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

Abstract

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.

Keywords: bio-enrichment; biofertilizer; domestic wastewater treatment plants; nitrogen fixing bacteria; sequential batch reactor.

Figure 1

Total nitrogen found in the bioreactors inoculated from Activated Sludge (ASI), Biodisc (BI) and Facultative Pond (FPI). “°” Boxplot outliers.

Figure 2

Biomass (gray) and effluent (black) N-NH₄⁺ content of (BI): Biodisc, (FPI): Facultative Pond and, (ASI): Activated Sludge.

Figure 3

Total N-NH₄ (dot plot) and Biomass (bar plot) content found in the bioreactors (a) ASI and (b) FPI. ASI-control: Activated Sludge Control. FPI-control: Facultative Pond Control.

Figure 4

Pearson–Phi correlation coefficient distance cladograms generated from the DGGE fingerprints profile of nifH gen of (1) FPI Phase 2-Initial time (T.0); (2) FPI Phase 2-end (T.35); (3) FPI Control-end (T.35); (4) ASI Phase 2-Initial time (T.0); (5) ASI Phase 2-end (T.35); (6) ASI Control-end (T.35). Detected by silver-nitrate staining.

Figure 5

Rarefaction plots of ASI on Phase 1 (gray) and Phase 2 (black).

Figure 6

Genus relative abundance in the bioreactor ASI at the end of Phase 1 and 2 (most representative < 1%).