Nitrogen removal is important for effective wastewater treatment. Since the first discovery in laboratory bioreactor in the Netherlands, this group of bacteria has been widely detected in different environments ad ecosystems for ubiquitous occurrence (Li and Gu 2011). Efforts have been made to enrich and re-design WWTPs for enrichment of anammox bacteria so that they can become the dominant ones to contribute inorganic N removal. A conventional WWTP under normal operational conditions was observed with visible accumulation of anammox bacteria as granules and then used subsequently for inoculation to other new WWTPs resulting in successful establishment of anammox bacteria (Meng et al. 2007). To understand microbial communities and the active biochemical N transforming processes for inorganic nitrogen removal in different WWTPs, the total microbial communities and microorganisms involved in nitrogen removal in four WWTPs with high ammonium concentrations and one WWTP with low ammonium concentration were analyzed using Illumina high-throughput sequencing method based on 16S rRNA gene at both DNA and RNA level in this study.

A total of sixteen samples were collected from the five WWTPs in December 2015, including 12 activated sludge samples (Meng et al. 2017). Activated sludge samples from aeration and anaerobic tanks were taken, preserved in RNAlater (MO-BIO, USA) on site and then stored at -80 °C. Total RNA isolation was completed within one week of sampling. Total DNA and RNA were extracted using PowerMax^® Soil DNA Isolation Kit (MO-BIO, USA) and RNA PowerSoil^® Total RNA Isolation Kit (MO-BIO, USA), respectively. All extracted DNA and RNA samples samples were stored at -80 °C before further processing and analysis. Sequencing libraries were generated using TruSeq^® DNA PCR-Free Sample Preparation Kit (Illumina, USA) and index codes were added. The library quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system. The library was sequenced on an Illumina HiSeq2500 platform and 250 bp paired-end reads were generated. Bioinformatics analysis of the sequence data has been described elsewhere for detailed description (Liu et al., 2008a, b).

After filtering the low-quality reads, trimming the adapters, barcodes, and primers, filtering out chimeras, at least 26,035 and 25,376 of DNA and cDNA short reads of 16S rRNA genes at V4 region for each of the 12 sample were yielded in this study. In all samples, Proteobacteria and Bacteroidetes were the most dominant phyla, accounting for 50-75% of the total reads in each sample. In Clusters I, II and III, Proteobacteria was the single dominant phylum, similar to most previous studies on activated sludges from different geographic locations. But in Cluster IV, Bacteroidetes surpassed Proteobacteria and became the dominant one. In Cluster III, the relative abundance of Bacteroidetes (8-16%) was lower compared to other clusters, but Chloroflexi and Planctomycetes occupied significantly higher proportion (20-40%). Proteobacteria was the most active phylum in activated sludge and could contribute to the biochemical function of activated sludge. In addition to Proteobacteria, Bacteroidetes, Actinobacteria, both Acidobacteria and Nitrospirae also showed high activity.

Up to 37 of the 49 genera belonged to the three main phyla, Proteobacteria, Firmicutes, and Bacteroidetes were resulted. Nitrososphaera was the unique archaea; and the remaining 48 genera were bacteria. Nitrososphaera is a genus of ammonia oxidizing archaea (AOA) in the phylum of Thaumarchaeota, but it was not observed in rRNA reads, suggesting that it might be inactive. Similar to Nitrososphaera, many genera in activated sludge were only detected in DNA, but not RNA level; and only 50-60% of the 49 genera were detected in the RNA level, indicating the transcriptionally active microorganisms were much less than those of DNA. Nitrosomonas was the dominant ammonia oxidation bacteria (AOB) detected with a relative abundance of 4.9-7.7%. Nitrospira was the unique nitrite oxidizing bacteria (NOB) with an abundance of 0.5-5.0%. Anammox bacteria play an important role in transforming ammonium and nitrite to dinitrogen gas. Until now, only five genera of anammox bacteria have been identified and named, including Brocadia, Kuenenia, Scalindua, Anammoxoglobus and Jettenia (Li and Gu 2011). Of which, Brocadia, Kuenenia, and Anammoxoglobus were widely observed in activated sludge. Brocadia was the dominant anammox bacteria with 8.0- 23.3% in these WWTPs, but Anammoxoglobus and Kuenenia accounted for ra elatively small fraction in these WWTPs.

