A PURPLE NON-SULFUR BACTERIUM PRODUCING POLYHYDROXYBUTYRATE AND THE CONSERVED REGION OF PHA SYNTHASE GENE

This study aimed to screen purple non-sulfur bacteria capable of accumulating granules or polyhydroxybutyrate (PHB) inside the cells, identify the potent strain, assay the enzyme or PHA synthase, and compare the PHB synthase gene with that of related strains. A total of 58 strains of purple non-sulfur bacteria were isolated from 108 samples of chicken feces in the chicken-egg farm of the Department of Animal Science, Faculty of Natural Resources at Prince of Songkla University, Hat Yai, Thailand. After cultivating the bacteria in glutamate malate (GM) medium without added glutamic acid under light (3,000 Lux) at 35 o C for 5 days, the intracellular biopolymer granules of the bacteria were observed by using a Confocal Laser Scanning Microscope (CLSM) with excitation and emission wavelength of 530 and 605 nm, respectively. Gas chromatography (GC) was carried out for quantitative analysis of PHB. There were five strains, CH12, CH52, CH72, CH90 and CH92, showed biopolymer granules under CLSM, and accumulated PHB 5, 1.7, 1.5, 1.4 and 1.8% (w w -1 ) of the cell dry weight (CDW), respectively. The 16S rDNA sequence analysis of CH12 strain showed a high homology of 100% correlation to that of Rhodopseudomonas palustris strain NCIB8288. Regarding the taxonomic characteristics and 16S rDNA sequence analysis, CH12 strain was identified as Rps. palustris NCIB8288. The PHA synthase activity of the crude extract from CH12 strain was 25 units/mL. The conserved regions could be aligned and selected among 5 strains of Rhodopseudomonas palustris (strains BisA53, TIE-1, CGA009, HaA2 and BisB18). The purified PCR product was obtained for further studies.


INTRODUCTION
Plastics are not environmentally friendly materials to the circumstances due to the long-term degradation.Although polyhydroxyalkanoate (PHA) is one of candidates for biodegradable plastics such as polylactides, aliphatic polyesters, polysaccharides, and the copolymers and/ or blends of these, it has attracted much interest because of its biodegradability and similar properties to conventional plastics (BYROM, 1994).There are about 150 kinds of hydroxyalkanoic acids as monomers of PHAs (STEINBüCHEL; VALENTIN, 1995) like inclusions or granules in the cell (DUNLOP; ROBARDS, 1973).The first PHA is the homopolymer poly(3-hydroxybutyrate) (PHB) found in 1925 by Maurice Lemoigne (JACKSON;SRIENC, 1994).PHAs are found in various kinds of microorganisms as energy and carbon reserve (SALEHIZADEH; LOOSDRECHT, 2004) under aerobic condition (GOBI;VADIVELU, 2014).They can be found in natural environments , for example, lake (ESTEVE et al., 1996), river (LOPEZ et al., 1995), knots of plant rhizoids (NDOYE et al., 1994), deep sea under high temperature (GUEZENNEC et al., 1998), and activated sludge (MINO et al., 1998).They can be observed in bacteria under unusual growth conditions or depletion of an essential nutrient other than the carbon source (ALDERETE et al., 1993).
Purple non-sulfur bacteria is a group of purple bacteria.They are heterotrophs using organic substrates, e.g.acetate, pyruvate and dicarboxylic acids.They are able to grow under aerobic, anaerobic and microaerobic conditions with light or without light.Nevertheless, they grow well under anaerobic to microaerobic condition with light.They also grow well in polluted environments (KOBAYASHI et al., 1978) which is well known for using them in wastewater treatments (KOBAYASHI; KOBAYASHI, 1995).The purple non-sulfur bacteria, for instance, Rhodobacter sphaeroides (KHATIPOV et al., 1998;BRANDL et al., 1991) and Rhodospirillum rubrum (BRANDL et al. 1991) are capable of producing PHBs.Thus, it is an expedient to get PHBs in purple non-sulfur bacteria using low-cost medium with environmental advantages.The aim of this study was to screen the potent strain which could produce PHBs from chicken feces, the PHA synthase assay and information of the conserved region of PHA synthase gene and primer design for the gene amplification.Further study on genetic manipulation to increase the PHB yield will have been carried out.

