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.

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Available from: Atsuko Michinaka, Jan 19, 2015
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    • "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. "
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    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.
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    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
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    ABSTRACT: One of the options enabling more economic production of polyhydroxyalkanoates compared to pure cultures is the application of mixed cultures. The use of a microbial community in a sequencing batch reactor has a few advantages: a simple process control, no necessity for sterile processing, and possibilities of using cheap substrates as a source of carbon. Nevertheless, while cultivation methods to achieve high PHAs biomass concentration and high productivity in wild and recombinant strains are defined, knowledge about the cultivation strategy for PHAs production by mixed culture and species composition of bacterial communities is still very limited. The main object of this study was to characterize on the molecular level the composition and activity of PHAs producing microorganism in activated sludge cultivated under oxygen limitation conditions. PHAs producers were detected using a PCR technique and the created PHA synthase gene library was analyzed by DNA sequencing. The obtained results indicate that PHAs-producers belonged to Pseudomonas sp., and possessed genes coding for mcl-PHA synthase. The kinetics of mcl-PHA synthase expression was relatively estimated using real-time PCR technology at several timepoints. Performed quantitative and qualitative analysis of total bacterial activity showed that there were differences in total activity during the process but differential expression of various groups of microorganisms examined by using DGGE was not observed.
    Journal of Industrial Microbiology and Biotechnology 05/2008; 35(8):805-14. DOI:10.1007/s10295-008-0352-7 · 2.44 Impact Factor
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