WO2011002270A1

WO2011002270A1 - A novel bacterium producing polyhydroxyalkanoates from palm oil mill effluent

Info

Publication number: WO2011002270A1
Application number: PCT/MY2010/000004
Authority: WO
Inventors: Mohd Aii Hassan; Mohd Raf Ein Zakaria; Suraini Abd. Aziz; Farinaz Leen Mohamad Ghazali; Yoshihito Shirai; Minato Wakisaka
Original assignee: Universiti Putra Malaysia; Kyushu Institute Of Technology; Felda Palm Industries Sdn. Bhd .
Priority date: 2009-06-29
Filing date: 2010-01-07
Publication date: 2011-01-06
Also published as: MY180619A

Abstract

The bacterial strain EB 172, isolated from digester treating palm oil mill effluent, was investigated by polyphasic taxonomic approach. The cells were rod-shaped, Gram- negative, non-pigmented, non-spore-forming and non-fermentative. Phylogenetic analyses using the 16S rRNA gene sequence showed that the strain was placed in the cluster of genus Comamonas; its closest neighbours were the type strains C. terrigena (96.8 %), C. koreensis (93.4 %), C. composti (92.9 %), and C. kerstersii (91.1 %). The ability of C. putranensis to produce polyhydroxyalkanoates (PHA) when supplied with organic acids made this bacterium is unique in Comamonas species. The bacterial strain was clearly distinguished from all of the existing strains using phylogenetic analysis, fatty acid composition data and a range of physiological and biochemical characteristics. The DNA G+C content of the genomic DNA was 59.1 mol%. It is evident from the genotypic and phenotypic data that strain Comamonas putranensis represents a novel species in the genus Comamonas, for which the name Comamonas putranensis sp. nov. is proposed.

Description

A Novel Bacterium Producing Polyhydroxyalkanoates From Palm Oil Mill Effluent

FIELD OF INVENTION

The invention relates to isolation of a novel bacterium and a process for polyhydroxyalkanoates (PHA)s production from anaerobically treated palm oil mill effluent and other organic wastes.

BACKGROUND OF INVENTION

Various scientific and scholarly articles are referred to throughout the specification. These articles are incorporated by reference herein to describe the state of the art to which this invention pertains. Polymeric materials occurring naturally or produced from renewable resources were recognized as extremely useful many years ago. These materials have become increasingly more interesting and attractive in recent years as alternatives to petrochemical-based polymers and plastics. Meanwhile, consumers and the plastics industry have shown a growing concern about the disposal of plastics and their environmental impact. The industry is actively looking for ways to minimize the unnecessary use of plastics to complement recycling and reuse programs. Others are working on new materials or modifications to old ones to reduce the environmental impact of plastics. Palm oil mill effluent (POME) is the most polluted organic residue generated from palm oil processing and comprises of high nutrient content mainly oil and fatty acids. POME is able to support bacterial growth with the degradation of the waste to reduce its pollution strength. Most of the mills in Malaysia adopt open lagoons and open digester tanks for POME treatments. Under anaerobic conditions, biogas will be generated and will mainly consist of methane gas. Other potential byproducts recovered from the anaerobic process of POME are volatile fatty acids, mainly acetic, propionic and butyric acids (Hassan et al., 1997). These acids then will be used for polyhydroxyalkanoate (PHA) production (Hassan et al., 2002). Several studies have reported on the isolation of PHA accumulating bacteria from the environments (Redzwan et al., 1997; Alias and Tan, 2005; Berlanga et al., 2006: Zakaria et al., 2008) utilizing oils, glucose and organic acids as carbon and energy source. Since organic acids are abundantly available from the anaerobic process of POME, and due to existing intellectual property or licensing in commercialize it in future, an attempt to screen and isolate the wild type strains from the anaerobic digester treating POME was carried out using conventional Nile Blue A staining method. The significance of this finding is that potential PHA producers were successfully isolated from oil palm wastewaters. The purified culture showed significant potential for PHBV accumulation utilizing those acids derived from anaerobically treated POME.

