High yield production of polyhydroxyalkanoates from soybean oil by Ralstonia eutropha and its recombinant strain Prihardi Kahar a , Takeharu Tsuge a, *, Kazunori Taguchi b , Yoshiharu Doi a,b a SORTS Group of Japan Science and Technology Corporation (JST), Department of Innovative and Engineered Materials, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8502, Japan b Polymer Chemistry Laboratory, RIKEN Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan Received 15 April 2003; accepted 14 May 2003 Abstract High yield production of polyhydroxyalkanoates (PHAs) by Ralstonia eutropha H16 and its recombinant strain PHB  4/ pJRDEE32d13 (a PHA-negative mutant harboring Aeromonas caviae PHA synthase gene, phaC Ac ) from renewable inexpensive soybean oil was investigated. The PHA production by the wild-type strain H16 was achieved with a high dry cells weight (118–126 g/l) and a high poly[(R)-3-hydroxybutyrate] [P(3HB)] content per dry cells of 72–76% (w/w). A copolymer of 3HB with 5 mol% (R)-3-hydroxyhexanoate, P(3HB-co-5 mol% 3HHx), could be produced from soybean oil as a sole carbon source by the recombi- nant strain PHB  4/pJRDEE32d13 with a high dry cells weight (128–138 g/l) and a high PHA content of 71–74% (w/w). The reproducible results of PHA production in the presence of soybean oil as a sole carbon source was obtained with a high yield at a range of 0.72 to 0.76 g-PHA per g-soybean oil used. # 2003 Elsevier Ltd. All rights reserved. Keywords: High yield production; Plant oils; Polyhydroxyalkanoates; Recombinant Ralstonia eutropha; Soybean oil 1. Introduction Polyhydroxyalkanoates (PHAs) are reserve polyesters that are accumulated as intracellular granules by many microorganisms [1–4]. Some bacteria are capable of accumulating PHA at levels exceeding 80% (w/w) of dry cells when a nutrient such as nitrogen, phosphorus and magnesium is limited for cell growth. Recently, PHAs have attracted much industrial attention because of their potential use as biodegradable thermoplastic [5]. Poly[(R)-3-hydroxybutyrate], P(3HB), is the most common bacterial PHA in nature. Although untreated P(3HB) is a brittle and stiff material, the P(3HB) film and fiber prepared by cold-drawing procedure have improved mechanical properties [6]. Also, PHA copoly- mers containing 3HB with a small amount of other monomer units are more flexible than the P(3HB) homopolymer. For example, a copolymer of 3HB and 4-hydroxybutyrate, P(3HB-co-4HB), is well known as a flexible material having appropriate properties for practical application [1,5]. Besides, instead of 4HB, introducing (R)-3-hydroxyhexanoate into P(3HB) sequence [to form P(3HB-co-3HHx) copolymer] leads an increase in polymer flexibility [7,8]. Much research has been focused on the efficient pro- duction of PHA using inexpensive carbon sources, because its production cost is still high in comparison with those of chemically synthesized plastics. Using sugars as inexpensive carbon sources, P(3HB) produc- tion with high productivity has been archived in an optimized fermentation process [9]. In spite of such many efforts, P(3HB) production from sugars has an unsolvable disadvantage on a low PHA yield from car- bon substrate. The yield of P(3HB) production from glucose is ranging within 0.30–0.40 g-P(3HB) per g-glu- cose used [10,11]. In face to the further reduction on the production cost, it is necessary to alter the carbon source from sugar to other inexpensive one, which allows bacteria to produce PHA with a high yield. Plant oils are desirable feedstocks for PHA produc- tion because they are also inexpensive carbon sources. In contrast to the other carbon sources, the theoretical 0141-3910/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0141-3910(03)00227-1 Polymer Degradation and Stability 83 (2004) 79–86 www.elsevier.com/locate/polydegstab * Corresponding author. Tel.: +81-45-924-5420; fax: +81-45-924- 5426. E-mail address: ttsuge@iem.titech.ac.jp (T. Tsuge).