Gottshall, E.*, Bryson, S.*, Cogert, K.*, Landreau, M.*, Sedlacek, C**., Stahl, D.*, Daims, H.**, and Winkler, M.*
*University of Washington, 616 NE Northlake Place, 476B Seattle, WA, 98105-5014
** University of Vienna, Universitätsring 1, 1010 Wien, Austria
Keywords: Comammox; Anammox; nitrification, symbiotic relationship, nitrogen Summary of key findings
We demonstrated that complete aerobic ammonia-oxidizing bacteria (Comammox) N. inopinata can establish a cooperative relationship with anaerobic ammonia-oxidizing bacteria (Anammox) in a synthetic granule format. The significance of this novel cooperation was documented by the low nitrate formation and the competitiveness of the Comammox organism over other aerobic ammonia- and nitrite-oxidizers. Since nitrate is an undesirable product of some engineered treatment systems, the Comammox-Anammox symbiosis may be of economic and ecological importance to reduce nitrogen contamination of receiving waters. In addition, our study also suggests that a similar cooperation of Comammox and Anammox may be important in natural aquatic or terrestrial habitats that are oxygen and ammonia deplete.
Background and relevance
Mathematical models have indicated that oxygen and ammonium concentrations are major niche differentiating factors for Anammox and ammonia-oxidizing aracheae (AOA) or Anammox and ammonia-oxidizing bacteria (AOB). For example, the association between AOB/AOA and Anammox is often disrupted by nitrite-oxidizing bacteria (NOB) competing with Anammox for nitrite (Kent et al., 2019; Third et al., 2001), resulting in a lower conversion of ammonia to N2 and an increase in nitrate formation. Thus, there is clear need to resolve competitive and cooperative relationships among Comammox, Anammox, AOA, AOB, and NOB in order to develop a better predictive understanding of their roles in the global nitrogen cycle and to better define possible applications in engineered systems (Straka et al., 2019). The Comammox relationship with Anammox is of specific interest since both bacteria utilize ammonia and nitrite and are active in low DO environments.
However, comparative laboratory analyses of organisms that function cooperatively at the boundary between oxic and anoxic environments have been limited for lack of appropriate experimental systems. Suspended microbial cultures do not form the oxygen gradient essential for partnership.
Chemostat studies must balance the conflicting nutrient requirements for cooperative growth and are prone to washout. We here use synthetic community assemblies of Comammox N. inopinata and Anammox (dominant species Brocadia anammoxidans) immobilized in hydrogel granules to evaluate environmental conditions supporting their partnership. The hydrogel format, which is a three-
dimensional network of hydrophilic polymers, has been successfully implemented to immobilize active nitrifying and denitrifying bacteria (Wijffels et al., 1995), denitrifying cultures (Xu et al., 2017),
anaerobes originating from hydrothermal vents (Landreau et al., 2016)(Ali et al., 2015) , and Anammox (Ali et al., 2015). The gels can also be formed to resemble naturally occurring biogranules (Flemming and Wingender, 2010; Flemming et al., 2007), in which Comammox and Anammox can be entrapped in a matrix of extracellular polymeric substances (EPS) that promotes the formation of stable nutrient gradients and microbial interactions on a micrometer scale. Using a synthetic hydrogel
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set-up to achieve conditions for both aerobic and anaerobic growth, we now demonstrate the formation of a stable partnership in which Comammox competitively excludes the AOB to supply nitrite to Anammox and, in turn, Anammox lowers nitrite to a non-inhibitory concentration. This is the first clear evidence for possible ecological relevance of an association that should be useful for various
biotechnological applications.
Results
Comammox and Anammox bacteria establish cooperation within synthetic granules in batch cultures.
