ComammoxNitrospiraare the dominant ammonia oxidizers in a mainstream low dissolved oxygen nitrification reactor

Abstract

Recent findings show that a subset of bacteria affiliated with Nitrospira, a genus known for its importance in nitrite oxidation for biological nutrient removal applications, are capable of complete ammonia oxidation (comammox) to nitrate. Early reports suggested that they were absent or present in low abundance in most activated sludge processes, and thus likely functionally irrelevant. Here we show the accumulation of comammox Nitrospira in a nitrifying sequencing batch reactor operated at low dissolved oxygen (DO) concentrations. Actual mainstream wastewater was used as influent after primary settling and an upstream pre-treatment process for carbon and phosphorus removal. The ammonia removal rate was stable and exceeded that of the treatment plant’s parallel full-scale high DO nitrifying activated sludge reactor. 16S rRNA sequencing showed a steady accumulation of Nitrospira to 53% total abundance and a decline in conventional ammonia oxidizing bacteria to <1% total abundance over 400+ days of operation. After ruling out other known ammonia oxidizers, qPCR confirmed the accumulation of comammox Nitrospira beginning around day 200, to eventually comprise 94% of all detected amoA and 4% of total bacteria by day 407. Quantitative fluorescence in-situ hybridization confirmed the increasing trend and high relative abundance of Nitrospira. These results demonstrate that comammox can be metabolically relevant to nitrogen transformation in wastewater treatment, and can even dominate the ammonia oxidizing community. Our results suggest that comammox may be an important functional group in energy efficient nitrification systems designed to operate at low DO levels.

High functional diversity among Nitrospira populations that dominate rotating biological contactor microbial communities in a municipal wastewater treatment plant

“…These results contrast with previous studies of full-scale activated sludge WWTPs that have reported comammox Nitrospira at lower relative abundances than AOA and other AOB [7, 18-480 21], albeit for systems with ammonium concentrations and operational parameters distinct from those in the RBCs. This current study, along with other more recent studies [12,15,17,18,22], shows that comammox Nitrospira can thrive in wastewater treatment systems. Although previous analyses of the Guelph WWTP did not test for these newly discovered ammonia oxidizers [28,29], the presence of comammox Nitrospira was predicted previously from activity 485 data and an abundance of Nitrospira cells that actively assimilated labelled bicarbonate when incubated with ammonium [29].…”

“…Consistent with a low ammonium niche, comammox Nitrospira have been detected in drinking water systems [7][8][9][10][11][12][13][14]. First identified from water treatment system metagenome sequences [5,6], most wastewaterassociated comammox Nitrospira belong to clade A [5, 7,12,[14][15][16][17][18], albeit with abundances generally lower than those reported for AOA and AOB [7,[18][19][20][21]. Nonetheless, amoA gene 60 abundances of comammox Nitrospira may outnumber those of AOB in activated sludge samples 4 in some WWTPs [12, 18,22], and comammox Nitrospira have been enriched in wastewater treatment reactors with low dissolved oxygen conditions, suggesting that comammox bacteria have an important role in ammonia oxidation in these reactors [15,17].…”

“…Additionally, the high abundance of amoA transcripts from comammox Nitrospira detected in activated sludge suggests 65 that these bacteria actively contribute to nitrification [23]. Previous studies examining comammox Nitrospira in WWTPs focused primarily on activated sludge secondary treatment systems and sequencing batch reactors [12,[15][16][17][18][19][20][21][22][23][24][25][26][27]. To date, no studies have investigated comammox Nitrospira in biofilm-based tertiary wastewater treatment systems.Rotating biological contactors (RBCs; Figure 1A) represent a full-scale tertiary system 70 treating municipal waste from ~132,000 residents of Guelph, Ontario, Canada.…”

Genomic profiling ofNitrospiraspecies reveals ecological success of comammoxNitrospira

“…This predominance of canonical nitrite oxidizers in WWTP is consistent with the proposition that nitrogen-rich environments select for division of labour because it maximizes growth rates 7,31 . However, in some full-scale WWTP 19,32 and in a lab-scale low nitrification reactor 33 comammox Nitrospira have been found more abundant than canonical ammonia oxidizers, suggesting that factors beyond nitrogen fluxes influence comammox Nitrospira fitness in these systems.…”

DNA and RNA-SIP revealNitrospira spp.as key drivers of nitrification in groundwater-fed biofilters

“…Our results provide the first in-situ physiological evidence of ecologically relevant NH 4 + oxidation by comammox Nitrospira in any environment. Other reports of i n situ activity are inferred from bulk observations (ammonium removal when comammox Nitrospira are more abundant than AOBs or AOAs (20, 51)) or from comammox-specific amoA transcript analysis (52). Our former observations that Nitrospira was more abundant than Nitrosomonas and the discovery that Nitrospira harbours the complete nitrification pathway in a full-scale RGSF microbiome (8, 10, 11) is thus directly linked to the ammonia oxidizing activity of Nitrospira in this environment.…”