Increased salinity triggers significant changes in the functional proteins of ANAMMOX bacteria within a biofilm community.

Anaerobic ammonium oxidation (ANAMMOX) processes can potentially be influenced by salinity related to variable salinity in water environment. Here, we used 16S rRNA sequencing analysis combining with iTRAQ-based quantitative proteomic approach to reveal the response of microbial community and functional proteins to salinity, which was increased from 0 to 20 g L-1 with a step of 5 g L-1 (designed as S5, S10, S15 and S20) compared to control reactor (without salinity stress desined as S0). The 16S rRNA sequencing analysis showed that a high salinity (20 g L-1, S20) decreased the abundance of genus Candidatus Jettenia but increased that of Candidatus Kuenenia. A total of 1609 differentially expressed proteins were acquired in the three comparison groups (S5:S0, S20:S0 and S20:S5). Of these, 39 proteins co-occurred in the three salt-exposed samples. Hydrazine dehydrogenase (HDH; Q1PW30) and nitrate reductase (Q1PZD8) were up-regulated more than 3-folds in the exposure of 20 g-NaCl/L. The functional enrichment analysis further showed that some proteins responsible for ion binding, catalysis and oxidation-reduction reaction were up-regulated, which explained the physiological resilience of ANAMMOX bacteria under salinity stress. Additionally, ANAMMOX bacteria responded to salinity by modulating the electron transport systems, indicating that the cells retained a high potential for proton pumping, as well as the ATP production. Furthermore, the over-expression of HDH which associated with ANAMMOX metabolism, was potentially related to the increased abundance of halophilic Candidatus Kuenenia. These findings provide a comprehensive baseline for understanding the roles of salinity stresses in shaping the functional proteins of ANAMMOX bacteria.

Characteristics of different molecular weight EPS fractions from mixed culture dominated by AnAOB and their role in binding metal ions.

Ultraviolet-visible (UV-Vis) absorbance spectra were adopted to quantify the binding of metal ions (e.g., Fe(III), Cu(II), Pb(II), and Cd(II)) on three MW fractions (> 100, 10~100, and < 10 k Da) of extracellular polymeric substances (EPS) extracted from mixed cultures dominated by anaerobic ammonium-oxidizing bacteria (AnAOB). The results showed that the AnAOB EPS with different MW size ranges all had strongest binding capability of Fe(III), and the lowest binding capability of Cd(II). The complexation ability of metal ions for the EPS of AnAOB with molecular weight < 10 kDa was stronger than EPS with >100 and 10~100 kDa, very likely because of the contribution of the tyrosine-, tryptophan-, and aromatic protein-like components. It was obvious that the different size fractions of EPS affect the metal binding ability. Essentially, the content of proteins, polysaccharides, TOC, and UVA254 distributed within various MW fractions of EPS from AnAOB were different, as well as the different fluorescent components and total functional groups.

Differential ultraviolet-visible absorbance spectra for characterizing metal ions binding onto extracellular polymeric substances in different mixed microbial cultures.

Ultraviolet-visible (UV-vis) absorbance spectra was adopted to quantify the binding of major metal ions (e.g., Na(I), Ca((II)), Fe(III), Cu(II), and Pb(II)) on extracellular polymeric substances (EPSs) extracted from different mixed cultures. The results showed that the differential absorbance spectra (DAS) provided discernible features for revealing the changes in optical properties of EPSs induced by metals, i.e., the intensity of DAS increased largely with incrementally increased metal concentrations (Fe(III), Cu(II), and Pb(II)). It can be assumed attributable to the changes in the conformations and inter-chromophores of the EPS biomolecules. In addition, the changes in spectral parameters of DSlope325-375 (spectral slope in the range of wavelengths 325-375 nm) and DA300 (differential absorbance at 300 nm) were found to be closely related to the amounts of metals bound onto all extracted EPSs, particularly for Fe(III) and Cu(II). The decreased SR (the ratio of slope275-295 to slope350-400) of the EPS solutions after dosage of metals suggested increased molecular weight or size of the EPS biomolecules. Deconvolution of the DAS yielded six Gaussian bands, which were present in all of the EPS samples with various metals. Moreover, the relative contributions of different Gaussian bands in the DAS were determined by the nature of EPS-metal ions interactions good correlated with the covalent-bonding index. This study concluded that DAS and selected spectral parameters (DA300, DSlope325-375 and SR) can be used to successfully characterize the binding of metals onto EPS at environmentally relevant concentrations.

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.