MATERIAL AND METHODS
A total of 108 samples of chicken feces in the chicken-egg farm of the Department of Animal Science, Faculty of Natural Resources at Prince of Songkla University, Hat Yai were used for isolation of purple non-sulfur bacteria.Glutamate-malate (GM) medium was prepared for growing the purple non-sulfur bacteria.12.5 mL of GM medium without added glutamic acid in a 15-mL screw-cap tube used for cultivation of the bacteria.The starter used was 5% (v v -1 ) and incubated under microaerobic condition with tungsten light intensity of 3,000 Lux at 35 o C for 48 h.It was then centrifuged at 12,000 rpm for 5 min.The cell pellet was washed with 0.85% saline solution and freeze-dried for further study.
The 5-day incubation time of the strain was smeared and heat fixed on a glass slide.1% aqueous solution of Nile blue A was used as a staining of PHB granules at 55 o C for 10 min.It was then washed with tap water and 8% acetic acid solution for 1 min, respectively.It was washed with distilled water and blotted dry.The slide was remoistened with distilled water and covered with glass cover slip before observing by Confocal Laser Scanning Microscope (CLSM) with excitation and emission wavelength of 530 and 605 nm, respectively.The procedure was followed using the Holt and Ostle method (HOLT;OSTLE, 1982).Azotobacter sp.TISTR 1094 was used as a reference strain.
The method of esterification of PHB for GC analysis was followed according to the protocol described by Riis and Mai (RIIS;MAI, 1988).20-40 mg of freeze dried bacterial cells was weighed and put into a 10 mL tightly sealable vial.It was added with 2 mL1, 2-dichloroethane (DCE), 2 mL npropanol containing hydrochloric acid (HCl) (1 volume of concentrated HCl mixed with 4 volumes of n-propanol) and 200 µL internal standard (two gram of benzoic acid in 50 mL n-propanol).The mixture was shaken intermittently in water bath at 85 o C for 4 h.It was then cooled to room temperature, added with 4 mL distilled water, and shaken for 20-30 sec, respectively.To determine the PHB content of the cells, the heavier phase or the DCE-propanol was collected and injected into the GC.The ratio of PHB to cell dry weight was defined as the PHB content.
The column used for determination of PHB by GC was HP-1 (HP 19091Z-413E, Crosslinked Methyl Siloxane, Hewlett Packard, USA) capillary column, 30 m in length with 0.32 mm inner diameter.The injection port and the flame ionization detector (FID) port was operated at 250 o C. The following temperature profile used was 5 min at 80 o C, followed by 7 o C/min rise to reach the final temperature of 200 o C. Nitrogen with the flow rate of 5 mL/min was used as the carrier gas.Injection volume was 1.0 µL and injection mode was splitless.A double flame ionization detector (FID) combined with a computer controlling gas chromatography (HP 6850) was used for analysis.To calibrate the GC, 200 mg of PHB was dissolved with a small volume of DCE by heating in a calibrated 10-mL flask.It was then cooled to room temperature and made up to 10 mL by distilled water.The solution was esterified as mentioned above, and injected to GC with the volume of 200; 400; 600; 800 and 1000 µL.
CH12 strain was identified based on the taxonomic characteristics according to the Bergey's Manual of Systematic Bacteriology (PFENNIG; TRUPER, 1989), and 16S rDNA sequence analysis.According to the standard method (SAMBROOK et al., 1989), the genomic DNA was extracted after cultivating the strain in GM medium under microaerobic condition with light (2,500 Lux) at 35 o C for 12 hr.Universal primers, position 170 for forward and 705 for reverse, were used to amplify the gene using GeneAmp PCR System 9600.The length of PCR product was 537 bp.It was sequenced using API 377 DNA sequencer.
After cultivating Rps.palustris CH12 for 3 days, the grown cells were harvested (0.62 g of wet cells from 100-mL culture).It was then suspended in 5.0 mL of 10 mM Tris-HCl buffer pH 7.5, and centrifuged at 10,000 rpm, 4 o C for 5 min.The pellet of cells was put on ice and added 1.5 mL of extraction buffer (10 mM Tris-HCl buffer pH 7.5 containing 10 mM EDTA, and 1 mM DTT).It was mixed well by vortex mixer, and then sonicated for 5 min, and paused every 15 sec for 5 sec.The sonicated mixture was centrifuged at 10,000 rpm, 4 o C for 5 min.The supernatant was kept at 4 o C for PHA synthase assay.

