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Cyanobacterium Microcoleus anatoxicus, isolated from a coastal stream in northern California, produces both anatoxin-a (ATX) and dihydroanatoxin- a (dhATX), responsible for dog deaths, but its environmental preferences are unknown. We tested the effect of environmentally relevant stressors, e.g., salinity enrichment and nitrogen (N) depletion, on mat formation and toxicity of M. anatoxicus during the stationary growth phase in culture. Microcoleus anatoxicus showed broad salinity tolerance and the potential to enter estuaries and produce toxins in mesohaline conditions. Maximum growth was observed in oligohaline waters with salinity of 4.6 ppt. Moderate salinity stress (up to 7.8 ppt) did not affect dhATX production significantly. In contrast, higher salinity above 9.3 ppt had a detrimental effect on cell growth and significantly suppressed dhATX production. Formation of a common polysaccharide sheath covering multiple filaments was characteristic with increased salinity and may provide protection against osmotic stress. Microcoleus anatoxicus grown for 40 days in N-depleted medium formed mats with significantly elevated dhATX and increased ATX concentrations. Phycobilisome degradation was a possible acclimation response to N-limitation, as indicated by distinctly keritomized and pale cells in these cultures. In both experiments, most of the anatoxins were extracellular,probably due to toxin leaking during the stationary growth phase.more » « less
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Soklida, Hong; Mari-KH, Winkler; Zhiwu, Wang; Goel, Ramesh (Ed.)This research studied integrated fixed film activated sludge (IFAS) technology to simultaneously remove N and P in real municipal wastewater by combining anammox biofilms with flocculent activated sludge for enhanced biological P removal (EBPR). The study was conducted in a sequencing batch reactor (SBR) operated as a conventional A2O (anaerobic-anoxic-oxic) process with an 8.8 h hydraulic retention time. After achieving steady-state operation, the reactor showed robust performance, with average removal efficiencies of 91.3±4.1% for total inorganic nitrogen (TIN) and 98.4±2.4% for phosphorus (P). Denitrifying polyphosphate accumulating organisms (DPAOs) were responsible for 15.9% of P uptake during the anoxic phase, while biofilms showed anammox activity in the aerobic step. The IFAS configuration with a low solid retention time (SRT) of 5 days prevented the washout of biofilm anammox bacteria and allowed selective washout of unwanted organisms. The results demonstrated the successful coexistence of anammox bacteria with other bacteria for efficient nutrient removal in real wastewater conditions.more » « less
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The mainstream application of anaerobic ammonium oxidation (anammox) for sustainable N removal remains a challenge. Similarly, with recent additional stringent regulations for P discharges, it is imperative to integrate N with P removal. This research studied integrated fixed film activated sludge (IFAS) technology to simultaneously remove N and P in real municipal wastewater by combining biofilm anammox with flocculent activated sludge for enhanced biological P removal (EBPR). This technology was assessed in a sequencing batch reactor (SBR) operated as a conventional A2O (anaerobic-anoxic-oxic) process with a hydraulic retention time of 8.8 h. After a steady state operation was reached, robust reactor performance was obtained with average TIN and P removal efficiencies of 91.3 ± 4.1% and 98.4 ± 2.4%, respectively. The average TIN removal rate recorded over the last 100 d of reactor operation was 118 mg/L⋅d, which is a reasonable number for mainstream applications. The activity of denitrifying polyphosphate accumulating organisms (DPAOs) accounted for nearly 15.9% of P-uptake during the anoxic phase. DPAOs and canonical denitrifiers removed approximately 5.9 mg TIN/L in the anoxic phase. Batch activity assays, which showed that nearly 44.5% of TIN were removed by the biofilms during the aerobic phase. The functional gene expression data also confirmed anammox activities. The IFAS configuration of the SBR allowed operation at a low solid retention time (SRT) of 5-d without washing out biofilm ammoniumoxidizing and anammox bacteria. The low SRT, combined with low dissolved oxygen and intermittent aeration, provided a selective pressure to washout nitrite-oxidizing bacteria and glycogen-accumulating organisms, as relative abundances.more » « less
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Anaerobic ammonium oxidation (anammox) has evolved as a carbon and energy-efficient nitrogen management bioprocess. However, factors such as inhibitory chemicals still challenge the easy operation of this powerful bioprocess. This research systematically evaluated the inhibition kinetics of sulfide, nitrite, and recalcitrant carbon under a genomic framework. The inhibition at the substrate and genetic levels of sulfide, nitrite and recalcitrant carbon on anammox activity was studied using batch tests. Nitrite inhibition of anammox followed substrate inhibition and was best described by the Aiba model with an inhibition coefficient KI,NO2 of 324.04 mg N/L. Hydrazine synthase (hzsB) gene (anammox biomarker) expression was increased over time when incubated with nitrite up to 400 mg N/L. However, despite having the highest specific nitrite removal (SNR), the expression of hzsB at 100 and 200 mg N/L of nitrite was more muted than in most other samples with lower SNRs. Sulfide severely inhibited anammox activities. The inhibition was fitted with a Monod-based model with a KI,S2 of 4.39 mg S/L. At a sulfide concentration of 5 mg/L, the hzsB expression decreased throughout the experiment from its original value at he beginning. Recalcitrant carbon of filtrate from thermal hydrolysis process pretreated anaerobic digester had a minimal effect on maximum specific anammox activity (MSAA), and thus the value of the inhibition coefficient could not be calculated. At the same time, its hzsB expression profile was similar to that in the control. Resiliency and recovery tests indicated that the inhibition of nitrite (up to 400 mg N/L) and recalcitrant carbon (in 100% filtrate) were reversible. About 32% of MSAA was recovered after repeated exposures to sulfide at 2.5 mg/L, while at 5 mg/L, the inhibition was irreversible. Findings from this study will be helpful for the successful design and implementation of anammox in full-scale applications.more » « less