SUMMARY OF THE INVENTION

The invention pertains to a method of the isolation of bacteria from digester tanks treating POME. The invention pertains to a method used in describing newly isolated bacterium by polyphasic approach. The bacterial strain is in the family Comamonadaceae and novel for their combination of biochemical and genetic properties. These bacterial strains have ability for PHBV production from organic acids derived from POME treatment. Published work on POME treatment and organic acids recovery were performed by previous invention (Hassan, 2006). The invention pertains to propose a new name of newly isolated bacterium as Comamonas putranensis sp. nov. The invention pertains to a process for production of polyhydroxyalkanoates (PHAs) from synthetic / organic acids derived from palm oil mill effluent (POME) treatment which comprises the step of incubating of bacterial strains for a sufficient period of time and specified conditions to produce PHA in a culture medium. The invention pertains to a method to improve culture growth and production of poly(3-hydroxybutyrate-co-3- hydroxyvalerate) [(P(3HB-co-3HV)] copolymers using single or combination of synthetic/ organic acids from POME. The invention pertains to a strategy to improve the incorporation of P(3HV) monomer units in the P(3HB-co-3HV) copolymer by controlling their culture conditions.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows the novel bacteria, Comamonas putranensis stained with Nile Blue A staining and were exposed under UV illumination. PHA containing cells exhibited pink-orange colour while colourless if no accumulation. Fig. 2 shows the growth profiles of C. putranensis when supplemented with different organic acids.

Fig. 3 shows the electron micrograph with negative stained cells of strain EB172 under growth phase. Bar represents 1.0 μm.

Fig. 4 shows the electron micrograph of strain EB172 grown under nitrogen limitation for PHAs accumulation. Bar represents 5 μm.

Fig 5 shows rooted neighbor joining tree (Saitou and Nei, 1987) based on 1 ,446 nucleotide positions of 16S rDNA sequences showing relationships between strain EB172 (C putranensis) and closely related taxa. R. eutropha was used as an out group. Percentages at the nodes indicate levels of bootstrap support based on neighbor-joining analyses of 1000 resampling datasets. Bar, 0.01 substitutions per nucleotide position.

Fig 6 shows 500 MHz 1 H-NMR spectrum of PHBV produced by C. putranensis when supplied with 10 g/L organic acids derived from POME treatment.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS Sampling

Sludge was obtained from an open digester tank (ODT) treating POME at the FELDA Selling HiMr mill located in the Negeri Sembilan, Malaysia. The ODT capacity was 3600 m³ which was able to treat POME at HRT 20 days daily. The treated effluent in the ODT was withdrawn and collected in the 1 L Schott bottles and kept on ice prior to use.

Screening and isolation process

Screening of PHA-producing bacteria from ODT was performed by direct plating and enrichment techniques. In direct plating technique, 50 ml. of activated POME sludge from ODT was homogenized by vortex at moderate speed and a dilution series up to 10x^'9 dilution was made to obtain a desired colony forming unit (c.f.u). Aliquots of 0.1 mL were plated onto nutrient agar (Merck, Germany) and the plates were incubated for 24 - 48 h at 30 or 37⁹C. Colonies developed on the agar were differentiated by color, elevation, form, and edge appearances (Dawes and Senior, 1973). For enrichment technique, the sludge was centrifuged at 3,500 rpm for 10 min at 4°C to remove residual oils. The supernatant was decanted and the pellet was transferred into enrich media containing 2 g/L of sodium acetate. The flask was agitated at 250 rpm for 2 weeks at 30 or 37O. Concentration of the sodium acetate was maintained at 2 g/L by weekly addition of sodium acetate solutions into the enriched media. Loopfuls of the culture media was withdrawn and streak onto the nutrient agar plates using spread plating technique. Selected isolates were purified by dilution-streaking to obtain single colonies. The isolation for PHA producing bacteria was performed by randomly picking out and culturing the colonies onto solid mineral salt medium (MSM) containing components as proposed by Berlanga et al., (2006). All MSM compositions were same except the concentration of NaCI. MSM was supplemented with 2.5 g sodium acetate/L. 0.5 mg Nile blue A (Sigma, St. Louis, MO, USA) (DMSO)/mL was added into the MSM media. The cultures were incubated for 4 - 5 days at 30 - 37 "C. The grown colonies from solid MSM were exposed under UV illuminator at the wavelength 280 - 360 nm. The orange fluorescence color observed from PHA positive colonies were different in brightness and it depends on the types of microorganisms able to store polymeric compounds (Fig. 1). The cultures from agar slants were then transferred in the 250 mL shake flask containing 50 mL NB. The cultures were incubated at 30⁰C for 18 h. The cells were centrifuged at 6,000 rpm for 5 min at 4⁰C. Then the cultures were transferred into 250 ml shake flask containing 50 mL nitrogen limiting media with 2.5 g/L sodium acetate. The compositions of the nitrogen limiting media were as mentioned above excluding agar. The cultures were incubated for 4- 5 days at 30 - 37⁰C. As for PHBV productions, the MSM medium was supplied with 10 g/L of acids mixture from anaerobically treated POME with ratio (acetic 5: propionic 3: butyric 2).

