Analysis of polyhydroxyalkanoate (PHA) synthase gene in activated sludge that produces PHA containing 3-hydroxy-2-methylvalerate

Institute of Environmental Studies, Graduate School of Frontier Sciences, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113-8656, Japan.
Biotechnology and Bioengineering (Impact Factor: 4.13). 04/2007; 96(5):871-80. DOI: 10.1002/bit.21085
Source: PubMed


The identification of phaC which encodes PHA synthase, that is involved in the formation of polyhydroxyalkanoate (PHA) containing 3-hydroxy-2-methylvalerate (3H2MV), was attempted. As of now, PHA containing 3H2MV has been reported to be produced only by mixed microbial cultures in activated sludge, but no pure bacterial cultures. A laboratory-scale activated sludge process was operated for 67 days. During the operation of the activated sludge process, its capacity to produce PHA containing 3H2MV, and the diversity of the partial phaC genes in the activated sludge microorganisms were monitored periodically. Analysis of the partial phaC genes was conducted by PCR followed by cloning and DNA sequencing, or by PCR followed by terminal-restriction fragment length polymorphism (T-RFLP). The cloning-sequencing of the 263 clones gave 11 distinct genetic groups (GGs). All of the genetic groups had similarities to known phaC higher than 48%, and one of them had similarity as high as 96% to that of Alcaligenes sp. The behavior of each of the genetic groups during the operation of the activated sludge process was monitored by the T-RFLP method. The restriction enzyme AccII, with the help of MboI, enabled the monitoring of each of the genetic groups. One of the genetic groups was found to have a strong correlation with the capability of the activated sludge to produce PHA containing 3H2MV, and its DNA sequence together with its amino acid sequence are reported.

Download full-text


Available from: Atsuko Michinaka, Jan 19, 2015
  • Source
    • "Thauera was present throughout the entire operational period, and at the end of the process, two other bands, with the highest similarity with Pseudomonas and Acinetobacter, also appeared. Regarding the phylogenetic analysis of PHA synthase genes in the acclimatized MMC, the same set of primers used by Michinaka et al. (2007) were applied to samples collected on the 9th day of operation. With a total of 80 phaC gene sequences obtained, 62 were embedded into 13 OTUs (clones with similarities higher than 97 % were classified as the same OTU), being 12 OTUs closer to class I PHA synthase and only one to class II. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Enrichment of mixed microbial cultures (MMCs) in polyhydroxyalkanoate (PHA)-storing microorganisms must take place to develop a successful PHA production process. Moreover, throughout the operational period of a MMC system, the population needs to be checked in order to understand the changes in the performance that eventually occurred. For these reasons, it is necessary to monitor the population evolution, in order to identify the different groups of microorganisms and relate them with the storage capacity and kinetics of the MMC. Regarding this particular process, several culture-independent molecular techniques were already applied, with the use of hybridization techniques such fluorescence in situ hybridization and also PCR-based methods like denaturing gradient gel electrophoresis, terminal restriction fragment length polymorphism, pyrosequencing, and quantitative PCR standing out. This review intends, thus, to look at the molecular methods currently applied in monitoring the PHA-storing population evolution and how they can be combined with the evolutionary engineering step in order to optimize the overall process.
    Applied Microbiology and Biotechnology 10/2015; DOI:10.1007/s00253-015-7010-6 · 3.34 Impact Factor
  • Source
    • "Lanes (from left to right) are the 1–10 day samples. which is consistent with previous reports on the investigation of the diversity of the phaC genes in activated sludge (Michinaka et al., 2007; Ciesielski et al., 2008). Moreover, the two main types of PHA produced by activated sludge are PHB and PHBV (Salehizadeh & Van Loosdrecht, 2004; Serafim et al., 2008), which indirectly provides evidence for the predominance of Class I PHA synthase in activated sludge. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Activated sludge is an alternative to pure cultures for polyhydroxyalkanoate (PHA) production due to the presence of many PHA-producing bacteria in activated sludge community. In this study, activated sludge was submitted to aerobic dynamic feeding in a sequencing batch reactor (SBR). During domestication, the changes of bacterial community structure were observed by terminal restriction fragment length polymorphism (T-RFLP) analysis. Furthermore, some potential PHA-producing bacteria, such as Thauera, Acinetobacter, and Pseudomonas, were identified by denaturing gradient gel electrophoresis (DGGE) analysis. The constructed PHA synthase gene library was analyzed by DNA sequencing. Of the total of 80 phaC genes obtained, 76 belonged to the Class I PHA synthase, and 4 belonged to the Class II PHA synthase. Gas chromatography-mass spectrometry (GC-MS) analysis shows that PHA produced by activated sludge was composed of three types of monomers, 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 3-hydroxydodecanoate (3HDD). This is the first report of production of medium-chain-length PHAs (PHAMCL ) containing 3HDD by activated sludge. Further studies suggested that a Pseudomonas strain may play an important role in the production of PHAMCL containing 3HDD. Moreover, a Class II PHA synthase was found to have a correlation with the production of 3HDD-containing PHAMCL . This article is protected by copyright. All rights reserved.
    FEMS Microbiology Letters 06/2013; 346(1). DOI:10.1111/1574-6968.12201 · 2.12 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: A new pretreatment method for the gas chromatographic determination of poly(3‐hydroxybutyrate) (PHB) was developed based on a combination of alkaline hydrolysis and acid esterification. The determination principle is as follows: PHB is hydrolyzed to its monomer 3‐hydroxybutyrate by alkaline solution, followed by the esterification with methanol to generate the methyl ester of 3‐hydroxybutyrate catalyzed by acid, which is detected by a gas chromatography. From the comparison of effects of alkali and acid on PHB hydrolysis and 3‐hydroxybutyrate esterification, alkali resulted in a better performance for the hydrolysis, while acid was better for the esterification. The pretreatment conditions for PHB were optimized and the determination performance was characterized.
    Analytical Letters 10/2007; 40(15-15):2915-2924. DOI:10.1080/00032710701603959 · 1.03 Impact Factor
Show more