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Exploring anammox bacteria ecology to improve nitrogen removal in wastewater treatment

Hugo Ribeiro1, 2, I Made Wijaya2, 3, Verónica Soares-Santos1, 2, Alexandra Santos2, Paula Salgado1, 2, Ana Machado1, 2, Sérgia Costa-Dias2, Adriano Bordalo1, 2 and Catarina Teixeira1, 2*
  • 1 Interdisciplinary Center for Marine and Environmental Research, Abel Salazar Institute of Biomedical Sciences, University of Porto, Portugal
  • 2 Abel Salazar Institute of Biomedical Sciences, University of Porto, Portugal
  • 3 Sepuluh Nopember Institute of Technology, Indonesia

PROBLEM The constant increase in population led to the intensification of agricultural practices, which consequently incremented the use of fertilizers, particularly nitrogen based. Over the last century, humans have substantially influenced the global nitrogen cycle by increasing both the availability and the mobility of nitrogen compounds in aquatic ecosystems (Camargo and Alonso 2006). The Directive 91/271/EEC highlighted the need of new biotechnological processes for water management solutions. The conventional wastewater treatment technology for nitrogen removal requires nitrification followed by denitrification. However, the procedure is not cost-effective (high oxygen demand, high sludge production, and requires an external electron donor supply), nor environment-friendly, since releases CO2 and N2O, contributing to global warming (Jetten et al. 2009; Kartal et al. 2010). CHALLENGES Anaerobic ammonium oxidation (anammox), an autotrophic process, has been considered as a promising alternative to convert ammonium directly to dinitrogen gas, using nitrite as an electron acceptor under anoxic conditions (van de Graaf et al. 1995). The anammox process can be applied to wastewater treatment instead of the conventional treatment, since requires no external carbon source, produces less sludge, and is more-cost effective (60 % reduction in costs), being environmentally friendly (90 % less CO2 emissions) (Kartal et al. 2010). Anammox bacteria are chemolithoautotrophs, belonging to a branch deeply within the Planctomycetes phylum (Oshiki et al. 2016). There are five known anammox genera within the order of Brocadiales: Candidatus “Brocadia”, Candidatus “Kuenenia”, Candidatus “Anammoxoglobus”, Candidatus “Scalindua”, and Candidatus “Jettenia”. Although anammox bacteria were first discovered in wastewater treatment plants and their applications studied worldwide in this context, they may account for more than 50% of N loss from natural environments playing a significant part of the biogeochemical N2 production (Francis et al. 2007). Presently, large-scale applications of anammox are limited by the long start-up period owing to the low growth rate and doubling time of anammox bacteria described so far (Jetten et al. 2009). Additionally, several variables can affect the efficiency of anammox bacteria activity, such as the availability of nitrite and ammonium, pH, temperature, organic matter content, salinity, and toxic compounds (Jin et al. 2012). These parameters can modify the anammox bacterial community structure and inhibit partially or totally the specific anammox activity (Gonzalez-Martinez et al. 2018). Nevertheless, the potential of anammox bacteria in the wastewater treatment application has attracted the scientific community attention to study the anammox ecology, phylogeny, and functional dynamics and nitrogen removal capacity (Gonzalez-Martinez et al. 2018, Oshiki et al. 2016). However, the interactions among all these aspects remains relatively unexplored. OBJECTIVE In this research project, we expect to contribute with fundamental knowledge about anammox ecology by exploring the relation between community structure and process activity. We intend to enrich anammox bacteria from natural environments, and ultimately to develop microbial inocula to be used as seeding sources for reactors. Our goal is to improve current available technology, and promote a wider application of anammox process in wastewater treatment. METHODOLOGY/EXPECTED RESULTS To attain the aims of this study, four main activities were planned and structured