing. A microorganism affiliated with the genus Azoarcus was the first to obviously incorporate (13)C: this microorganism was thought to have utilized phenol directly. The microorganisms affiliated with the genera Microbulbifer, Pelagiobacter, Pseudomonas, and Thauera were the next to incorporate (13)C. Although these microorganisms were involved in phenol degradation, whether they assimilated (13)C-labeled phenol directly or indirectly, could not be determined. Some Azoarcus and Thauera strains have previously been reported to degrade phenol under denitrifying conditions, but no strains of the other three genera have been reported to do so.

Isolation, characterization of bacteriophages specific to Microlunatus phosphovorus and their application for rapid host detection

Aims:  To isolate and characterize lytic‐bacteriophages specific to Microlunatus phosphovorus, and prepare fluorescently labelled phages (FLPs) for the rapid detection of the host bacterium in activated sludge.

PHA-accumulating microorganisms in full-scale wastewater treatment plants.

A study was conducted to clarify phylogenetic affiliations of polyhydroxyalkanoate (PHA)-accumulating microorganisms in full-scale activated sludge processes. Activated sludge samples obtained from three full-scale activated sludge processes were aerobically incubated with excess acetate to increase their PHA content. The buoyant density separation method was applied to selectively collect PHA-accumulating cells, which were then analysed by the group-level FISH and the PCR-DGGE-sequencing methods, and possible PHA-accumulating microbial groups were screened. A set of oligonucleotide probes targeting the microbial groups suspected to accumulate PHA was introduced, and seven oligonucleotide probes were newly designed for this purpose. PHA accumulation of probe-positive cells was confirmed by the post-FISH PHA staining method, wherein PHA staining with Nile Blue A (NBA) was applied after FISH. As a result, the following seven bacterial groups were found to have PHA: Dechloromonas, Accumulibacter, Thauera, Zoogloea, Comamonas, Competibacter and a novel cluster in Beta-proteobacteria. Based on the results of the post-FISH PHA staining method, these seven bacterial groups were estimated to account for around four-tenths to two-thirds of total PHA-accumulating microorganisms.

Microbial Community Composition of Polyhydroxyalkanoate-Accumulating Organisms in Full-Scale Wastewater Treatment Plants Operated in Fully Aerobic Mode

The removal of biodegradable organic matter is one of the most important objectives in biological wastewater treatments. Polyhydroxyalkanoate (PHA)-accumulating organisms (PHAAOs) significantly contribute to the removal of biodegradable organic matter; however, their microbial community composition is mostly unknown. In the present study, the microbial community composition of PHAAOs was investigated at 8 full-scale wastewater treatment plants (WWTPs), operated in fully aerobic mode, by fluorescence in situ hybridization (FISH) analysis and post-FISH Nile blue A (NBA) staining techniques. Our results demonstrated that 1) PHAAOs were in the range of 11–18% in the total number of cells, and 2) the microbial community composition of PHAAOs was similar at the bacterial domain/phylum/class/order level among the 8 full-scale WWTPs, and dominant PHAAOs were members of the class Alphaproteobacteria and Betaproteobacteria. The microbial community composition of α- and β-proteobacterial PHAAOs was examined by 16S rRNA gene clone library analysis and further by applying a set of newly designed oligonucleotide probes targeting 16S rRNA gene sequences of α- or β-proteobacterial PHAAOs. The results demonstrated that the microbial community composition of PHAAOs differed in the class Alphaproteobacteria and Betaproteobacteria, which possibly resulted in a different PHA accumulation capacity among the WWTPs (8.5–38.2 mg-C g-VSS−1 h−1). The present study extended the knowledge of the microbial diversity of PHAAOs in full-scale WWTPs operated in fully aerobic mode.

Reconstruction Following the 2011 Tohoku Earthquake Tsunami: Case Study of Otsuchi Town in Iwate Prefecture, Japan

The 2011 Tohoku Earthquake and Tsunami caused great devastation throughout the northeastern coast of Japan. Following the disaster important reconstruction efforts are currently ongoing to improve the safety and resilience of coastal communities against future events. To do so the government is investing considerable resources in the creation of a true modern multi-layer safety system, involving reclassifying coastal areas into “Disaster Hazard Areas” where only businesses and public use areas are allowed, and residential areas where people should live. These residential areas are being elevated throughout the region, theoretically ensuring that people living in them should be safer in case coastal defenses are overcome by a tsunami in the future. Important efforts are also being made to promote cultural awareness about the need to evacuate and to improve evacuation routes. The present chapter will discuss various reconstruction issues, including the ever-present dilemma inherent to any disaster reconstruction process where the need to improve disaster resilience is confronted with the desires of survivors to rebuild their houses and livelihoods as quickly as possible. The authors will use Otsuchi Town in Iwate Prefecture as a case study, as this was one of the settlements worst hit by the disaster and can serve to highlight the particular socio-economical and demographic challenges facing the region.

Challenges in Build-Back-Better Housing Reconstruction Programs for Coastal Disaster Management: Case of Tacloban City, Philippines

Following typhoon Haiyan in November 2013, the government of the Philippines organized massive housing reconstruction programs that are currently facing complex implementation issues. The present study investigated the mechanisms of three types of housing reconstruction programs (i.e. owner-driven on-site reconstruction, community-driven off-site relocation and contractor-driven off-site relocation) and discussed sustainability challenges by assessing the gaps between community needs and program performance, measured through the level of beneficiary satisfaction. The study conducted semi-structured questionnaire surveys with beneficiary households and key informant interviews with government officials and nongovernment organization representatives in March 2015. Results showed that on-site reconstruction was delayed due to insufficient and poorly implemented assistance schemes relating to reconstruction (e.g. materials, skills training), while off-site relocation was delayed by prolonged land acquisition and subcontracting issues. Disruption of critical infrastructure, such as water utility services, and lack of livelihood opportunities significantly affected the satisfaction levels of respondents with the recovery progress. The study also found that the no-dwelling-zone policy was not strictly enforced as houses were still being rebuilt in high-risk areas near the coast. Finally permanently relocated residents continue to struggle, especially with regard to their source of livelihood, as relocation sites are inaccessible and located away from employment opportunities.

Rapid quantification of total viral DNA in the supernatants of activated sludge samples with the fluorescent dye PicoGreen

Aims:  To develop a rapid and simple method for quantifying viral DNA concentrations and determining viral quantities in activated sludge.

Identification of predominant microbial populations in a non-phosphate removing anaerobic aerobic bioreactor fed with fermented products

Publisher Summary Activated sludge processes remove organic pollutants and nutrients from the wastewater stream. The sludge is sampled from an anaerobic–aerobic sequence batch reactor (A/O SBR) fed with a mixture of a fermented substrate of acetate, yeast extract, peptone, magnesium, potassium, and calcium. Predominant microbial populations are identified using 16S rDNA cloning analysis, and 34 different sequences are found among 161 clones screened. Cloning analysis reveals that microbial populations in the beta-subdivision of the Proteobacteria, Cytophagales division, and the alpha subdivision of the proteobacteria are the predominant ones. The abundance of major phylogenetic groups is further evaluated using fluorescent in situ hybridization (FISH) with group-specific rRNA probes. The bacteria of the beta-subdivision of the division proteobacteria are the most dominating ones and responsible for the substrate metabolism. FISH analysis further detects bacteria from alpha- and gamma-subdivisions, but not from Cytophagales and actinobacteria divisions.