A hydrogel formulation was developed to entrap high numbers of the only available Comammox pure culture N. inopinata and an Anammox enrichment (dominant organism Brocadia anammoxidans) in synthetic Polyvinyl alcohol – sodium alginate (PVA-SA) hydrogel beads. Comammox-Anammox beads demonstrated a constant rate of ammonium consumption over the study period of 86 days, resulting in complete nitrogen removal from the system (Fig. 1.1). Since no external nitrite was supplied, Comammox activity was the sole source of nitrite for Anammox. Comammox-Anammox bead consumption of 1mM ammonium was about 3-fold slower than observed for beads containing only Comammox (0.021 mM NH4/day versus 0.064 mM NH4/day).
The bacterial abundance, as measured by qPCR targeting the amoA genes of Comammox and betaproteobacterial AOB and 16S rRNA gene of Anammox demonstrated that Comammox and Anammox bacteria dominated the ammonia-oxidizer population in the hydrogel beads (Fig. 2.1). In contrast, betaproteobacterial AOB were near the limit of detection in all hydrogel samples and persisted only in the fraction of non-immobilized planktonic bacteria (Fig. 2.1).
Figure 1.1 Comammox and Anammox cooperation within hydrogel beads. Immobilized Comammox and Anammox hydrogel beads demonstrated cooperation at 4 mgO2/L and with ammonium as sole source of energy, electrons, and nitrogen in three biological replicates (n=3).
qPCR and 16S rRNA sequencing supports Anammox and Comammox cooperation in hydrogel beads The bacterial abundance, as measured by qPCR targeting the amoA genes of Comammox and betaproteobacterial AOB and 16S rRNA gene of Anammox demonstrated that Comammox and Anammox bacteria dominated the ammonia-oxidizer population in the hydrogel beads (Fig. 2.1). In contrast, betaproteobacterial AOB were near the limit of detection in all hydrogel samples and persisted only in the fraction of non-immobilized planktonic bacteria (Fig. 2.1).
Figure 2.1. Abundance of Comammox, Anammox, and canonical AOB in hydrogel beads and in the planktonic fraction
determined by qPCR of amoA genes (Comammox, AOB) and 16S rRNA genes (Anammox). Gene copy numbers per ng of total extracted DNA are shown for samples taken at the start of the incubation (day 0) and after 86 days. Genetic material
corresponding to the initial biomass (day 0) was obtained directly after immobilization through the same extraction method as the day 86 biomass samples. Gene copy numbers were adjusted according to their average occurrence.
The community composition of PVA-SA hydrogels at the beginning and end of incubation was
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investigated using amplicon sequencing of the V4-V5 region of the 16S rRNA gene. The Comammox OTU accounted for 20.3% reads at the beginning of the incubation and averaged 5.15% (± 7.73% S.D., n=3) at termination. Canonical AOB, AOA, and NOB accounted for only 0.060%, 0.010%, and 0.020%
of the reads initially and remained low throughout the experimental period. Together this indicated that Comammox was responsible for partial nitrification in support of Anammox. The observed apparent loss of Anammox in PVA-SA hydrogels after 86 days (7.68% of the reads in the initial beads and 0.32% (± 0.05% S.D., n=3) of the final biomass) likely reflected a combination of degradative loss in the nutrient-limited interior and by oxygen exposure near the outer boundary of the granules, increased heterotroph abundance, and poor extraction of DNA from Anammox cells embedded deep within hydrogel granules. Additionally, PCR biases for the v4-v5 16S rRNA gene primers may preferentially amplify other taxa that increased in abundance during incubation, skewing relative abundance data (Orschler et al., 2019).
Comammox and Anammox species are spatially segregated within hydrogel beads.
Given the efficient removal of nitrogen by this community, we further examined the population structure using rRNA-targeted FISH imaging and qPCR. Fluorescent probe AMX368-hybridized Anammox bacteria were detected in the initial bead samples as well as at the end of the incubation period (Fig. 3), confirming Anammox persistence within hydrogel beads. Following initial bead fabrication, cells were dispersed in the polymer matrix while they mainly appeared on the peripheral layer of the beads after 86 days of incubation (Fig. 3.1). While the Comammox organisms N.
inopinata (green) was randomly distributed just after immobilization, it formed a thin layer at the surface following the incubation period. In particular, Comammox cells resided at the oxygenated edge of the hydrogel beads, whereas Anammox cells were situated in a deeper (presumably anoxic) layer. Probe Nso1225 (specific for betaproteobacterial AOB) did not produce a detectable fluorescent signal (Fig. 3.1).
