Enrichment of Nitrogen-Fixing Bacteria in a Nitrogen-Deficient Wastewater Treatment System

Abstract

Anthropogenic nitrogen fixation is essential to sustain a global population of 7.7 billion. However, there has been a long-standing desire to find cheaper and more environmentally friendly alternatives to the Haber−Bosch process. In this study, we developed a new strategy of nitrogen fixation by enriching free-living N 2 -fixing bacteria (NFB) in reactors fed with low nitrogen wastewater, analogous to those usually found in certain industrial effluents such as paper mills. Our reactors fixed appreciable quantities of nitrogen with a rate of 11.8 mg N L −1 day −1 . This rate is comparable to recent "breakthrough" nitrogen-fixing technologies and far higher than observed in low C/N reactors (fed with organic matter and nitrogen). NFB were quantified using quantitative polymerase chain reaction (qPCR) of the nif H (marker gene used to identify biological nitrogen fixation) and 16S rRNA genes. The nif H gene was enriched by a factor of 10 in the nitrogen-fixing reactors (compared to controls) attaining 13% of the bacterial population (1:4.2 copies of nif H to 16S rRNA). The Illumina MiSeq 16S rRNA gene amplicon sequencing of reactors showed that the microbial community was dominated (19%) by Clostridium pasteurianum. We envisage that nitrogen-enriched biomass could potentially be used as a biofertilizer and that the treated wastewater could be released to the environment with very little post-treatment.

Valorization of pulp and paper industry wastewater using sludge enriched with nitrogen‐fixing bacteria

“…Negative results of the acetylene reduction test on day 62 were further confirmed with the 15 N 2 method following Bellenger et al, (2014). The amount of N 2 fixed during the 48 h of operation of the batch reactors was estimated in 1.24 and was calculated using the acetylene reduction rates obtained in this study and the R ratio (acetylene reduction rate to 15 N 2 fixation rate) obtained previously in Ospina-Betancourth et al, (2020).…”

“…The acetylene reduction assay as specified elsewhere (Ospina-Betancourth et al, 2020) was used to indirectly measure nitrogen-fixing activity for 48 h in batch reactors during day 62 and 114 of operation. Briefly, duplicated crimp-sealed 160-ml serum bottles were inoculated with 30 ml of mixed liquor from each SBR reactor collected at the beginning of the HRT.…”

“…The physicochemical parameters of the paper mill effluent (obtained after the second month of operation) and the synthetic wastewater, used in Ospina-Betancourth et al, (2020) to previously enrich the sludge with NFB, were compared (Table 1). The COD/N/P ratio of the paper mill wastewater ranged between 100:0.2:0.07 and 100:0.8:0.5.…”

“…In previous work, we developed a new strategy for nitrogen fixation, using sequencing batch reactors (SBR) fed with synthetic high C/N wastewater, to enrich an activated sludge with NFB (Ospina-Betancourth et al, 2020). These reactors contained sludge enriched by 13% with NFB and were able to fix nitrogen at an average rate of up to 11.8 mg of N L −1 day −1 .…”

High Resistance of a Sludge Enriched with Nitrogen-Fixing Bacteria to Ammonium Salts and Its Potential as a Biofertilizer

“…Bioengineering 2021, 8, 55 2 of 13 NFB have been reported in different WWTP [16,17] and isolated from municipal WWTP [18]. Municipal wastewater is characterized by high concentrations of ammonium (>50 mg L −1 NH 4 + ) and salts (>2 dS m −1 ) [19].…”