Characterization of magnetite assisted anammox granules based on in-depth analysis of extracellular polymeric substance (EPS).

Magnetite can be considered as an iron-rich carrier particles that can be ionized into Fe2+ and Fe3+ which improves the activity and aggregation of anammox bacteria. Three samples from this carrier assisted granulation reactor with size groups including Flocs, FL (0-300 µm), Small Granules, SG (300-500 µm) and Large Granules, LG (500-1000 µm) were used in this study. It was observed that as the granule size increased, the iron-rich carrier content increased, and their active crystals improved the microbial cell density. Specific anammox activity (SAA) was 34.63 ± 5.02, 55.29 ± 5.14, and 63.81 ± 7.50 mg-N/g-VSS/d for FL, SG and LG, respectively. In addition, in heme c content of LG was 31.5 % higher than SG and 62.9 % higher than FL. An in-depth study into the extracellular polymeric substances (EPS) showed that the secretion intensity of essential proteins followed the order of FL < SG < LG in loosely bound EPS and FL > SG > LG in tightly bound EPS. Functional group analysis confirmed that the hydrophobic CN and NH stretching vibration band had almost 3.5 times higher transmittance intensity in LG than the other sizes and the corresponding ratio of α-helix/(ß sheet + random coil) in secondary derivative proteins analysis showed tightness in the protein structures of FL. The relative abundance of Brocadia Sinica increased from 0 % in FL to a high of 20.46 % in LG. This study aims to communicate the essence of in-depth EPS analysis beyond the usual EPS yield and major contents of proteins (PN) and polysaccharides (PS) analysis.

The role of magnetite (Fe3O4) particles for enhancing the performance and granulation of anammox.

In this study, two lab-scale sequencing batch reactors each with an effective volume of 2.3 L were operated as C-AMX (no carrier addition) and M-AMX (magnetite carrier added) for 147 days with synthetic wastewater at an NLR range of 0.19-0.47 kgN/m3/d. The long-term effect of magnetite on the granulation and performance of anammox bacteria in terms of nitrogen removal and other essential parameters were confirmed. In phase I (1-24 days), M-AMX took approximately 12 days to obtain a nitrogen removal rate (NRR) above 80 % of the initial input nitrogen. Although free nitrous acid inhibited the reactor at a high concentration at the onset of phase III, the NRR of M-AMX recovered about 3.7 times faster than that of C-AMX. In addition, it was confirmed that the M-AMX granules had a dense and compact structure compared to C-AMX, and the presence of the carrier promoted the development of these resilient granules. While the measured microbial stress gradually increased in C-AMX reactor, a vice versa was observed in the M-AMX reactor as granulation proceeded. Compared to other alternative iron-based carrier particles, the stable crystal structure of magnetite as a carrier created a mechanism where filamentous bacteria groups were repelled from the granulation hence the microbial stress in the M-AMX in the final phase was 61.54 % lower than that in the C-AMX. The iron rich environment created by the magnetite addition led to Ignavibacteria, (a Feammox bacteria) increasing significantly in the M-AMX bioreactor.

Distinctive differences in the granulation of saline and non-saline enriched anaerobic ammonia oxidizing (AMX) bacteria.

The growing interest in the anaerobic ammonium oxidizing (AMX) process in treating high nitrogen containing wastewaters and a comprehensive study into the granulation mechanism of these bacteria under diverse environmental conditions over the years have been unequal. To this effect, the distinctive differences in saline adapted AMX (S_AMX) and non-saline adapted AMX (NS_AMX) granules are presented in this study. It was observed that substrate utilisation profiles, granule formation mechanism, and pace towards granulation differed marginally for the two adaptation conditions. The different microbial dominant aggregation types aided in splitting the 471 days operated lab-scale SBRs into three distinct phases. In both reactors, phase III (granules dominant phase) showed the highest average nitrogen removal efficiency of 87.9% ± 4.8% and 85.6% ± 3.6% for the S_AMX and NS_AMX processes, respectively. The extracellular polymeric substances (EPS) quantity and major composition determined its role either as a binding agent in granulation or a survival mechanism in saline adaptation. It was also observed that granules of the S_AMX reactor were mostly loosely and less condensed aggregates of smaller sub-units and flocs while those of the NS_AMX reactor were compact agglomerates. The ionic gradient in saline enrichment led to an increased activity of the Na+/K+ - ATPase, hence enriched granules produced higher cellular adenosine triphosphate molecules which finally improved the granules active biomass ratio by 32.96%. Microbial community showed that about three to four major known AMX species made up the granules consortia in both reactors. Proteins and expression of functional genes differed for these different species.

From freshwater anammox bacteria (FAB) to marine anammox bacteria (MAB): A stepwise salinity acclimation process.

An investigation into the effect of stepwise saline introduction (3-20 g·L-1 NaCl) on the anaerobic ammonium oxidation (anammox) process in a lab-scale sequencing batch reactor was carried out for 252 days by evaluating the changes in influent and effluent nitrogen concentrations, conductivity, microbial extracellular polymeric substances' (EPS) ionic content, as well as stresses due to salinity, via microbial ATP analysis. It was observed that, effluent nitrogen concentrations remained stable at low saline levels of 3 g·L-1 to 10 g·L-1. Nonetheless, midway through 10 g·L-1 and the preliminary phase of 15 g·L-1 salinity presented a very unstable, highly fluctuating as well as deteriorating effluent nitrogen concentrations. A more satisfactory nitrogen removal efficiency of 83.7 ± 5.9% was obtained at higher saline concentrations implying that, the adaptation mechanism to tolerate increasing salinity was taking place. Saline induced stress, which measures the variation in viable anammox bacteria, was correlative to the formation of EPS and changes in its cationic contents along the increasing salinity. Although the specific anammox activity (SAA) dropped by approximately 15% from the beginning of the process to the midpoint, the drop in SAA after the midpoint was not as drastic as the initial phase. A change in microbial aggregation and dominance proved the existence of new saline-dependent species that can withstand high saline stresses. Recovery from abrupt high saline shocks in batch experiment was seen to be almost impossible.

Saline conditions effect on the performance and stress index of anaerobic ammonium oxidizing (anammox) bacteria.

In this study, a lab-scale sequencing batch reactor dominated by freshwater anammox bacteria (FAB) was used to study the performance and stress index of the anammox bacteria at various saline conditions. The reactor with an effective volume of 1.8 L was operated for about 160 days. The nitrogen-loading rate was maintained at 0.364 kg-N m-3d-1 throughout the operational period. At the start-up phase, the seed biomass acclimation to the lab bioreactor showed an inconsistent performance. However, a stable performance was observed after day 38. The average substrate removal efficiency was 92% during most of the operational period. Anammox stress index; a ratio of dissolved Adenosine Triphosphate (dATPamx) to total Adenosine Triphosphate (tATPamx) showed an irrefutable correlation between NaCl concentration, anammox stress and microbial community. A drop in the biomass cellular ATP at 5 g L-1 salinity led to a significant decrease in the Specific Anammox activity. Candidatus Brocadia was identified as the main anammox species and its relative abundance reduced along the stepwise salinity increment.