accordingly to Figure 1. The first activity intends to explore the presence of anammox bacteria in different ecological niches, such as estuarine sediments, freshwater sediments, groundwater, and agriculture soils. Locations were selected based on environmental characteristics, and several environmental parameters were monitored, namely the temperature, conductivity, pH, dissolved oxygen, oxidation-reduction potential, nitrate, nitrite, ammonium, and organic matter. The presence, abundance, and diversity of anammox bacteria was characterized by phylogenetic analysis and quantitative PCR targeting anammox bacteria, and specific functional genes (such as hydrazine oxidoreductase, hzo; and hydrazine synthase, hzs), using previously successfully described primer sets (Harhangi et al. 2012). The use of the 16S rRNA and functional genes has become a key procedure providing insights into the ecological role of anammox bacteria and their nature and constrains in several environments (e.g. Dang et al 2013). In the scope of the second activity, anammox biomass was enriched in batch cultures as well as laboratory scale bioreactor systems, such as Up-flow Biofilter and Anaerobic Baffled Reactor. For this, environmental samples with the most anammox potential were selected as biomass sources starter for enrichment with a synthetic nutrient medium (ammonium and nitrite), under anoxic conditions (van de Graaf et al. 1996). Nitrogen compounds were periodically monitored by standards methods as previously described in Teixeira et al. (2016). Several operational parameters were screened, including a range of different temperatures, conductivity, pH, dissolved oxygen, oxidation-reduction potential, nitrate, nitrite, and ammonium, and organic matter. Afterwards, optimum growth conditions and nitrogen removal efficiency were evaluated. Quantitative PCR targeting functional genes (hzo and hzs) was used to detect and as an indicator for growth of the anammox population (van der Star et al. 2007). Anammox activity was measured using the N isotope pairing technique that involves performing incubations amended with different 15N-14N isotopic mixtures and the different N masses (28N2, 29N2 and 30N2), quantified by isotope-ratio mass spectrometry (IRMS), following the procedures described in Teixeira et al. (2016). The microbial community structure was assessed under the scope of the third main activity in environmental samples (activity 1), as well as in enriched samples (activity 2), using automated rRNA intergenic spacer analysis, as described in Teixeira et al. (2014). Based on the level of the obtained diversity, bacterial community composition of selected samples was further investigated using next generation sequencing (NGS) Illumina MiSeq platforms. Bioinformatic analysis of raw reads was performed using QIIME Illumina pipeline for the metagenomic data analysis (e.g. Ribeiro et al. 2018). This analysis provided insights into the phylogeny of the communities, and the presence of catabolic genes present in each environmental sample (activity 1), and in the enrichment cultures (activity 2). All collected data has been analyzed by means of several statistical methodologies, including multivariate data analysis to explore relationships between molecular microbial data, operational parameters, and nitrogen removal activities. FUTURE PERSPECTIVES Successfully enriched cultures will be maintained, and the links established between enriched cultures and function will be matched with wastewaters with specific properties. This will allow the use of enriched cultures as seed sludges for anammox reactors for wastewater treatment, as planned in the fourth activity of the projected study. FINAL REMARKS The findings of this study will improve the currently available knowledge concerning the relationships among environmental parameters, phylogeny/functional taxa, anammox ecology in order to explore microbial inocula to be used as seeding sources for anammox reactors. Therefore, these results will be useful for optimizing parameters in a rapid startup and enhanced bioreactor efficiency, contributing to a wider and efficient application of anammox process in wastewater treatment.

