Application of Halophilic Archaea for the Production of Polyhydroxyalkanoates (bioplastics)

Biodegradable plastics or bioplastics have raised great interest globally since the continuous decline of fossil fuel reserves used in manufacturing petroleum-derived plastics and their recalcitrant nature is slowly degrading the environment. Amongst the bio-based polymers, polyhydroxyalkanoates (PHAs) are accumulated as intracellular reserve materials by many microorganisms when carbon sources are present in excess and other nutrients like nitrogen, phosphorus, oxygen are limited in supply. PHAs are completely bio-synthesized and bio-polymerized, possessing over 150 monomer variations having different material properties. However, widespread commercialization of PHAs is still striving due to their higher production costs compared to that of synthetic plastics. While the price of polypropylene and polyethylene is US$ 1.32-1.91/kg, PHA cost is estimated as US$ 4.95-6.05/kg. In recent times, production of PHAs by halophilic archaea have gathered huge attention as they have many advantages like rapid growth using cheap carbon sources and waste products, less chance of contamination, no requirement of medium sterilization, re-use of medium salts, use of seawater as the salt-containing medium, thus reducing fresh water consumption and PHA recovery by easy lysis of cells with water. Here an overview of the work done in our laboratory is presented. Previous studies using ethanol industry waste, vinasse (molasses-based) and stillage (rice-based) as substrates to produce PHA from halophiles have been reported by our laboratory (Pramanik et al. 2012; Bhattacharyya et al. 2012, 2014, 2015). Haloarcula marismortui cultivated using 100% pre-treated vinasse resulted in 30% poly(3-hydroxybutyrate) (PHB) accumulation with 0.02 g/l-h volumetric productivity. Later, the study was improved using a more potent archaeon, Haloferax mediterranei, where 25% and 50% pre-treated vinasse were used to yield a more elastic biopolymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) amounting 70% and 66% with productivity of 0.21 and 0.18 g/l-h, respectively. 100% thin stillage was later used by the same archaeon resulting in 71% PHBV accumulation with productivity 0.17 g/l-h. 96% medium salts of the spent stillage were recovered using a temperature-dependant directional solvent, decanoic acid. The production was upgraded in a 14 l simple plug-reactor producing 63% PHBV with productivity of 0.136 g/l-h and almost 99.3% medium salts were recovered. The cost of PHBV was estimated as US$ 2.05/kg when the annual production was simulated as 1890 tons. In addition, from our laboratory, Mahansaria et al. (2015) discovered 14 new haloarchaeal and halobacterial species as novel PHA producers from the salt producing multi-pond solar salterns in the states of West Bengal and Gujarat in India via a polymerase chain reaction-based screening method developed to detect the presence of the enzyme PHA synthase (phaC). The method was also tested on 9 type strains yielding 5 phaC-positive species. The method was developed as the existing techniques for detection of PHAs in environmental halophilic archaea/bacteria are either imprecise or require prior PHA production before screening. The newly developed method involved amplification of approximately 280-300 bp conserved region of Class III phaC gene of halophiles using primers codehopCF and codehopCR, thus eliminating the false positives seen with traditional Nile Red staining procedure and can be used universally to detect PHA producers.