Microbial community structure in a full-scale anaerobic treatment plant during start-up and first year of operation revealed by high-throughput 16S rRNA gene amplicon sequencing
Introduction
The aeration ponds of the biological treatment plant at a wood and pulp factory operated by Borregaard Ind. Ltd., a world’s leading supplier of lignin-based chemicals, were shut down in September 2008 due to high concentrations of Legionella spp. bacteria in the ponds (Blatny et al., 2008, Nygård et al., 2008, Olsen et al., 2010). Aeration ponds in activated sludge plants may provide a favorable growth environment for pathogenic microorganisms, including Legionella spp. and freshwater amoeba which can serve as host organisms for amoeba-resistant Legionella spp. (Fykse et al., 2014, Olsen et al., 2010). A concern when operating biological treatment plants is therefore the potential growth of pathogenic Legionella spp. and other microbial pathogens, and in particular the release of pathogens to the open environment through discharge of liquid effluent (e.g. to rivers) or aerosolization to the atmosphere (Blatny et al., 2011, Fykse et al., 2013).
In 2013, a new full-scale closed anaerobic treatment plant was established at Borregaard to reduce the potential for future releases of Legionella spp. and other potential human pathogens into the open atmosphere. The anaerobic treatment plant also has other advantages such as reduced discharge of organic material to the nearby river Glomma and allowing waste to be used for biogas production. The full-scale anaerobic reactor is based on the Biothane Biobed Expanded Granular Sludge Bed (EGSB) technology.
Anaerobic reactors contain highly complex microbial communities which play critical roles in the degradation processes which mainly proceed in four metabolic steps: hydrolysis, fermentation, acetogenesis, and methanogenesis (Zinder, 1984). Multiple types of bacteria, including Eubacteria and Archaea, contribute in a complex chain of events. During anaerobic fermentation, large organic molecules are broken down into hydrogen and acetic acid, which can be used in methanogenic respiration (Sandoval Lozano et al., 2009). During the initial startup of an anaerobic reactor it is common to seed the reactor with biomass from another reactor to establish a microbial community that are able to quickly start digestion as a response to wastewater infeed (Ahring, 2003) to avoid buildup of harmful intermediate such as for example volatile fatty acids (VFA) (acidosis), which inhibits the methane production (Goux et al., 2015, Nelson et al., 2012). A link between microbial diversity and process robustness has been shown (Goux et al., 2015, Sundberg et al., 2013, Werner et al., 2010, Ziganshin et al., 2013) and knowledge about the microbial communities in anaerobic reactors may lead to increased awareness of factors that are important for efficient/optimal and stable operation as well as increased understanding of the potential for amplification and release of pathogenic microorganisms to the environment.
The microbial communities of anaerobic reactors have in the past been investigated using various methods, including: conventional microbiological methods (Roest et al., 2005), Sanger sequencing of 16S rRNA gene cloning libraries, denaturing gradient gel electrophoresis, and fluorescent in situ hybridization (Nelson et al., 2011, Pereira et al., 2006, Rivière et al., 2009). It is, however, assumed that only a small fraction of the microbial community present in an anaerobic reactor can be cultured under laboratory conditions (Amann et al., 1995). High throughput sequencing has therefore gained popularity in recent years as these techniques allow for a deeper and more convenient analysis of complex microbial communities without the need for time-consuming and labor-intensive Sanger sequencing and cloning processes or culture analysis. High-throughput sequencing has been used in several metagenomics studies to characterize the microbial community in anaerobic reactors, both as high-throughput amplicon sequencing of the 16S rRNA gene (Li et al., 2013, Shu et al., 2015, Sundberg et al., 2013, Wong et al., 2013) and direct high-throughput shotgun sequencing (Guo et al., 2015). From these studies, sequences belonging to the phyla Proteobacteria, Bacteroidetes, Firmicutes and Chloroflexi were most abundantly identified. Deeper understanding of the microbial community structure in full-scale anaerobic reactors, including community dynamics and adaptation in response to environmental changes such as changes in wastewater composition, is important to optimize the performance of anaerobic treatment plants (Goux et al., 2015).
Few studies using high throughput sequencing to characterize the microbial community dynamics during the start-up phase of an anaerobic reactor have been performed (Goux et al., 2016, Solli et al., 2014). The main objective of the present study was to characterize the microbial community in a full-scale anaerobic reactor (EGSB) at a Norwegian wood and pulp factory using high throughput amplicon sequencing. The V3–V5 hypervariable region of the bacterial 16S rRNA gene was used to characterize the microbial community in the biomass used to seed the anaerobic reactor (inoculum) and comparison to the microbial community in the reactor and discharged effluent after six as well as 12 months of operation, respectively. Bioinformatic analysis was used to examine the alpha diversity and the taxonomy of the samples. Additionally, the secondary objective of this work was to investigate the potential presence (growth/amplification) of human pathogenic microorganisms with emphasis on Legionella spp. in the anaerobic treatment plant.
Section snippets
Wastewater treatment plant and sample collection
The surveyed wastewater treatment plant was a full-scale anaerobic treatment plant operated by Borregaard located in Sarpsborg, Norway. The anaerobic reactor was based on Biothane Biobed EGSB technology. Two wastewater streams from Borregaard’s production process, condensates from ethanol and alkacell production, were combined in a buffer tank before fed into a conditioning tank. The organic content of the ethanol condensate was as mainly acetic acid, ethanol, methanol, aldehydes, and amyl
Operational performance of the full-scale anaerobic reactor
This full scale anaerobic reactor was fed with two wastewater streams from the production processes at Borregaard (condensates from ethanol production and alkacell production, respectively) as described in material and methods. The composition of the wastewater streams was kept relatively constant throughout the study period. In general, the ethanol production condensate contributed about 80% (Table 2) throughout the entire test period. On average the Chemical Oxygen Demand (COD) of the
Conclusions
High-throughput amplicon sequencing of six biomass samples from a full-scale anaerobic treatment plant during start-up and first year of operation was performed in this work. A more diverse microbial community was observed in the inoculum biomass compared to the six and 12 months samples. The dominant bacterial communities were affiliated to the phyla Bacteroidetes, Chloroflexi, Firmicutes, Proteobacteria and the Spirochaetes. The observed community structure changes probably reflect an
Acknowledgements
The work was funded by Borregaard ind. Ltd. and by the Norwegian Defence Research Establishment (FFI). The authors acknowledge Simen Settem Simonsen, Rune Gunnar Bergh and Viggo Waagen at Borregaard for valuable help and support including providing the samples and the operational performance data of the reactor.
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