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

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 N2-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 nifH (marker gene used to identify biological nitrogen fixation) and 16S rRNA genes. The nifH 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 nifH 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.

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

The increasing use of chemical fertilizers causes the loss of natural biological nitrogen fixation in soils, water eutrophication and emits more than 300 Mton CO2 per year. It also limits the success of external bacterial inoculation in the soil. Nitrogen fixing bacteria can be inhibited by the presence of ammonia as its presence can inhibit biological nitrogen fixation. Two aerobic sludges from wastewater treatment plants (WWTP) were exposed to high ammonium salts concentrations (>450 mg L-1 and >2 dS m-1). Microbial analysis after treatment through 16S pyrosequencing showed the presence of Fluviicola sp. (17.70%), a genus of the Clostridiaceae family (11.17%), and Azospirillum sp. (10.42%), which were present at the beginning with lower abundance. Denaturing gradient gel electrophoresis (DGGE) analysis based on nifH genes did not show changes in the nitrogen-fixing population. Nitrogen-Fixing Bacteria (NFB) were identified and associated with other microorganisms involved in the nitrogen cycle, presumably for survival at extreme conditions. The potential use of aerobic sludges enriched with NFB is proposed as an alternative to chemical fertilizer as this bacteria could supplement nitrogen to the plant showing competitive results with chemical fertilization.

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

Nitrogen-fixing bacteria (NFB) can reduce nitrogen at ambient pressure and temperature. In this study, we treated effluent from a paper mill in sequencing batch reactors (SBRs) and monitored the abundance and activity of NFB with a view to producing a sludge that could work as a biofertilizer. Four reactors were inoculated with activated sludge enriched with NFB and fed with a high C/N waste (100:0.5) from a paper mill. Though the reactors were able to reduce the organic load of the wastewater by up to 89%, they did not have any nitrogen-fixing activity and showed a decrease in the putative number of NFB (quantified with qPCR). The most abundant species in the reactors treating high C/N paper mill wastewater was identified by Illumina MiSeq 16S rRNA gene amplicon sequencing as Methyloversatilis sp. (relative abundance of 4.4%). Nitrogen fixation was observed when the C/N ratio was increased by adding sucrose. We suspect that real-world biological nitrogen fixation (BNF) will only occur where there is a C/N ratio ≤100:0.07. Consequently, operators should actively avoid adding or allowing nitrogen in the waste streams if they wish to valorize their sludge and reduce running costs. PRACTITIONER POINTS: Efficient biological wastewater treatment of low nitrogen paper mill effluent was achieved without nutrient supplementation. The sludge was still capable of fixing nitrogen although this process was not observed in the wastewater treatment system. This high C/N wastewater treatment technology could be used with effluents from cassava flour, olive oil, wine and dairy industries.