Discussion
Figure 3.1. Comammox Nitrospira and Anammox demonstrate spatial segregation within hydrogel beads. Simultaneous in situ hybridization of a bead section with Cy3-labeled probe AMX368 (red; Anammox), fluorescein-labeled probe Ntsp662 (green; Nitrospira), and Cy5-labeled probe Nso1225 (blue;
betaproteobacterial AOB). The bar represents 100 μm.
Here we used a unique hydrogel format to pair Comammox and Anammox in a granule-like structure engineered to promote the formation of nutrient gradients theorized to foster their co-occurrence within natural habitats. The hydrogel format promotes a similar to natural biofilms relationship in a controlled laboratory setting. Therefore, we fabricated synthetic hydrogel granules harboring a single species of Comammox, Nitrospira inopinata, and an enrichment of Anammox related to Brocardia. The high total nitrogen removal was consistent with Anammox being sustained by nitrite produced by the
co-immobilized N. inopinata, suggesting that very little nitrite was further oxidized to nitrate in the synthetic community. Together these results suggest that nitrite was consumed by Anammox and not further oxidized by Comammox. This is in alignment with reported nitrite affinities of Km(app), NO2 ≈ 48 µM for Anammox and Km(app), NO2 ≈ 449.2 μM for N. inopinata, suggesting that a higher rate of nitrite consumption by Anammox provided it a competitive advantage. 16S rRNA analysis of microbial populations from the initial beads versus those incubated for 86 days beads revealed that N.
inopinata remained the major aerobic ammonia oxidizer. In addition to confirming by qPCR the retention of Comammox and Anammox, and an insignificant contribution of AOB and NOB, we demonstrated their expected spatial relationship using fluorescent probes specific to the associated bacterial families. Low nitrate accumulation sets the cooperation described here apart from previously
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studied systems of AOB/AOA and Anammox or AOB/AOA and NOB, in which significant nitrate was produced. The stability of this association is suggestive of other interactions, such as the release of small organic compounds (e.g., formate) from autotrophic Anammox (Freitag et al., 1987; Koch et al., 2015; Lawson et al., 2017) serving as electron donors for heterotrophs or possible mixotrophic
lifestyle of Comammox. A Comammox-Anammox partnership may therefore be beneficial in engineering applications that aim to eliminate nitrogen species using autotrophic microbial populations. While the NH3 affinity of AOB is moderate to low, AOA and Comammox have high affinities for NH3, which is beneficial for better N removal in mainstream WRRFs. However, AOA are generally less abundant in WRRFs possibly due to organic materials inhibition leading to poor bioavailability of copper in these settings (Gwak et al., 2020). Comammox, in contrast, can be quite prevalent in WRRFs (Spasov et al., 2020; Yang et al., 2020). From this perspective, Comammox may be better adapted than AOA for engineered systems and could therefore be paired with Anammox for N removal in those systems.
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Presenting Author
Dr. Gottshall
Post-doctoral Researcher University of Washington
Is the presenting author an IWA Young Water Professional? N (i.e. an IWA member under 35 years of age)
Bio: I grew up and studied biology as an undergraduate student in Russia in a coal minining heavily polluted area of Southern Siberia. Aspiration to study microbial world and to explore different cultures brought me to the United States where I completed my PhD in molecular biology at the University of Wyoming studying cellular structure and functions of Planktomycetes Gemmata
obscuriglobus. I become a mother of two boys and was fortunate to spend time with them as my family and I moved to Hong Kong. After returning to the United States I joined my current postdoctoral laboratory to futher study enigmatic Planktomycetes cell physiology along with the newely discovered Comammox bacteria however with an engeneering and ecological aspects.
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