Microbial ecology of intermittently aerated sequencing batch reactors (IASBRs) for the treatment of dairy processing wastewaters

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Gil Pulido, Beatriz
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University College Cork
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This thesis was conducted as part of the Dairy Water Project, funded by the Department of Agriculture, Food and the Marine, Ireland (Ref.: 13‐F‐507). The Dairy Water project was a multi-stakeholder research collaboration focussed on evaluating opportunities to increase environmental sustainability within the Irish Dairy processing sector. The research scope of the Dairy Water project included: water consumption in the Irish dairy industry and potential water reuse and/or rainwater harvesting; nanomaterial based disinfection technologies and dairy wastewater characterisation; secondary treatment via Intermittently Aerated Sequencing Batch Reactor (IASBR) technology and microbial ecological profiling of systems from laboratory to pilot-scale. In this context, the work presented in this thesis focuses on the microbial characterization of the IASBR technology applied to dairy processing wastewaters bioremediation, which was operated by engineering collaborators in the National University of Ireland, Galway. Ireland is one of Europe’s largest producers and exporter of milk and the dairy processing industry is a key component of the national economy. Following abolition of the European milk quotas in 2015, the dairy industry in Ireland significantly increased its milk production and exports annually. However, processing of milk into secondary products, (e.g. ingredients, cheeses, milk powders, etc.), generates significant volumes of high nutrient-load wastewaters. These require extensive remediation prior to release into natural environments to meet permitted discharge license limits regulated by the Environmental Protection Agency. Traditional biological treatments are often applied to dairy processing wastewater remediation, but typically require chemical precipitant use, high energy inputs and separate bioreactor units with significant infrastructural/capital demand. As a result, there is a continual drive within the wastewater treatment sector to develop cost-effective, high performance, low footprint technologies. IASBR systems are an emerging technology offering an economical and sustainable solution for co-nutrient removal in a single reactor. IASBRs have been successfully applied at laboratory scale to the treatment of domestic and slaughterhouse wastewaters, however the nature of the microbial ecology of these systems is poorly understood. In biological wastewater treatments, microorganisms are the key players and the success in removing pollutants from wastewaters is dependent on their capabilities to remediate such substances. Hence, knowledge of the bacterial community (e.g. overall diversity, stability, dominant relative abundances etc.) in the different remediation processes may contribute to optimisation of bioreactor performance. The study presented here seeks to address the existing knowledge gap in relation to the microbial community structure of IASBRs via next generation sequencing approaches (454 pyrosequencing, MiSeq). Furthermore, high throughput sequencing techniques were combined with comprehensive in silico and statistical analyses to assess the functional gene diversity and dynamics of bacterial populations within representative biomass samples in laboratory and pilot-scale settings. Such knowledge is critical to the understanding and optimisation of IASBR technologies for application to the dairy processing sector and potentially in the treatment of other industrial wastewaters. To position this work within the broader context of wastewater biological treatment technologies, Chapter 1 presents an overview of the scope and scale of dairy industry and wastewater generation, traditional biological treatment design and operation, emerging technologies, key microbial phyla and the biochemical capacities being exploited and modern molecular approaches to profiling same within mixed culture reactor systems. In Chapter 2, the microbial community structure of a laboratory-scale IASBR system operating at 11˚C was analysed using 454-pyrosequencing technique and in silico analyses. The microbial ecology of the IASBR receiving synthetic wastewater was linked with nutrient removal performance within the bioreactor under three different aeration rates, 0.4, 0.6 and 0.8 Litres of oxygen per minute (LPM). In addition, metabolic profiles of the bacterial community were analysed to predict the contribution to nitrogen and phosphorus genes and to identify potential, key contributors to nutrient bioremediation. A key finding was the strong dominance of the family Comamonadaceae (>80% relative abundance) in parallel with optimal nitrogen and phosphorus removal efficiencies over 90% during the aeration of 0.6 LPM, which was not maintained at 0.4 or 0.8 LPM aeration rates. Chapter 3 compared the microbial ecology of two laboratory-scale IASBRs when they were fed with synthetic and industrial wastewaters, respectively. The bioreactors were initially fed with biomass from an industrial plant and were operated under 0.6 LPM and 11˚C. Metagenomic studies were also combined with comprehensive in silico analyses to assess the functional gene diversity within respective biomass samples. In order to gain a greater understanding of population dynamics, statistical analyses were applied to evaluate the impact of wastewater type (synthetic Vs. industrial) on observed communities by means of multivariate redundancy analyses (RDA). Taxonomical analyses revealed the dominance of the Comamonadaceae family and members under controlled conditions (synthetic). However, under industrial wastewater influent feeding, bacterial diversity was observed to be more distributed among Comamonadaceae and other different families. Functional gene prediction analyses carried out in Chapter 2 and Chapter 3, revealed Comamonadaceae family and members as key contributors of nitrogen and phosphorus metabolism genes (nirK, nosZ, norB, ppK, ppX and phaC) during laboratory-scale trials. The investigations carried in Chapter 2 and Chapter 3 of the current thesis provide theoretical support for the currently emerging profile in the literature of Comamonadaceae members as potentially significant contributors to nitrogen and phosphorus remediation processes in the wastewater sphere, under controlled conditions. In Chapter 4, the gained knowledge on microbial communities of laboratory-scale IASBR system was expanded with the investigation of the bacterial ecology structure and dynamics in a pilot-scale IASBR located at an Irish dairy processing plant operated over a five-month period. Metagenomic 16S rRNA gene analyses of pilot scale IASBR reactor samples via Illumina Miseq sequencing revealed a more complex and diverse bacterial community profile in the pilot-scale IASBR than those observed previously in laboratory-scale settings. Although some of the predominant phyla and orders identified were shared with the systems reported in Chapter 2 and Chapter 3, such as Bacteroidetes and Proteobacteria phyla, a number of distinct bacterial groups were observed in the pilot-scale system. Interestingly, the main difference observed at pilot-scale was the absence of Comamonadaceae family and members. Overall, the composition of the bacterial community in the pilot-scale system operated over an extended period was stable in parallel with high nutrient removal performance within the bioreactor (>95% for both nitrogen and phosphorus). The three different trial systems investigated in the current thesis displayed differing bacterial community profiles despite consistent optimal nutrient performance. Thus, IASBR reactors, as operated in the current study, do not appear to be strictly dependent on the dominance of any particular genus for high performance. This demonstrates the potential versatility in diverse wastewater remediation applications for this biological treatment technology.
Wastewater , Biological wastewater treatments , Microbial ecology , Nutrient bioremediation , Dairy processing wastewaters , Intermittently aerated sequencing batch reactors
Gil Pulido, B. 2019. Microbial ecology of intermittently aerated sequencing batch reactors (IASBRs) for the treatment of dairy processing wastewaters. PhD Thesis, University College Cork.