Characterization of P(3HB-co-3HV) producer

Biochemical characterization, Quinones, 16S rDNA sequencing, DNA base composition, cellular fatty acids analysis and DNA-DNA hybridization were performed to determine the identity of the isolate, C. putranensis.

Morphological features were studied using a phase-contrast microscope (Olympus BH-2). For the determination of cell shape, size and the detection of flagella, cells were negatively stained with 1% (w/v) phosphotungstic acid according to the method of Cole & Popkin (1981 ) and observed with a CM-20 Philips transmission electron microscope at a voltage of 100 kV. Staining of polysaccharides was performed using Sudan black and staining of inclusion bodies with Nile blue A (Ostle and Holt, 1981 ). Oxidase and catalase activities were analyzed according to Gerhardt et al., (1981 ) and antibiotic sensitivity tests were performed using antibiotic sensitivity discs (OXOID, England) on Muller-Hilton Agar (MHA) (Merck, Germany). Saline tolerance was investigated using NB medium and NA with the addition of NaCI (Merck) at required concentration.

Biochemical characteristics of test strain were determined using the API 20NE, API 50 CH and API ZYM (BioMerieux) following the manufacturer's protocols. Screening for carbon substrates utilized was performed with the BIOLOG GN2 microplate system release 4.0. The pH range for growth was checked using a 250 ml flask containing 50 ml NB with a pH of 5-9 at 30 ⁰C. Growth was checked by monitoring optical density at 600 nm.

Quinones were characterized by HPLC using an EcoCart 125-3 (Lichrospher; RP- 18, 5 μm) column and acetonitrile/2-propanol (65:35, v/v) as mobile phase at a flow rate of 0.5 mUmin. The column was kept at 40 °C. The ubiquinone was detected by a UV detector at 254 nm (Kroppenstedt, 1985). The analyses were performed at the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany).

For fatty acids analysis, fatty acid methyl esters were obtained by saponification, methylation and extraction using the method as described by Kuykendall et al. (1988). The fatty acid methyl esters mixtures were separated using Sherlock Microbial Identification System (MIS) (Microbial ID, Newark, DE 1971 1 USA.) which consisted of a Hewlett-Packard Model 5980 gas chromatograph fitted with a 5% phenyl-methyl silicone capillary column (0.2 mm ^• 25 m), a flame ionization detector, Hewlett-Packard model 7673A automatic sampler, and a Hewlett- Packard model KAYAK XA computer (Hewlett- Packard Co., Palo Alto, California, USA). Peaks were automatically integrated and fatty acid names and percentages calculated by the MIS Standard Software (Microbial ID). For the G+C content determination genomic DNA was prepared according to the procedure of Wilson (1987). The HPLC technique (Mesbah et al., 1989) was used for the determination of the G+C content of strain C. putranensis and the control strain, C. terrigena (DSM 7099^T) was used. DNA-DNA hybridization by the initial renaturation rate was carried out under consideration of the modifications described by Huss et al. (1983) using spectrophotometer (Cary 100 Bio UV/VIS) equipped with a Peltierthermostatted 6X6 multicell changer and a temperature controller with in situ temperature probe (Varian). The hybridization temperature was 70¹C and the reaction was carried out in 2X standard saline citrate (SSC) and 10% formamide. Each value was the mean of at least two hybridization experiments.

