Spectroscopic characterization of extracellular polymeric substances from a mixed culture dominated by ammonia-oxidizing bacteria

Extracellular polymeric substances (EPS) of aerobic (AerAOB) and anaerobic ammonium-oxidizing bacteria (AnAOB) are expected to have a significant impact on the performance of autotrophic nitrogen removal in engineered systems. However, there are a few investigations of the EPS of AerAOB and AnAOB, and the results are contradictory. In this study, photometric measurements indicated that the EPS of AerAOB- (31.74 ± 1.48 mg/g-VSS, volatile suspended solids) and AnAOB-enriched cultures (30.12 ± 1.52 mg/g-VSS) contained more polysaccharides than did conventional activated sludge from a municipal wastewater treatment facility (10.76 ± 0.83 mg/g-VSS). In addition, the EPS of the AnAOB-enriched culture was dominated by proteins, leading to a considerably higher protein/polysaccharide ratio (2.64 ± 0.12) than those of the AerAOB-enriched culture (0.56 ± 0.03) and conventional activated sludge (1.96 ± 0.09). Characterization using Fourier transform infrared spectroscopy (FTIR) revealed the dominance of amide bands and/or polysaccharide-associated bands in the EPS of AnAOB and AerAOB. These results corroborate the data from the photometric measurements. In addition, the EPS of AnAOB (23.1% ± 1.2%) and AerAOB (21.9% ± 1.1%) had a higher portion of α-helices, which is the key protein secondary structure that determines flocculation or cell aggregation, in the amide I band than that of activated sludge (16.7% ± 0.8%). X-ray photoelectron spectroscopy (XPS) characterization also revealed significantly different functionalities among the EPS of the three mixed cultures; e.g., O-(C,H), which indicates the presence of polysaccharides, was richer in the EPS of AerAOB, whereas protonated amines, which are commonly found in amino acids and amino sugars, accounted for a large portion of the EPS of AnAOB. The results of this study can potentially expand our knowledge of the microbial aggregates responsible for autotrophic nitrogen removal.