RESULTS AND DISCUSSION
A total of 58 strains of purple non-sulfur bacteria could be isolated from 108 samples of chicken feces under micro-aerobic conditions with light.After cultivating 58 strains of purple nonsulfur bacteria in GM medium without added glutamic acid under micro-aerobic conditions under light (3,000 Lux) at 35 o C for 5 days, only five strains: CH12, CH52, CH72, CH90 and CH92, showed biopolymer granules under fluorescence microscopy and accumulated PHB 5; 1.7; 1.5; 1.4 and 1.8% (w w -1 ) of the CDW, respectively, analysed by using GC.The results do not correlate to those of other purple non-sulfur bacteria, for example, Rhodospirillum rubrum and Rhodobacter sphaeroides (Rba.sphaeroides) are able to accumulate PHA under nitrogen-limited source conditions 45% and 60-70%, respectively (BRANDL et al. 1989;BRANDL et al. 1991), and Rba.sphaeroides accumulate PHB higher in lactateglutamate than in lactate-ammonium medium (KHATIPOV et al. 1998).However, PHB accumulation of the five strains was higher than that of the type strain, Azotobacter sp.TISTR 1094, 1.1% (w w -1 ) of the CDW.The CH12 strain was selected as the most potent strain for further study.
The CH12 strain was identified as Rhodopseudomonas palustris (Rps.palustris) strain NCIB8288 based on the taxonomic characteristics (Table 1) and 16S rDNA sequence analysis (Figure 1).To increase the expression of PHB synthesizing genes of CH12 strain, genetic manipulation need to be carried out for further study.Firstly the synthase was assayed and determined with standard curve of coenzyme A (CoA).The activity of PHA synthase of crude enzyme was 0. 111 (A 412 ) or 25.0 unit/mL.A standard curve of CoA versus A 412 was shown in Figure 3. Rhodopseudomonas palustris BisA53 (A53), Rhodopseudomonas palustris BisB18 (BisB18), Rhodopseudomonas palustris TIE-1 (TIE-1), Rhodopseudomonas palustris CGA009 (CGA009), and Rhodopseudomonas palustris HaA2 (HaA2).The star represents the highly conserved region shown in panel.
The conserved regions in sequence alignments were used for designing primer.The forward and reverse primers were GGCAAGGTGATCTTCCAGAACG and CGCAGCATGTTGAASGCCATCG, respectively.Amplification of the conserved region between positions 766 and 1304 resulted in 538 bp products (Table 2).After purification of the PCR product and performed on 1.0% agarose gel electrophoresis, the 538 bp of purified PCR product was obtained.Thus, the designed primers had a good match the DNA template of CH12 strain.This result correlate to the report on phaC synthases play an important role in PHB production (AMARA; MOAWAD, 2011).

CONCLUSIONS
A purple non-sulfur bacterium capable of accumulating PHB inside the cells could be screened from chicken feces.It was named CH12 and identified as Rhodopseudomonas palustris based on taxonomic characteristics and 16S rDNA sequence analysis.
The CH12 strain had PHA synthase activity of 25.0 units/mL.The conserved region of phaC gene of CH12 strain could be aligned and selected among 5 related strains.

ACKNOWLEDGEMENT
This work was supported by the budget revenue of Faculty of Science, Prince of Songkla University, and the government budget of Prince of Songkla University.

Figure 2 .
Figure 2. The evolutionary tree between CH12 and other Rhodopseudomonas palustris strains The percentage shown in parentheses indicated the similarity of 16S rDNA sequence of CH12 to those of others.