16S rDNA sequence analysis and phylogenetic tree construction

The extraction of genomic DNA from strain C. putranensis was performed using genomic DNA extraction kit (Vivantis Tech., Malaysia) by following manufacturer's instruction. Bacterial universal primers used for PCR were: forward pAF δ^'-AGAGTTTGATCCTGGCTCAG-S^" and reverse pAR 5^{^}-AAGGAGGTGATCCAGCCGCA -3\ PCR reaction mixture contained 25 μl_: 10 X PCR buffer (Fermentas, USA), 0.5 μl_: 10 mM dNTP mix (Fermentas, USA), 2.5 μL: 25 mM MgCI (Fermentas, USA), 0.5 μL: of each primer (10 μM) (1^st Base, Malaysia), 0.2 μL: 5U/ μL Taq DNA polymerase (Fermentas, USA), 0.5 μL: total DNA sample and 17.8 μL: sterile de- ionized water to make total reaction mixture 25 μL. The amplification was performed by PCR machine; model T Gradient Thermoblock, (Biometra, Germany) with PCR condition as follows: initial denaturation 94⁰C, 3 min: denaturation 94⁰C, 40 s: annealing 54 °C, 40 s: elongation 72⁰C, 1 min 30 s and 72⁰C, 10 min: 35 cycles. An expected PCR product of approximately 1500 bp was purified using a gel extraction kit (Qiagen, Germany) and the PCR products were sent to Medigene Sdn. Bhd. (Malaysia) for sequencing. The sequences generated in this work were all searched for similarity against GenBank using the advanced gapped blast option (Altschul et al., 1997) at the NCBI home page (http:// www.ncbi.edu) and Ribosomal Database Project Il (RDP) (Maidak et al., 1997). Sequence alignment was performed with the multiple sequence alignment using CLUSTAL W on the website http://www/ebi.ac.uk/embl/. Phylogenetic analyses were conducted by MEGA 3.1 software (Kumar et al., 2004). Phylogenetic distances between the cultures were calculated based on a Kimura 2-parameter (Kimura, 1980) substitution model. Phylogenetic trees were generated from the distance matrixes using a neighbour joining tree-building algorithm (Saitou and Nei, 1987). Statistical support for the branching nodes was obtained by bootstrap (1000 replicates).

Production of Polyhydroxyalkanoates (PHA) Bacterial strain

C. putranensis used in this study was isolated from open digester treating POME collected in Serting Hilir palm oil mill located in the Negeri Sembilan, Malaysia. For preparation of inocula, the culture was grown at 30⁰C under aerobic condition in nutrient broth (5 g of peptone, 3 g of meat extract, 1 g of yeast extract, 2.5 g of NaCI and 3 g of sodium acetate in 1 I distilled water) for 14 hours. The cultures were stored at -80 ⁰C in 25 % (v/v) glycerol for maintenance purposes.

Growth and PHA biosynthesis by one- step cultivation Precultured cells were transferred into 50 ml mineral salts medium (MSM) in 250 mL flasks containing (per liter of distilled water) KH₂PO₄, 6.7 g ; (NH₄)SO₄, 1 g ; MgSO₄, 0.2 g, 0.02 CaCI₂ and 0.1 mL microelements solution as reported by Hassan et al. (2002). The initial pH of the medium was adjusted to 7.0. Synthetic organic acids and their mixtures (total acids 5 g/L) were added into the medium and were extrapolated to the desired ratio. The cultures were incubated at 3O⁰C for 48 hours under aerobic condition with agitation speed 200 rpm. Cell growth was monitored by measuring the optical density (OD) of the broth at 600 nm and correlation of OD versus cell dry weight (CDW) was established from this study.

Analytical procedures PHA content and composition in the lyophilized cell material were determined using gas chromatography (Agilent, Series 2000) and nuclear magnetic resonance (¹H NMR) analyses. In GC analysis (Braunegg et a/., 1978), approximately 15 mg of lyophilized cell was subjected to methanolysis in the presence of methanol and sulfuric acid [85%:15% (v/v)]. The reaction mixture was incubated at 100⁰C for 2 h. The organic layer containing the reaction products was separated, dried over Na₂SO₄, and analyzed by GC.

