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Granular Sludge Session
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Genome-centric metagenomics of saline anaerobic granular sludge reactors identifies microbial drivers for granulation and adaptation towards halotolerance
Sampara P.3*, Gagliano, C.G. 1,2*, Plugge, C.M.1,2, Temmink, H.1,4, Sudmalis, D.4, Ziels, R.M.3 Wetsus – European Centre of Excellence for Sustainable Water Technology, Oostergoweg 9, 8911MA Leeuwarden, the Netherlands
Laboratory of Microbiology, Wageningen University and Research, Stippeneng 4, 6708 WE, Wageningen, the Netherlands
Civil Engineering, University of British Columbia, 2002 - 6250 Applied Science Lane, Vancouver, BC, V6T 1Z4, Canada
Department of Environmental Technology, Wageningen University and Research, Bornse Weilanden 9, 6708 WG, Wageningen, the Netherlands
(*) authors contributed equally
Keywords: Salinity adaptation; Metagenomics; Anaerobic Digestion Summary of key findings
Genome-centric metagenomic analysis was conducted on two anaerobic granular sludge systems treating synthetic wastewater with different salinities (5 g/L and 20 g/L Na+) to identify microbial drivers of granulation and metabolic features for osmoprotection. Methanothrix_A was the predominant methanogenic genus in the salinity reactors. Mechanisms to produce and uptake multiple osmolytes (N- acetyl-beta-lysine, ectoine, and glycine betaine) that could abate osmotic stress were identified in metagenome-assembled genomes (MAGs) classified as Methanothrix_A. N-glycosylation and extracellular polymeric substance (EPS) production pathways were also detected in Methanothrix_A MAGs, and could have played a role in granular sludge formation in addition to osmoprotection under highly saline conditions. A pangenomic analysis revealed novel gene clusters in the Methanothrix_A MAGs encoding for isoprenoid synthesis, which could be commercially valuable. Based on these findings, Methanothrix_A appears to play a key role in adaptation towards effective anaerobic treatment of saline industrial wastewater using granular bioprocesses.
Background and relevance
Upflow anaerobic sludge blanket (UASB) reactors are of interest for anaerobic treatment of saline wastewater due to their higher energy recovery, lower sludge production, and an ability to handle high volumetric loads compared to aerobic processes1. Approximately 5 % of industrial wastewater discharges are saline or highly saline (> 2% w/v NaCl), and elevated salinity has been shown to be inhibitory to UASB treatment process2,3. Elevated salinities can result in EPS disintegration, leading to reduced granule (spherical biofilms) strength and stability, washout of granules, and overall process deterioration4,5. Thus, there is a need to understand microbial dynamics under high salinitiy conditions, to characterize salinity adapted community members, and to probe into their functional roles in forming stable granules. It was shown that Methanothrix spp. play an important role in EPS production and granulation in UASB reactors, under normal and saline conditions, and thereby influencing the stability of UASB reactors6. Thus, understanding metabolic potential for salinity adaptation in the UASB microbiome, particularly salinity adaptation by Methanothrix, could lead to better engineering control strategies for UASB reactors that promote robust treatment of saline wastewaters.
In this study, genome-centric metagenomics was applied to identify abundant community members within two UASB reactors, one operated at high-salinity (HS 20, g/L Na+) and the other at low salinity (LS 5, g/L Na+)). Pathway analysis was conducted to identify the genomic potential of salinity adapted Methanothrix and Methanothrix_A MAGs using Pathway Tools7 and HMM models after annotating recovered MAGs using the MicroScope8 platform. Further, pangenomic analysis of recovered
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Methanothrix and Methanothrix_A MAGs along with representative Methanothrix and other halotolerant archaeal genomes identified metabolic adaptations and novel gene clusters within the MAGs from the saline UASB reactors.
Results
A total of 298 dereplicated MAGs with over 70% completion and below 10% redundancy were recovered via composition-based and differential coverage binning of 10 DNA samples collected from each UASB community throughout the 217-day study period (n=20 samples total). Of the recovered MAGs, 20 were classified as Archaea. Methanothrix_A MAG_279 and MAG_280, and Methanothrix MAG_281 were identified as the dominant methanogenic archaeal MAGs within the saline UASB reactors.
Methanothrix_A MAGs were highly abundant in both the low and high salinity reactors, while the abundance of Methanothrix MAG_281 became negligibly abundant by the end of the study.
