Production of biodegradable plastics in the form of polyhydroxyalkanoates (PHA) especially from renewable substrates is gaining interest. The present work mainly aims to investigate the influence of substrate load and nutrient concentration (nitrogen and phosphorous) on PHA production using wastewater as substrate and mixed culture as biocatalyst. PHA accumulation was high at higher substrate load [OLR3, 40.3% of dry cell weight (DCW)], low nitrogen (N-1, 45.1% DCW) and low phosphorous (P-1, 54.2% DCW) conditions. With optimized nutrient conditions production efficiency increased by 14%. Fractional composition of PHA showed co-polymer [poly(beta-OH) butyrate-co-poly(beta-OH) valerate, P3(HB-co-HV)] contains PUB (88%) in more concentration compared to PHV (8%). Dehydrogenase and phosphatase enzymatic activities were monitored during process operation. Good substrate degradation (as COD) of 75% was registered during PHA production. The phylogenetic profile of 16S rRNA sequencing showed the dominance of Firmicutes (71.4%) and Proteobacteria (28.6%), which are known to involve in PHA accumulation and waste treatment. (C) 2012 Elsevier Ltd. All rights reserved.