In NMR analysis, the PHA was extracted from freeze-dried cells. 1 .0 g freeze-dried cells were stirred in 100 mL of chloroform for 24 h at 30°C. The extract was filtered to remove cell debris, and the chloroform was concentrated to a volume of about 15 mL using a rotary evaporator. The concentrated solution was then added drop-wise to 150 mL of rapidly stirred methanol to precipitate the dissolved PHA. The precipitated PHA was recovered by filtration using a 0.45 μm PTFE membrane and dried overnight at room temperature. The purified PHA was dissolved in deuterated chloroform (CDCI₃) and subjected to the 400 MHz ¹H and 300 MHz ¹³C NMR analyses.

Results:

Screening and Isolation of PHBV producer

Approximately 200 colonies were randomly screened for their ability to accumulate polyhydroxybutyrates (PHB). All colonies were transferred onto the selective medium containing the Nile Blue A dye with limiting nitrogen source. After several days of incubation, the viable colonies that showed a bright orange color under UV light were selected for further processed indicated the isolates were able to accumulate PHA. All the colonies that have native colors (orange or yellow) were discarded during the screening process because this color will influence the orange fluorescence exhibited and all the color strains tested previously did not promoted the PHB accumulation. Only the white colonies were selected for further analysis. Eleven colonies were successfully isolated from the palm oil wastewaters using acetate as sole carbon and energy sources. The isolates namely as PHB 5 showed a good PHB accumulation and later designated as C. putranensis. Growth study of C. putranensis was performed using various organic acids intended to obtain suitable acids for PHB and PHBV production. As showed in Fig. 2, propionic acid is the best carbon source for growth of C. putranensis as determined by CDW, followed by acetic and butyric acids. The results obtained showed that this bacterium was able to consume propionic acids and possibly could produce higher hydroxyvalerate (HV) monomer units during PHA accumulation with POME acids mixture. Morphological studies

C. putranensis is actively motile, aerobic, nonfermentative, Gram- negative rod, 0.71 by 2.64 μm size. Colonies on NA (nutrient agar) plates developed after 24 h incubation period were circular (1 -2 mm diameter) and transparent cream- colour. Electron micrograph of Negative staining showed that presented of flagella in multipolar tufts during growth stage as showed in Fig. 3. All validly reported Comamonas strains were having flagella and motile except C. koreensis (Chang ef al., 2002). Thin sections electron micrograph of strain C. putranensis revealed typical PHA granules clearly show the existence of intracellular PHA granules (Fig. 4). There were several Comamonas strains showed capabilities of PHA accumulations: C. denitrificans (Gumaelius et al., 2001 ), C. testosteroni (Thakor ef al., 2003), and C. composti (Young et al., 2008). Other unique features that differed C. putranensis with other reported Comamonas species are described in later section.

DNA-DNA hybridization

The DNA-DNA hybridization was performed to investigate the DNA relatedness between C. putranensis with reference strain C. terrigena (DSM 7099). The DNA-DNA relatedness between those strains were high (90%) and this value is above the cut-off point recommended for assignment of the strains to the genomic species (Wayne et al., 1987). It was clear that from the results obtained that C. putranensis belongs to C. terrigena species. However, other properties like biochemical and phenotypic features are also should take into consideration in describing new species. Studies conducted by Wauters et al. (2003), showed that C. aquatica and C. kerstersii were the subgroups of the C. terrigena after performing additional analysis like, SDS-PAGE, biochemical analysis, and immunodiffusion analysis. These three bacteria was previously named as C. terrigena and later amended to new name after those analyses was carried out.

G+C content For G+C content calculations, the DNA sample was prepared in duplicate and degraded enzymically into nucleosides as described by Mesbah ef al. (1989). The obtained nucleoside mixture was then separated with a HPLC system. The G+C content of C. putranensis was 59.1 ±1.0 mol%, which was a little bit lower in comparison with other DNA G+C contents previously reported for Comamonas species (60.8-66.3 mol%) (Young et al., 2008). This is another feature that differed this microorganism with other existing Comamonas species.

Analysis of quinone and cellular fatty acids

C. putranensis contained ubiquinone Q-8 (98%) as the predominant component of the quinone system while menaquinone or rhodoquinone was not detected. Fatty acid methyl esters were prepared, separated and identified according to the instructions of Microbial Identification System (MIDI; Microbial ID). Predominant fatty acids of C. putranensis were 16: 0, 18 : 1/ 18: 1w 7c and summed feature 3. The fatty acid pattern of the C. putranensis is shown in Table 1 in comparison with other representative Comamonas species. The fatty acid profile of C. putranensis was in good agreement with data obtained for other members of the genus Comamonas (Chang ef al., 2002; Wauters ef al., 2003; Tago &Yokota, 2004; Chou ef al., 2007) (Table 1).

