Evaluation of Simultaneous Nutrient and COD Removal with Polyhydroxybutyrate (PHB) Accumulation Using Mixed Microbial Consortia under Anoxic Condition and Their Bioinformatics Analysis

Simultaneous nitrate-N, phosphate and COD removal was evaluated from synthetic waste water using mixed microbial consortia in an anoxic environment under various initial carbon load (ICL) in a batch scale reactor system. Within 6 hours of incubation, enriched DNPAOs (Denitrifying Polyphosphate Accumulating Microorganisms) were able to remove maximum COD (87%) at 2g/L of ICL whereas maximum nitrate-N (97%) and phosphate (87%) removal along with PHB accumulation (49 mg/L) was achieved at 8 g/L of ICL. Exhaustion of nitrate-N, beyond 6 hours of incubation, had a detrimental effect on COD and phosphate removal rate. Fresh supply of nitrate-N to the reaction medium, beyond 6 hours, helped revive the removal rates of both COD and phosphate. Therefore, it was apparent that in spite of a high carbon load, maximum COD and nutrient removal can be maintained, with adequate nitrate-N availability. Denitrifying condition in the medium was evident from an increasing pH trend. PHB accumulation by the mixed culture was directly proportional to ICL; however the time taken for accumulation at higher ICL was more. Unlike conventional EBPR, PHB depletion did not support phosphate accumulation in this case. The unique aspect of all the batch studies were PHB accumulation was observed along with phosphate uptake and nitrate reduction under anoxic conditions. Bioinformatics analysis followed by pyrosequencing of the mixed culture DNA from the seed sludge revealed the dominance of denitrifying population, such as Corynebacterium, Rhodocyclus and Paraccocus (Alphaproteobacteria and Betaproteobacteria). Rarefaction curve indicated complete bacterial population and corresponding number of OTUs through sequence analysis. Chao1 and Shannon index (H’) was used to study the diversity of sampling. “UCI95” and “LCI95” indicated 95% confidence level of upper and lower values of Chao1 for each distance. Values of Chao1 index supported the results of rarefaction curve.