Sulfide-Munching Microbes Team Up With Anammox to Strip Nearly All Nitrogen From Wastewater

Every year, treatment plants around the world process staggering volumes of wastewater that carry ammonium, the nitrogen compound at the heart of eutrophication, fish kills, and drinking water contamination. Conventional nitrogen removal relies on energy-hungry aeration and dosing of organic carbon, costs that strain municipal budgets and inflate the carbon footprint of sanitation. Now, a team of environmental engineers from the National University of Singapore and Zhejiang University has engineered a microbial partnership that removes almost all nitrogen from sulfide-rich, carbon-poor wastewater using two bacterial guilds that feed each other’s strengths. The study, published in Frontiers of Environmental Science & Engineering, reports a laboratory model system that achieved 99.4 percent total nitrogen removal while slashing the sulfate byproduct that has long plagued sulfur-based approaches.

The core of the innovation lies in combining two well-known but notoriously difficult-to-pair processes. The first is anammox, short for anaerobic ammonium oxidation, in which specialized bacteria convert ammonium and nitrite directly into inert nitrogen gas without oxygen or organic carbon. The second is sulfide-dependent autotrophic denitrification, or S-SADN, in which sulfur-oxidizing bacteria use sulfide as an electron donor to reduce nitrate and nitrite. Each process alone has limitations: anammox bacteria are slow-growing and sensitive to sulfide toxicity, while conventional autotrophic denitrification with sulfide generates excessive sulfate and competes with anammox for nitrite. The new work shows that a carefully tuned mixotrophic design, in which the denitrifying partner also consumes a small amount of organic carbon, resolves these conflicts.

Lead author Yifan Zhang and colleagues integrated an anammox-enriched culture designated KAS1 with Thauera sp. AutoDN2, a sulfide-oxidizing denitrifying bacterium previously identified by the same group. Crucially, AutoDN2 is not a strict autotroph; it can use both sulfide and acetate, allowing the researchers to maintain a very low carbon-to-nitrogen ratio of just 0.8. Under these conditions, the coupled system removed 98.1 percent of ammonium and 99.4 percent of total nitrogen, performance figures that rival or exceed the best reported values for similar coupled systems while requiring far less external carbon than heterotrophic denitrification would demand.

Long-term operation in fed-batch mode revealed how the workload was divided between the two guilds. Anammox accounted for 71.2 to 77.1 percent of the total nitrogen removed, confirming that it remained the dominant pathway throughout extended operation. The mixotrophic S-SADN component provided a complementary route, polishing nitrate produced by anammox and handling sulfide oxidation. This division of labor proved stable over repeated feeding cycles, a critical finding because many attempted couplings of anammox with sulfur-driven denitrification have collapsed under sulfide inhibition or nitrite starvation of the anammox population.

One of the most striking outcomes concerns sulfate, the typical end product of sulfide oxidation. In conventional sulfide-based autotrophic denitrification systems, sulfide is fully oxidized to sulfate, which accumulates in the effluent, corrodes infrastructure, and raises salinity in receiving waters. In the mixotrophic system, sulfate yields were 63 to 68 percent lower than in purely autotrophic counterparts. The mechanism appears to be stoichiometric: when acetate is available, the denitrifiers require less sulfide per unit of nitrate reduced, and the reduced sulfide oxidation load shifts the sulfur balance away from complete oxidation. In effect, the organic co-substrate absorbs part of the electron-donation burden that sulfide would otherwise carry alone.

To verify that both processes were genuinely active rather than merely coexisting, the researchers tracked transcript levels of key functional genes. Stable expression of hzsA and hzsB, which encode hydrazine synthase subunits essential to the anammox metabolism, demonstrated that the anammox bacteria maintained their central catabolic machinery. Simultaneously, steady transcription of narG and napA, genes encoding nitrate reductases in the denitrification pathway, confirmed that AutoDN2 was actively respiring nitrogen oxides. The synchronized activity of these gene sets provides molecular evidence of metabolic synergy rather than competitive exclusion, and it suggests the partnership could be monitored in real time at full-scale plants through transcriptomic or genomic surveillance of activated sludge.

Equally notable is what the system did not emit. Across the experimental campaign, the researchers detected no nitrous oxide, a greenhouse gas roughly 265 times more potent than carbon dioxide over a century and a notorious byproduct of conventional nitrification-denitrification. The authors attribute this to the high nitrite affinity of anammox bacteria, which scavenge nitrite so efficiently that denitrifiers are rarely pushed toward the nitric oxide reductase steps that leak nitrous oxide. For utilities facing tightening greenhouse gas accounting rules, an ammonium treatment train that emits essentially no nitrous oxide represents a significant compliance advantage alongside its energy savings.

The practical implications extend to several wastewater streams where sulfide and ammonium co-occur with little biodegradable carbon. Anaerobic digester liquors, landfill leachate, tannery effluents, petrochemical wastewater, and sidestream returns from sludge treatment all fit this profile. In such streams, sulfide is usually treated as a nuisance to be stripped or precipitated before biological nitrogen removal, adding cost and complexity. The coupled platform instead treats sulfide as a free electron donor, converting a pollutant into a process resource. Because anammox does not require aeration and the denitrifying partner needs only a whisper of organic carbon, the system avoids the aeration and carbon-dosing costs that dominate conventional treatment economics.

The authors caution that translating a fed-batch laboratory model to continuous full-scale operation will require attention to process control, particularly maintaining the delicate nitrite balance that both guilds depend upon and managing sulfide loading to keep concentrations below anammox inhibition thresholds. Nevertheless, the demonstration that strategic mixotrophy can simultaneously mitigate sulfide toxicity, suppress sulfate overproduction, stabilize integrated carbon-nitrogen-sulfur cycling, and deliver near-complete nitrogen removal marks a substantial advance. As water utilities worldwide seek low-carbon pathways to meet stricter nitrogen discharge limits, this anammox-mixotrophic denitrification partnership offers a compelling blueprint: two microbial metabolisms, each compensating for the other’s weaknesses, working in concert to turn some of wastewater’s most stubborn pollutants into harmless nitrogen gas.

Subject of Research: Coupling anammox with mixotrophic sulfide-driven autotrophic denitrification for extensive biological nitrogen removal from sulfide-rich, carbon-limited wastewater

Article Title: Synergistic coupling of anammox and mixotrophic sulfide-driven denitrification for extensive nitrogen removal

Article References: Synergistic coupling of anammox and mixotrophic sulfide-driven denitrification for extensive nitrogen removal. (n.d.). https://doi.org/10.1007/s11783-026-2281-y

Image Credits: AI Generated

DOI: 10.1007/s11783-026-2281-y

Keywords: anammox, autotrophic denitrification, sulfide oxidation, wastewater treatment, nitrogen removal, Thauera, mixotrophic metabolism, nitrous oxide, sulfate reduction, carbon-nitrogen-sulfur cycling, environmental biotechnology, low-carbon sanitation