Figure 1
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Acknowledgements

This study was supported by the Strategic Funding UID/Multi/04423/2019 and by the Project UNNOWN (PTDC/BTA-BTA/31098/2017), co-financed by COMPETE 2020, Portugal 2020 and the European Union through the ERDF, and by FCT through national funds.

References

Camargo J.A., and Alonso Á. (2006). Ecological and toxicological effects of inorganic nitrogen pollution in aquatic ecosystems: a global assessment. Environment International 32, 831-849. Dang H., Zhou H., Zhang Z., Yu Z., Hua E., Liu X.S., and Jiao N.Z. (2013). Molecular Detection of Candidatus Scalindua pacifica and Environmental Responses of Sediment Anammox Bacterial Community in the Bohai Sea, China. Plos One 8:e61330. Francis C.A., Beman J.M., and Kuypers M.M.M. (2007) New processes and players in the nitrogen cycle: the microbial ecology of anaerobic and archaeal ammonia oxidation. ISME Journal 1, 19-27. Gonzalez-Martinez A., Muñoz-Palazon B., Rodriguez-Sanchez A., and Gonzalez-Lopez J. (2018). New concepts in anammox processes for wastewater nitrogen removal: recent advances and future prospects. FEMS microbiology letters 365(6), fny031. Harhangi H.R., Le Roy M., van Alen T., Hu B-I., Groen J., Kartal B., Tringe S.G., Quan Z-X, Jetten M.S.M., and Op den Camp H.J.M. (2012). Hydrazine Synthase, a unique phylomarker with which to study the presence and biodiversity of anammox bacteria. Applied and Environmental Microbiology 78 (3), 752-758. Jetten M.S., Niftrik L.V., Strous M., Kartal B., Keltjens J.T., and Op den Camp H.J. (2009). Biochemistry and molecular biology of anammox bacteria. Critical reviews in biochemistry and molecular biology 44, 65-84. Jin R.C., Yang G.F., Yu J.J., and Zheng P. (2012). The inhibition of the Anammox process: a review. Chemical Engineering Journal 197, 67-79. Kartal B., Kuenen J.G., and van Loosdrecht M.C.M. (2010). Sewage treatment with anammox. Science 328, 702–703. Oshiki M., Satoh H., and Okabe S. (2016). Ecology and physiology of anaerobic ammonium oxidizing bacteria. Environmental microbiology 18, 2784-2796. Ribeiro H., de Sousa T., Santos J.P., Sousa A.G.G., Teixeira C., Monteiro M.R., Mucha A.P., Almeida C.M.R., Torgo L., and Magalhães C. (2018). Potential of dissimilatory nitrate reduction pathways in polycyclic aromatic hydrocarbon degradation. Chemosphere 199, 54-67. Teixeira C., Almeida C.M.R., Bordalo A.A., and Mucha A.P. (2014). Development of autochthonous microbial consortia for enhanced phytoremediation of Cd on salt-marsh sediments. Science of the Total Environment 493: 757-765. Teixeira C., Magalhães C., Joye S.B., and Bordalo A.A. (2016). Response of anaerobic ammonium oxidation to inorganic nitrogen fluctuations in temperate estuarine sediments, Journal of Geophysical Research – Biogeosciences 121, 1829-1839. van de Graaf A.A., Mulder A., de Bruijn P., Jetten M.S.M., Robertson L.A., and Kuenen J.G. (1995). Anaerobic oxidation of ammonium is a biologically mediated process. Applied and Environmental Microbiology 61, 1246-1251. van de Graaf A.A., de Bruijn P., Robertson L.A., Jetten M.S.M., and Kuenen J.G. (1996). Autotrophic growth of anaerobic ammonium-oxidizing micro-organisms in a fluidized bed reactor. Microbiology 142, 2187-2196. Van der Star W.R., Abma W.R., Blommers D., Mulder J.W., Tokutomi T., Strous M., Picioreanu C., and van Loosdrecht M.C. (2007). Startup of reactors for anoxic ammonium oxidation: experiences from the first full-scale anammox reactor in Rotterdam. Water Research 41, 4149-4163.

Keywords: Anammox, bioreactor, microbial ecology, nitrogen removal, wastewater treatment

Conference: XX Iberian Symposium on Marine Biology Studies (SIEBM XX) , Braga, Portugal, 9 Sep - 12 Sep, 2019.

Presentation Type: Poster Presentation

Topic: Ecology, Biodiversity and Vulnerable Ecosystems

Citation: Ribeiro H, Wijaya I, Soares-Santos V, Santos A, Salgado P, Machado A, Costa-Dias S, Bordalo A and Teixeira C (2019). Exploring anammox bacteria ecology to improve nitrogen removal in wastewater treatment. Front. Mar. Sci. Conference Abstract: XX Iberian Symposium on Marine Biology Studies (SIEBM XX) . doi: 10.3389/conf.fmars.2019.08.00090

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Received: 14 May 2019; Published Online: 27 Sep 2019.

* Correspondence: Mx. Catarina Teixeira, Interdisciplinary Center for Marine and Environmental Research, Abel Salazar Institute of Biomedical Sciences, University of Porto, Matosinhos, 4450-208, Portugal, catarina@icbas.up.pt