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Polymer Degradation and Stability
journal homepage: www.elsevier.com/locate/polydegstab
Isolation of polyhydroxyalkanoate-producing bacteria from a polluted soil and characterization of the isolated strain Bacillus cereus YB-4
Kouhei Mizuno a, *, Aya Ohta a, Manami Hyakutake b, YousukeIchinomiya b, Takeharu Tsuge b
Division of Biochemical Engineering, Department of Materials Science and Chemical Engineering, Kitakyushu National College of Technology, 5-20-1 Shii, Kokuraminami-ku, Kitakyushu 802-0985, Japan b Department of Innovative and Engineered Materials, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8502, Japan
a r t i c l e i n f o
Articlehistory: Received 1 December 2009 Received in revised form 23 January 2010 Accepted 27 January 2010 Available online 6 February 2010 Keywords: Polyhydroxyalkanoate Bacillus Molecular weight change Polluted soil Isolation
a b s t r a c t
We describe the characterization of polyhydroxyalkanoate (PHA)-producing bacteria isolated from an ammunition-polluted soil in Kitakyushu City, Japan. Over 270strains were evaluated for PHA accumulation based on a colony staining method using Nile red. Of these, nine strains were selected based on the intensity of Nile red ﬂuorescence and the cells were quantitatively analyzed for PHA by gas chromatography. PHA accumulation was observed in ﬁve strains, all of which are inferred to be close to the Bacillus cereus group according to 16S rDNA sequenceanalysis. Interestingly, these strains produced a PHA copolymer, poly(3-hydroxybutyrae-co-3-hydroxyvalerate) [P(3HB-co-3HV)], with a 3HV fraction up to 2 mol% with glucose as a carbon source. Further characterization was performed on one isolate, B. cereus YB-4. Gel permeation chromatography analysis revealed that the number of average molecular weights of PHA accumulated in B. cereus YB-4 drasticallychanged from 722,000 to 85,000 over a 72-h cultivation period. Furthermore, the PHA synthase genes were cloned and the deduced amino acid sequences were determined. This study provides new insights into PHA biosynthesis by members of the B. cereus group. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Polyhydroxyalkanoates (PHAs) are biopolyesters synthesized by bacteria as storagecompounds for energy and carbon, normally under nutrient-limiting conditions with excess carbon. PHAs are biodegradable thermoplastics that can be obtained from renewable resources such as sugars and vegetable oils [1,2]. They are water insoluble, non-toxic, biocompatible, and have recently received attention because of their applications in the packaging industry, medicine, agriculture, and foodindustry. Currently, there are plans to produce some PHAs on an industrial scale employing gram-negative bacteria such as Ralstonia eutropha and recombinant Escherichia coli [3,4] because these bacteria show better growth and higher accumulation of PHAs than others, including gram-positive bacteria. However, PHAs isolated from gram-negative bacteria contain outer membrane lipopolysaccharide (LPS)endotoxins, which induce a strong immunogenic reaction in humans. Hence, these PHAs are undesirable, particularly for biomedical applications. Gram-positive bacteria have thick
* Corresponding author. Tel./fax: þ81 93 964 7303. E-mail address: email@example.com (K. Mizuno). 0141-3910/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.polymdegradstab.2010.01.033
outermembranes that do not contain LPS, and therefore, are potentially better sources of PHAs for use in biomedical applications . In addition, some studies have addressed gram-positive bacteria as preferable candidates for host cells of PHA production. For example, members of the genus Rhodococcus which frequently resides in arid sites like deserts permits the cells to survive in stressful...