Microbial community was different using DNA or RNA for Illumina analysis, suggesting active population shall be obtained meaningfully with RNA. Proteobacteria was the dominant and most active phylum in activated sludge. Microorganisms responsible for anammox, nitrification and denitrification existed in these WWTPs, and abundance of N transforming microorganisms was significant correlation with ammonium concentration. N-15 dilution results confirmed that contribution of anammox bacteria to the overall N₂ production can be as high as more than 95%. Anammox bacteria cooperated with nitrification and denitrification for inorganic nitrogen removal in 2 WWTPs of this study while the conventional nitrification-denitrification process was responsible in the other three WWTPs

References

Li M, Gu J-D (2011) Advances in methods for detection of anaerobic ammonium oxidizing (anammox) bacteria. Appl Microbiol Biotechnol 90: 1241-1252. DOI: 10.1007/s00253-011-3230-6

Liu Y-F, Galzerani DD, Mbadinga SM, Zaramela LS, Gu J-D, Mu B-Z, Zengler K (2018a) Metabolic capability and in situ activity of microorganisms in an oil reservoir. Microbiome 6: 5 DOI: 10.1186/s40168-017-0392-1

Liu Y, Zhou Z, Pan J, Baker BJ, Gu J-D, Li M (2018b) Comparative genomic inference suggests mixtrophic life style for Thoarchaeatoa. ISME J DOI: 10.1038/s41396-018-0060-x

Meng H, Yang Y, Lin J-G, Li M, Denecke M, Gu J-D (2017) Occurrence of anammox bacteria in a traditional full-scale wastewater treatment plant and successful inoculation for new establishment. Intern Biodeter Biodegr 120: 224-231. DOI: 10.1016/j.ibiod.2017.01.022

Ji-Dong Gu is currently a Full Professor of the Environmental Science and Engineering Research Group, Guangdong Technion - Israel Institute of Technology in Shantou, China; and concurrently of Technion - Israel Institute of Technology in Haifa, Israel. His h-index is 77, i10-index 382, and a total citation of 21,412 (GoogleScholar). He has been on the world’s top 1% scientists list by WoS since 2013 and is ranked one of the Highly cited scientists in Environmental Science and Engineering in China in 2020.

He received B.Sc. degree from Heilongjiang August First Land Reclamation University (P.R. China), M.Sc. from University of Alberta (Canada), and Ph.D. from Virginia Tech (USA). After post-doctoral research at University of Massachusetts – Lowell briefly and then Harvard University for 6 years, he joined The University of Hong Kong as an Assistant Professor in 1999 and then promoted to Associated Professor in 2003. In the summer of 2020, he resigned from his job in Hong Kong and joined Guangdong Technion – Israel Institute of Technology (GTIIT).

His recent research interest includes: 1) microbiology of cultural heritage; 2) oil field microbiology for enhanced oil recovery and pollution remediation; and 3) carbon and nitrogen cycling, including anaerobic ammonium oxidation and nitrite-dependent anaerobic methane oxidation.

He has published in the areas of applied and environmental microbiology with more than 400 refereed scientific journal papers, 41 book chapters. He co-edited with Ralph Mitchell the book ‘Environmental Microbiology’ (2nd ed, John Wiley-Blackwell. 2010), and wrote a book on ‘Biosusceptibility of Polymers and Fiber-reinforced Composites and Testing Methods’ (Springer, 2023), and additionally edited/co-edited more than 12 special topic issues in International Biodeterioration & Biodegradation, Ecotoxicology, International Journal of Molecular Sciences, and Frontiers in Microbiology.

He is the Editor-in-Chief (EiC) for International Biodeterioration & Biodegradation (2015– ), and founding EiC and Co-EiC for Applied Environmental Biotechnology; Assistant Chief Editor for Frontier in Microbiology: Microbiotechnology Ecotoxicology and Bioremediation (before 2016); Associate Editor for Ecotoxicology, Journal of Environmental Engineering & Ecological Science, and International Journal of Environmental Science and Technology, and as editorial board member for Applied and Environmental Microbiology, Biodegradation, Environmental Geochemistry and Health, Journal of Polymers and the Environment, and Microbes and the Environment (2007-2010).

He also serves as International Board Member of International Society for Subsurface Microbiology (2016– ); and International Board Member, International Biodeterioration & Biodegradation Society; previously ambassador of International Society for Microbial Ecology.