Influence of ammonia and NaCl on nitrifying community and activity: Implications for formulating nitrifying culture augmentation.

Bioaugmentation of nitrifying cultures can accelerate nitrification during startup and transition periods of recirculating aquaculture system (RAS) operations. To formulate nitrifying cultures for RASs, impacts of ammonia and salinity (NaCl) on culturing nitrifying microorganisms were comprehensively investigated by including currently known groups of nitrifying microorganisms (ammonia oxidizing bacteria (AOB), ammonia-oxidizing archaea (AOA), comammox, Nitrospira, and Nitrobacter). By varying ammonia loading rate (ALRs of 1.6, 8, 20, 40, 60 and 150 mgN/L/d) of continuous-flow bioreactors fed with inorganic medium experimented for culture preparation, cultures containing distinct patterns of nitrifying communities were produced. Operating the reactors at the ALRs of ≤40 mgN/L/d, resulting in the effluent total ammonia nitrogen (TAN) and nitrite concentrations of ≤2.64 and ≤0.53 mgN/L, respectively, delivered the consortia consisting of a broad spectrum of substrate affinity nitrifying microorganisms. At the lower ranges of these ALRs (≤8 mgN/L/d), the most desirable consortia comprising comparable numbers of AOB, AOA, and comammox could be produced (the effluent TAN concentrations of ≤0.20 mgN/L), which would be resilient for applying in various RAS types. Enriching the cultures at the ALRs of ≥60 mgN/L/d allowed only the nitrifying microorganisms with low substrate affinity to dominate, incongruent with the consortia found in actual RASs. AOB were adaptable at all salinity studied (2, 15, and 30 g/L), while AOA and comammox were sensitive to elevated salinity (15 and 30 g/L, respectively). The ammonia removal rate of a culture prepared at 2 g/L salinity decreased largely when applied at 15 and 30 g/L. In contrast, those prepared at 15 and 30 g/L were more robust to different salinity. Separately preparing the cultures at different salinity for uses in freshwater-low salinity and brackish-marine RASs is recommended. The findings of this work enhance our understanding on how to formulate nitrifying culture augmentation for used in different RAS types.

Substrate loading rates conducive to nitritation in entrapped cell reactors: performance and microbial community structure.

This study aimed to elucidate the boundaries of ammonia and organic loading rates that allow for nitritation in continuous flow phosphorylated-polyvinyl alcohol entrapped cell reactors and to clarify the community structure of microorganisms involving nitrogen transformation in the gel bead matrices. At operating bulk dissolved oxygen concentration of 2 mg/L, nitritation was accomplished when the total ammonia nitrogen (TAN) loading rate was ≥ 0.3 kgN/m3/d. At TAN loading rates of ≤ 0.2 kgN/m3 /d, complete oxidation of ammonia to nitrate took place. Nitritation performance dropped with increased chemical oxygen demand (COD) loading rates indicating limitation of nitritation reactor operation at some COD loading conditions. 16S rRNA gene amplicon sequencing revealed that the uncultured Cytophagaceae bacterium, Arenimonas, Truepera, Nitrosomonas, Comamonas, unclassified Soil Crenarchaeotic Group, and uncultured Chitinophagaceae bacterium were highly abundant taxa in the reactors' gel bead matrices. qPCR with specific primers targeting amoA genes demonstrated the coexistence of ammonia-oxidizing bacteria (AOB), ammonia-oxidizing archaea, and Comammox in the gel bead matrices. AOB was likely the main functioning ammonia-oxidizing microorganisms due to the amoA gene being of highest abundance in most of the studied conditions. Nitrite-oxidizing microorganisms presented in less relative abundance than ammonia-oxidizing microorganisms, with Nitrobacter rather than Nitrospira dominating in the group. Results obtained from this study are expected to further the application of nitritation entrapped cell reactors to real wastewater treatment processes.

Incorporation of 13C-HCO3- by ammonia-oxidizing archaea and bacteria during ammonia oxidation of sludge from a municipal wastewater treatment plant.

