Two-stage process for ammonium and nitrate removal and polyhydroxyalkanoate production by rhodobacter sphaeroides ADZ101

Abstract

The application of photosynthetic bacteria in bioremediation is an eco-friendly technique that remains untapped. For the aquaculture industries that are based on high density protein feeding, elevated levels of ammonium and nitrate had been reported. High concentrations of ammonium can cause coma, convulsion and death to aquatic organisms besides eutrophication causing oxygen depletion in water bodies, increasing its harmful effect to aquatic organisms. In view of this, fundamental aspects of nitrogen removal were studied using synthetic medium. The ability of Rhodobacter sp. ADZ101, a denitrifying phototrophic bacterium which was successfully isolated and identified using 16S rRNA analysis was investigated for the removal of ammonium and nitrate. Different initial concentrations of ammonium and nitrate were used to determine the nitrogen removal and its reaction kinetics using the Michaelis-Menten rate expression. Results showed that 71% of nitrate was removed at initial concentration of 85 mg/L and 62% of ammonium at initial concentration of 52 mg/L under photoheterotrophic and anoxic dark conditions respectively. The kinetic coefficients of nitrate were determined as: k = 4.5×10-2 g NO3- g L-1 DCW d-1, Km = 0.55 g L-1, and that of ammonium as: k = 4.5×10-3g NH4-N g L-1 DCW d-1, Km = 0.52 g L-1. The yield coefficient of nitrate (YN) was 0.15 mg DCW mg L-1NO3- and that of ammonium was 0.3 mg DCW mg L-1 NH4-N. Analysis and amplification of the possible genes that are involved in denitrification revealed the presence of both nitrate reductase (napA) and nitrite reductase (nirK) genes. Rhodobacter sp. ADZ101 was also found to produce PHA. Using different carbon and nitrogen sources, acetate and ammonia chloride showed the highest accumulation of PHA of 46% (DCW) with C:N ratio of 32.5 at pH.7. The structural analysis via NMR and GCMS of PHA produced under optimised condition showed that the polymer consisted of PHB/V with methyl esters of butyrate, dodecanoic, hexadecanoic, and heptadecanoic acids as well as oxirane, 2-methyl 2-phenyl, Phenol 2,5 bis (1,1 dimethyl ethyl)-, and benzenepropanoic acid, 3,5-bis (1,1-dimethylethyl)-4-hydroxyl as major monomers. The PHA has molecular weight of 628.55 kDa and maximum decomposition temperature of 395ºC and 454ºC. To incorporate nitrogen removal and production of PHA, a two-stage fermentation process was selected. The two-stage process revealed that the biomass produced during ammonium and nitrate removal enhanced the production of PHA up to 35%. This is the first report of two-stage process of ammonium and nitrate removal with PHA production using Rhodobacter sp. ADZ101.