Table 1 Comparison of the fatty acid compositions of strain EB172 and other Comamonas species.

Taxa: 1 , strain EB172 (C. putranensis sp. nov.); 2, C. composti; 3, C. odontotermitis Dant3-8^T; 4, C. koreensis DSM 18232^T; 5, C. testosteroni DSM 50244^T; 6, C. terrigena LMG 1253^T; 7, C. denitrificans DSM 17887^T; 8, C. nitrativorans DSM 13191^T; 9, C. aquatica LMG 2370^T; 10, C. kestersii LMG 3475^T ; 1 1 , C. badia DSM 17552^T. All the reference data was obtained from Young et al., (2008). Values are percentages of total fatty acids; -, fatty acids representing les than 0.5%. The position of the double bond in the unsaturated fatty acids is obtained by counting from the methyl (ω) end of the molecule.

Fatty acid 1 2 3 4 5 6 7 8 9 10 11

10 : 03- 4.3 5.6 3.8 3.5 4.8 5.3 4.2 5 5 4.5 2.4

OH

12:0 3 3.2 2.7 2.3 2.4 2.8 3 2.9 3 2.6 2.9

14:0 4.5 1.4 - 1 1 3.3 3.2 3.4 3.9 2.9 1.5

15:0 0.51 - - 9.4 1 3.7 - - - -

15 : 02- 0.6 - - OH

16:0 31.9 33.3 33.6 29.9 30.4 27.5 17.8 21.2 25.2 23.4 33.3

16 : 02- - - 2.5 2.2 2 - - - - - 2.3 OH

16 : 12- . . . . 0.6 - - OH

17:0 - 1 - 2.6 0.8 1.5 - - - 0.6

17 : 0 4.7 1.4 5.9 12.3 3.8 2.4 - - - 0.7 cyclo

17:1 0.7 - - -

18 : 1/ 14.8 12.9 16.2 17.9 14.9 22.4 23.8 19 36.1 13.1 18: Iw 7c

19 : 0 - . . . . . cyclo

20: Oiso - - - - -

Summed 32.5 48.6 42.9 28.2 41.9 feature

3* Phylogenetic analysis

Almost complete 16S rDNA sequence of C. putranensis, consisting 1 ,446 nucleotides have been determined. The resulting sequence was checked for similarity against deposited data in GenBank for the top 50 hits, including described and noncultivated organisms were retrieved and merged into the alignment. Phylogenetic analysis in which this sequence was compared with corresponding sequences of representatives of the family Comamonadaceae indicated this strain belongs to the genus Comamonas (Fig. 5). The 16S rDNA sequence similarities between C. putranensis and its nearest neighbors, C. kestersii (91.1%), C. terrigena (96.8%), C. koreensis (93.4%), C. testosteronii (90.2%), C. composti (92.9%), and the type strains of other Comamonas species are low enough to categorize C. putranensis as a species that is distinct from previous described Comamonas species. None of the validly described species showed more than 97% 16S rDNA similarity (Stackebrandt and Goebel, 1994). Bootstrap resampling showed that it possesses a statistically significant association with C. kerstersii. Therefore, on the basis of this data, strain C. putranensis should be placed in the genus Comamonas as a novel species, for which we propose the name Comamonas putranensis sp. nov.

Biosynthesis of PHA during one-step cultivation

The ability of C. putranensiss to accumulate PHA during one- step cultivation process was investigated using different types of carbon sources. Carbon sources such as acetic, propionic, butyric, lactic valeric acid, and glucose were used as solely or in combination with suitable ratios to observe C. putranensis growth and accumulation of polyesters. It was found that C. putranensis was able to produce homopolymer P(3HB) and copolymer P(3HB-co-3HV) when grown on mainly fatty acids as their carbon and energy sources. Table 2 shows PHA content, CDW and monomer compositions were significantly different observed through out the study when supplied with various carbon sources. P(3HB) monomer was produced when acetic, lactic and butyric acid was used sole carbon sources. Table 2 Cell dry weight, PHA content and PHA monomer compositions produced fro m various carbon sources by Comamonas putranenstf.