The metabolic potential to synthesize multiple osmolytes was identified in these methanogenic archaeal MAGs. Osmolytes are compounds that can confer tolerance to osmotic stress9. The metabolic potential to synthesize N-acetyl-beta-lysine and ectoine (Figure 1), and uptake glycine betaine was observed in the Methanothrix_A MAGs from the high salinity reactor. Experimental evidence indicating the presence of N-acetyl-beta-lysine in the high salinity reactor supports the hypothesis that the Methanothrix_A MAGs may have the necessary metabolic potential for the synthesis of these compounds. The Methanothrix MAG_281 obtained from the low salinity reactor, and that was washed out of the UASB reactor by the end of the study, did not encode for N-acetyl-beta-lysine (Figure 1). Methanothrix_A MAGs (279 and 280) were also found to encode for N-glycosylation with mannose as the sugar. Lectin staining and FISH imaging confirmed the presence of mannose as a sugar in the EPS surrounding methanogenic archaea in the UASB reactors. CoroNa Red staining indicated that EPS glycoconjugation by Methanothrix_A could have reduced the localization of Na+, and thus further reduced salinity stress. The Methanothrix MAG_281, however, did not seem to encode for N- glycosylation. A pangenomic analysis of the methanogenic MAGs from this study along with halotolerant methanogenic archaea revealed novel gene clusters in the Methanothrix_A MAGs that have the potential to encode for isoprenoid synthesis, such as lycopene and phytoene. Particularly, Methanothrix_A MAG_280 had the metabolic potential for partial carotenoid synthesis (Figure 1). Additionally, an average nucleotide identity (ANI) analysis with 60 Methanothrix MAGs from NCBI, and our recovered MAGs revealed that Methanothrix_A MAG_280 was a potentially novel species, with a maximum shared ANI of 88.9 % with available Methanothrix genomes.
Discussion
The successful operation of a UASB reactor heavily depends on its microbiome. A microbial community analysis using genome-resolved metagenomics in this study indicated that Methanothrix_A members may be key for efficient operation of UASB reactors for saline industrial wastewater treatment.
Methanothrix_A MAGs were identified to have the metabolic capacity to promote halotolerance by utilizing multiple osmolytes, such as N-acetyl-beta lysine, ectoine, and glycine betaine. Particularly, N- acetyl-beta-lysine could be key for survival under high salinities, as Methanothrix MAG_281 which does not have the potential to produce this osmolyte, became negligible in abundance under elevated salinities.
Additionally, N-glycosylation and EPS glycoconjugates seem to abate salinity stress at elevated salinities.
EPS production may also help in granulation, in addition to reducing localization of Na+ and thereby relieving salinity stress. These metabolic features supporting halotolerance likely provide an advantage to Methanothrix_A for survival under elevated salinities. Additionally, their key role in granulation, along with their metabolic potential to synthesize commercially valuable isoprenoids, underscore the importance of Methanothrix_A in UASB reactor microbiomes. Engineering UASB bioreactors to select for a higher abundance of Methanothrix_A organisms could potentially be a strategy to improve the operational robustness of UASB reactors treating industrial high salinity wastewaters.
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Figure 1 Presence and absence of genes encoding N-acetyl-beta-lysine synthesis, ectoine synthesis, and carotenoid synthesis in 19 Methanothrix genomes (NCBI accession numbers in parantheses) and three MAGs from this study (indicated in red). Methanothrix genomes from NCBI are indicated with blue color, Methanothrix and Methanothrix_A MAGs from this study are indicated in red color. Methanosarcina mazei Go1 (light green), Methanococcus maripaludis C5 (dark green), Haloferax volcanii DS2, Haloferax denitrificans ATCC 35960 (orange) are also included in the analysis. Absence of gene encoding a protein is indicated in white. The genomes are arranged based on hierarchal clustering of their average nucleotide identities. The heatmap on the left shows the presence or absence of genes encoding N-acetyl-beta-lysine. The heatmap in the middle shows the presence and absence of genes encoding ectoine biosynthesis. The heatmap on the right shows the presence and absence of genes for isoprenoid synthesis.
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51 Presenting Author
Pranav Sampara Graduate student
The University of British Columbia
Is the presenting author an IWA Young Water Professional? Y/N Y
Bio: Pranav is a third-year Ph.D. student in the Ziels Lab at UBC. Pranav’s primary research is to identify and characterize microbial metabolic potential using stable isotope probing and multi-omics. Before joining UBC, he received is MS in Civil and Environmental Engineering from Virginia Tech. Apart from research, Pranav enjoys reading fiction, hiking, and strumming the guitar.
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