Valorisation of nitrogen-deficient wastewater treatment systems using sludge enriched with nitrogen-fixing bacteria

Abstract

We live in a world highly dependent on the use of nitrogen fertilisers produced with the Haber-Bosch process (HBP) but their production and deployment are unsustainable. Finding cheaper and greener alternative technologies that can sustain global agricultural production is therefore a research priority. Biological nitrogen fixation (BNF) is a process carried out by some bacteria in which molecular nitrogen (N2) is converted to ammonia (NH3) and is an example of sustainable nitrogen fixation. The energy source for biological nitrogen fixation can, in principle, be obtained from industrial or domestic wastewater. In this study a new strategy of nitrogen fixation was developed using bench scale wastewater treatment plants, treating waste with a high carbon-to-nitrogen ratio, to enrich wastewater sludge with N2-fixing bacteria (NFB). This residual sludge enriched with NFB could contribute to the valorisation of low nitrogen wastewater treatment systems as it can be potentially used as a biofertiliser. Initially, NFB found in the sludge from industrial and domestic wastewater treatment plants and from an anaerobic digester were used as an inoculum to build replicated nitrogen-fixing sequencing batch reactors (SBR). After 85 days of operation, these reactors were able to fix nitrogen at an average rate of up to 11.8 mg of N L-1 day-1 and treat the wastewater with a COD removal efficiency of 73%. Additionally, the reactors contained sludge that was enriched with NFB, attaining 13% of the total bacterial population (1:4.2 copies of nifH to 16S rRNA quantified with qPCR). Though the relationship between nitrogen content and the efficiency of wastewater treatment has been investigated in low nitrogen wastewater, there are no estimates of the inhibition of nitrogen fixation under different ammonia concentrations. Therefore, the nitrogen fixation rates of this sludge enriched with NFB were measured under the effect ammonium in concentrations of up to 78 mg of N L−1. The sludge fed with high C:N wastewater was resistant to inhibition by ammonia with a half maximal inhibitory concentration (IC50) of 54 mg of N L−1. This result suggests that it is reasonable to expect BNF in high C:N wastes that are contaminated with ammonia and that it seems at least plausible that the NFB in the sludge could work as a biofertiliser by fixing nitrogen in soils with modest quantities of nitrogen-based chemical fertilisers. Finally, the feasibility of using real effluent from pulp and paper mills as a source of carbon to culture NFB in sequencing batch reactors (SBR) was evaluated. Effluent from this industry is produced in large volumes and contains high organic loads with iii little to no nitrogen. 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. The reactors were able to reduce the organic load of the wastewater by up to 89% after 114 days of operation but did not have any nitrogen-fixing activity and showed a decrease in the putative number of NFB (quantified with qPCR). Nitrogen fixation was only observed when sucrose in concentration of 3 g L-1 was added as a further supplement of carbon source. It is likely 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 valorise their sludge and reduce running costs. This biotechnology demonstrates the scope and magnitude of nitrogen fixation in wastewaters and provides fundamental insight into a novel green method that can treat pulp and paper mill wastewater using less resources (compared to traditional high C/N wastewater treatment systems) and existing facilities. In addition, this technology has the potential to valorise the sludge from the treatment system by producing NFB which could be used as a sustainable biofertiliser. This investigation sets the basis for future work which should be aimed to demonstrate and quantify the benefits of the nitrogenfixing sludge as a biofertiliser.