Carbon PHA PHA composition sources Cell dry content (mol%)^b

weight

(g/L) (wt.%)^b 3HB 3HV

2.1 + 16 +

Acetic acid 0.15 1.2 100 0

3.8 + 50 +

Butyric acid 0.42 17.0 100 0

Propionic 1.1 + 15 + 68 + 29 ± acid 0.32 2.0 9.3 14.9

3.0 + 66 +

Lactic acid 0.35 7.1 100 0

1 .5 + 10 + 42 + 58 ±

Valeric acid 0.45 4.5 9.6 9.6

Acetic + 3.4 + 27 + 56 + 44 ± Valeric 0.70 5.7 1 1 .8 11.8

Glucose + 2.9 + 19 + 35 + 65 ± Valeric 0.90 6.0 4.2 4.2

Glucose + 2.0 + 34 + 79 + 21 ± Propionic 0.85 14.8 0.6 0.6

Acetic + 3.0 + 20 + 91 +

Propionic 0.10 3.5 5.0 9 ± 5.0

aFermentation was conducted by single or in combination of carbon sources with total 5 g/L of substrates, 30⁹C, 200 rpm for 48 h. bDetermination by GC On the other hand, P(3HB-co-3HV) was produced when propionic and valeric acids were used as carbon sources. Other carbon sources like glycerol and fructose also was carried out but there was no good results obtained (results not shown). The highest CDW (3.8 g/L) was produced when butyric acid was used as the carbon source and PHB accumulation 50 wt.%. Lactic acids, showed the highest PHB accumulation (66 wt.%) but with moderate CDW produced (3.0 g/L). An inhibition on cell growth was observed when propionic or valeric acids were fed into MSM media and resulted lower amount of CDW produced with 1.1 g/L and 1.5 g/L, respectively. However, combination of propionic and valeric acids with acetic, and glucose resulted in improvement of CDW, PHA content and mol % of HV units. Copolymers with a broad range of 3HV molar fractions (9-65 mol %) were obtained when the medium were co-fed with propionic and valeric acids.

P(3HB-co-3HV) copolymer production using acetic : propionic acid at different ratios

In order to obtain a P(3HB-co-3HV) copolymer containing varying mol % P(3HV) units, a study was conducted using acetic and propionic acids at different ratio. From the results obtained (Table 3), combination of substrates used resulted in better cell biomass formation and P(3HB- co-3HV) accumulations. The highest CDW and PHA content were obtained when acetic: propionic ratio (2:3) was supplied into the MSM medium resulted 2.8 g/L and 30 wt%, respectively. Thus suggesting the optimal acids mixture for acetic and propionic for P(3HB-co- 3HV) formation. The results also showed that the mol% of HV units formed were proportional to propionic acids concentration in which P(3HV) monomers in the P(3HB-co-3HV) copolymers increasing when supplemented with higher propionic acids concentration. The highest P(3HV) monomer (17 mol%) was detected when the ratio of 1 :4 (acetic: propionic) supplemented into the medium.

Table 3 Cell dry weight, PHA content and PHA monomer compositions produced from combination of acetic and propionic acids at different ratio^a.

PHA

Carbon sources Cell dry content PHA composition (mol%)^b

Acetic: weight

Propionic (g/L) (wt.%)^b 3HB 3HV

(5:0) 1.5 ± 0.2 15 ± 1.5 100 0

(4:1) 2.2 ± 0.3 18 ± 1.5 91 ± 2.1 9 ± 2.1

(3:2) 2.5 ± 0.1 23 ± 1.5 90 ± 1.0 10 ± 1.0

(2:3) 2.8 ± 0.3 30 ± 5.9 85 ± 2.5 15 ± 2.5

(1 :4) 2.6 ± 0.1 21 ± 2.1 83 ± 2.5 17 ± 2.5

aFermentation was conducted by combination of carbon sources with total 5 g/L of substrates, 3O⁰C, 200 rpm for 48 h.

bDetermination by GC.