Ammonia-oxidizing archaea (AOA) have recently been proposed as potential players for ammonia removal in wastewater treatment plants (WWTPs). However, there is little evidence directly showing the contribution of AOA to ammonia oxidation in these engineered systems. In this study, DNA-stable isotope probing (DNA-SIP) with labeled 13C-HCO3- was introduced to sludge from a municipal WWTP. Quantitative PCR demonstrated that AOA amoA genes outnumbered AOB amoA genes in this WWTP sludge. AOA amoA gene sequence analysis revealed that AOA present in this WWTP were specific to one subcluster within the group 1.1b Thaumarchaeota. When ammonia was supplied to DNA-SIP incubation, the DNA-SIP profiles demonstrated the incorporation of the 13C into AOA and AOB. However, the 13C was not found to be assimilated into both microorganisms in the incubation without ammonia. Specific primers were designed to target amoA genes of AOA belonging to the subcluster found in this WWTP. Applying the primers to DNA-SIP experiment revealed that AOA of this subcluter most likely utilized inorganic carbon during ammonia oxidation under the studied conditions.

Contribution of ammonia-oxidizing archaea and ammonia-oxidizing bacteria to ammonia oxidation in two nitrifying reactors.

In this study, two laboratory nitrifying reactors (NRI and NRII), which were seeded by sludge from different sources and operated under different operating conditions, were found to possess distinct dominant ammonia-oxidizing microorganisms. Ammonia-oxidizing archaeal (AOA) amoA genes outnumbered ammonia-oxidizing bacterial (AOB) amoA genes in reactor NRI, while only AOB amoA genes were detectable in reactor NRII. The AOA amoA gene sequences retrieved from NRI were characterized within the Nitrososphaera sister cluster of the group 1.1b Thaumarchaeota. Two inhibitors for ammonia oxidation, allylthiourea (ATU) and 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO), were applied individually and as a mixture to observe the ammonia-oxidizing activity of both microorganisms in the reactors' sludge. The results indicated that AOA and AOB jointly oxidized ammonia in NRI, while AOB played the main role in ammonia oxidation in NRII. DNA-stable isotope probing with labeled 13C-HCO3- was performed on NRI sludge. Incorporation of 13C into AOA and AOB implied that both microorganisms may perform autotrophy during ammonia oxidation. Taken together, the results from this study provide direct evidence demonstrating the contribution of AOA and AOB to ammonia oxidation in the nitrifying reactors.

Differences in nitrite-oxidizing communities and kinetics in a brackish environment after enrichment at low and high nitrite concentrations.

Nitrite accumulation in shrimp ponds can pose serious adverse effects to shrimp production and the environment. This study aims to develop an effective process for the enrichment of ready-to-use nitrite-oxidizing bacteria (NOB) inocula that would be appropriate for nitrite removal in brackish shrimp ponds. To achieve this objective, the effects of nitrite concentrations on NOB communities and nitrite oxidation kinetics in a brackish environment were investigated. Moving-bed biofilm sequencing batch reactors and continuous moving-bed biofilm reactors were used for the enrichment of NOB at various nitrite concentrations, using sediment from brackish shrimp ponds as seed inoculum. The results from NOB population analysis with quantitative polymerase chain reaction (qPCR) show that only Nitrospira were detected in the sediment from the shrimp ponds. After the enrichment, both Nitrospira and Nitrobacter coexisted in the reactors controlling effluent nitrite at 0.1 and 0.5 mg-NO2(-)-N/L. On the other hand, in the reactors controlling effluent nitrite at 3, 20, and 100 mg-NO2(-)-N/L, Nitrobacter outcompeted Nitrospira in many orders of magnitude. The half saturation coefficients (Ks) for nitrite oxidation of the enrichments at low nitrite concentrations (0.1 and 0.5 mg-NO2(-)-N/L) were in the range of 0.71-0.98 mg-NO2(-)-N/L. In contrast, the K(s) values of NOB enriched at high nitrite concentrations (3, 20, and 100 mg-NO2(-)-N/L) were much higher (8.36-12.20 mg-NO2(-)-N/L). The results suggest that the selection of nitrite concentrations for the enrichment of NOB inocula can significantly influence NOB populations and kinetics, which could affect the effectiveness of their applications in brackish shrimp ponds.