Biosynthesis of medium chain length poly(3-hydroxyalkanoates) (mcl-PHAs) by Comamonas testosteroni during cultivation on vegetable oils

Abstract

Comamonas testosteroni has been studied for its ability to synthesize and accumulate medium chain length poly(3-hydroxyalkanoates) (mcl-PHAs) during cultivation on vegetable oils available in the local market. Castor seed oil, coconut oil, mustard oil, cotton seed oil, groundnut oil, olive oil and sesame oil were supplemented in the mineral medium as a sole source of carbon for growth and PHAs accumulation. The composition of PHAs was analysed by a coupled gas chromatography/mass spectroscopy (GC/MS). PHAs contained C₆ to C₁₄ 3-hydroxy acids, with a strong presence of 3-hydroxyoctanoate when coconut oil, mustard oil, cotton seed oil and groundnut oil were supplied. 3-Hydroxydecanoate was incorporated at higher concentrations when castor seed oil, olive oil and sesame oil were the substrates. Purified PHAs samples were characterized by Fourier Transform Infrared (FTIR) and ¹³C NMR analysis. During cultivation on various vegetable oils, C. testosteroni accumulated PHAs up to 78.5–87.5% of the cellular dry material (CDM). The efficiency of the culture to convert oil to PHAs ranged from 53.1% to 58.3% for different vegetable oils. Further more, the composition of the PHAs formed was not found to be substrate dependent as PHAs obtained from C. testosteroni during growth on variety of vegetable oils showed similar compositions; 3-hydroxyoctanoic acid and/or 3-hydroxydecanoic acid being always predominant. The polymerizing system of C. testosteroni showed higher preference for C₈ and C₁₀ monomers as longer and smaller monomers were incorporated less efficiently.

Introduction

After first detection of a short chain length PHA (scl-PHA); poly(3-hydroxybutyrate) (PHB), as bacterial storage material by Lemoigne (1926), intensive research has been carried out on the physiology, biochemistry and molecular genetics of poly(3-hydroxyalkanoates) (PHAs) metabolism in various PHAs producing bacteria during the last two decades (Steinbüchel and Hein, 2001, Rehm and Steinbüchel, 2002). PHAs are intracellular polymers synthesized by a wide variety of bacteria via different pathways (Steinbüchel and Füchtenbusch, 1998, Lee et al., 1999, Steinbüchel, 2001, Rehm and Steinbüchel, 2002). Naturally, PHAs are synthesized from coenzyme A thioesters of the hydroxyalkanoic acids, which are synthesized during fatty acid metabolism (Steinbüchel and Füchtenbusch, 1998, Witholt and Kessler, 1999, Steinbüchel, 2001, Steinbüchel and Lütke-Eversloh, 2003). More than 140 different hydroxyalkanoic acids have been identified as constituents of microbial PHAs when bacteria were cultivated under nitrogen limited conditions and if a suitable carbon source was provided in excess (Steinbüchel and Valentin, 1995).

Intracellular degradation of PHAs comprises the hydrolysis of the endogenous storage material mobilizing the carbon/energy reservoir by the accumulating cell itself when supply of limiting nutrient is restored. PHAs degrading microorganisms occur in many ecosystems, such as soil or compost and can excrete specific hydrolyzing PHA depolymerases, which can degrade PHAs and the degradation products can be used as carbon/energy source (Jendrossek et al., 1996, Jendrossek, 2002).

Due to similarities of physical and material properties with conventional plastics, PHAs can be recommended for application in various areas. They are thermoplastic and/or elastomeric, in-soluble in water, enantiomerically pure, non toxic, biocompatible, piezoelectric and exhibit a high degree of polymerization and have molecular weights of up to several million Da (Müller and Seebach, 1993, Hocking and Marchessault, 1994). The physical properties of the PHB are similar to those of polypropylene, i.e. melting points, crystallinity and glass transition temperatures, representing it as a stiff and brittle material (Hocking and Marchessault, 1994). A polymer containing mainly of 3-hydroxyoctanoic acid is synthesized by Pseudomonas oleovorans and was the first example for PHAs consisting of medium-chain-length hydroxyalkanoic acids, mcl-PHAs obtained from an axenic culture (de Smet et al., 1983). Pseudomonads belonging to rRNA homology group I are able to synthesize mcl-PHAs when they are cultivated on various aliphatic alkanes or aliphatic fatty acids (Huisman et al., 1989, Witholt and Kessler, 1999) but only a few exceptions are able to synthesize mcl-PHAs from glucose and other structurally unrelated carbon sources (Haywood et al., 1989, Timm and Steinbüchel, 1990).

Metabolic engineering approaches have been used to expand the spectrum of utilizable substrate and to improve PHAs production (Steinbüchel, 2001, Steinbüchel and Lütke-Eversloh, 2003); for PHAs production from structurally unrelated carbon source (Klinke et al., 1999) and also for production of PHAs having both short and medium chain length 3-hydroxyalkanoates (Matsusaki et al., 2000). There are only few reports on production of PHAs using cheap renewable carbon sources like palm oil (Tan et al., 1997), tallow (Cromwick et al., 1996), oil remaining from biotechnological rhamnose production (Füchtenbusch et al., 2000), whey (Park et al., 2002) and triglycerides (Ashby and Foglia, 1998).

There are several reports on simultaneous production of scl–mcl PHAs by Pseudomonas oleovorans, Pseudomonas resinovorans other Pseudomonas sp. and Aeromonas caviae (Ashby et al., 2002, Cromwick et al., 1996, Chung et al., 1999, Doi et al., 1995, Kato et al., 1996, Kang et al., 2001). In our previous study we have reported C. testosteroni for production of PHB during cultivation on naphthalene (Thakor et al., 2003). In the present study we have reported the ability of the same strain to accumulate mcl-PHAs and characterization of the PHAs obtained during growth on various vegetable oils.