Effect of pH on P(3HV) unit in the P(3HB-co-3HV) copolymers using sodium valerate as substrate precursor The study using sodium valerate as precursor for P(3HB-co-3HV) copolymer production was carried out. Despite the ability of C. putranensis to consume and accumulates P(3HB-co-3HV) copolymer, the incorporation of P(3HV) unit monomer in the P(3HB-co-3HV) copolymers was improved by selecting initial medium pH. Table 4 showed initial medium pH plays an important role in biomass production, PHA accumulation as well as incorporation of P(3HV) unit monomers. By selecting the initial medium pH (6.5 - 8.0), production of P(3HB-co-3HV) copolymers with varying P(3HV) monomer units in the range of 45- 86 mol % P(3HV) was obtained. pH 7.5 was observed the best pH for biomass production (2.1 g/L), meanwhile pH 7.0 is the best pH for PHA accumulation with 59 wt.%. Thus this result indicates the neutral pH is the best condition for biomass and PHA accumulations. The most interesting finding was P(3HV) monomer units were observed in increasing trends towards the alkaline condition (86 mol % P(3HV)), however reduced in PHA accumulation. This finding was contradictory to the study reported by Loo et al. (2004), in which P(3HV) units formation increased towards lower pH. However, P(3HV) monomer units produced in this study were in the reported range and comparable to other studies (Loo et al., 2004).

Table 4 Effect of pH on biosynthesis of P(3HB-cσ-3HV) copolymers production using sodium valerate by C. putranensis³.

PHA

Cell dry content PHA composition (mol%)^b weight

PH (g/L) (wt.%)^b 3HB 3HV

6.5 0.3 ± 0.2 15 ± 3.5 55 ± 1.5 45 ± 1.5

7.0 1.9 ± 0.4 59 ± 3.2 30 ± 1.5 70 ± 1.5

7.5 2.1 ± 0.2 26 ± 3.2 23 ± 2.1 77 ± 2.1

8.0 1.7 ± 0.2 30 ± 2.5 14 ± 1.5 86 ± 1.5

aFermentation was conducted at 3O⁰C, 200 rpm for 48 h.

bDetermination by GC

¹H NMR

It was expected that the PHA produced from the cells is a P(3HB-co-3HV) copolymer due to the feedstock used during fermentation. In order to confirm this hypothesis, the extracted PHA sample was analyzed by ¹H NMR and the spectrum is shown in Fig. 6. The assignments of P(3HB-co-3HV) signals have been reported previously (Doi et al., 1986; Kamiya et al., 1990). Based on the reports and by the estimation of ¹H NMR chemical shifts using a ChemNMR program in a CS ChemDraw Ultra version 6.0, the peaks in Figure 4 were assigned. The assignments of the ¹H NMR signals revealed that the PHA produced by C. putranensis was a P(3HB-co-3HV) copolymers and hence, confirmed the speculation.

Claims

1 ) A process for production of polyhydroxyalkanoat.es (PHAs) from synthetic acids / organic acids derived from palm oil mill effluent (POME) treatment which comprises the step of incubating of a biologically pure bacterial strain for a sufficient period of time and under specified conditions to produce said PHA in a culture medium comprising synthetic acids / organic acids derived from POME or other organic wastes.

2) A biologically pure bacterial strain as claimed in claim 1 which is isolated from digester- treated palm oil mill effluent (POME) identified by polyphasic approach.

3) A biologically pure bacterial strain as claimed in claim 1 which belongs to Comamonadaceae family in the genus of Comamonas in which proposed as Comamonas putranensis sp. nov. 4) The process as claimed in Claim 1 in which the synthetic acids are acetic, butyric, lactic, propionic, valeric acids and other organic acids.

5) The process as claimed in Claim 1 in which the polyhydroxyalkanoates are selected from the group consisting of polymer of a hydroxybutyric acid, hydrovaleric acid or other hydroxyl acids and copolymers thereof.

6) The copolymer as claimed in claim 5 which is a poly(3-hydroxybutyrate-co-3- hydroxyvalerate) [P(3HB-co-3HV)]. 7) The process according to Claim 1 , wherein the PHA produced from the bacterial strain is capable of producing 10 - 66 % PHA by cell dry weight

8) The process according to Claim 5, wherein the said P(3HB-co-3HV) copolymer produced from the selected bacterial strain with P(3HV) monomer units is in the range of 0-86 mol %. 9) A biologically pure bacterial strain with 16S rDNA sequences as deposited in NCBI accession number of EU 847238.