J Polym Environ (2010) 18:584–592 DOI 10.1007/s10924-010-0204-1
ORIGINAL PAPER
Isolation and Characterization of a Burkholderia sp. USM (JCM15050) Capable of Producing Polyhydroxyalkanoate (PHA) from Triglycerides, Fatty Acids and Glycerols Jiun-Yee Chee • Yifen Tan • Mohd-Razip Samian Kumar Sudesh
•
Published online: 25 May 2010 Ó Springer Science+Business Media, LLC 2010
Abstract A consortium of microorganisms from oil polluted wastewater sample was cultivated to promote polyhydroxyalkanoate (PHA) accumulation before subjecting the mixed cultures to sucrose density gradient ultracentrifugation. This resulted in the fractionation of the bacterial cells according to their physical features such as size, morphology and/or densities. An isolate was identified as Burkholderia sp. USM (JCM15050), which was capable of converting palm oil products [crude palm kernel oil (CPKO), palm olein (PO), palm kernel acid oil (PKAO), palm stearin (PS), crude palm oil (CPO), palm acid oil (PAO) and palm fatty acid distillate (PFAD)], fatty acids and various glycerol by-products into poly(3-hydroxybutyrate) [P(3HB)]. Up to 70 and 60 wt% of P(3HB) could be obtained when 0.5%(v/v) CPKO and glycerol was fed, respectively. Among the various fatty acids tested, lauric acid followed by oleic acid and myristic acid gave the best cell growth and PHA accumulation. Compared to Cupriavidus necator H16, the present isolate showed better ability to grow on and produce PHA from various glycerol by-products generated by the palm oil industry. This study demonstrated for the first time an isolate that has the potential to utilize palm oil and glycerol derivatives for the biosynthesis of PHA.
J.-Y. Chee Y. Tan M.-R. Samian K. Sudesh (&) School of Biological Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia e-mail:
[email protected]
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Keywords PHA Ultracentrifugation Palm oil Glycerol Burkholderia sp.
Introduction Polyhydroxyalkanoate (PHA) is a family of bio-based and biodegradable polymers synthesized by a wide variety of naturally occurring microorganisms [1, 2]. PHA serves as carbon and energy storage compound for these microorganisms. Approximately 150 types of monomers have been identified as PHA constituents which are produced via various metabolic pathways using different carbon sources. PHA containing short-chain-length monomers such as 3-hydroxybutyrate (4C) and/or 3-hydroxyvalerate (5C) are termed SCL-PHA. The PHA made of medium chain-length monomers such as 3-hydroxyhexanoate (6C), 3-hydroxyoctanoate (8C), 3-hydroxydecanoate (10C), 3-hydroxydodecanoate (12C) and 3-hydroxytetradecanoate (14C) are termed MCL-PHA. SCL and MCL-PHA are often synthesized by different types of bacteria although some bacteria are capable of synthesizing both types of PHA [3, 4]. PHA is stored in the form of water insoluble granules in the cell cytoplasm. It is known that the density of SCL-PHA granules is higher than that of MCL-PHA granules due to the presence of longer side chains in MCLPHA structure that might cause loose packing of the bulky MCL-PHA polymers in the granules [5]. There is much interest in PHA as a class of bio-based and biodegradable polymers that can be produced using renewable resources such as sugars and vegetable oils [6–9]. In addition, the ability to produce PHA from industrial and domestic wastes is also gaining much importance as this approach can minimize waste disposal problems while at the same time reduce the production cost
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of PHA. Among the various fermentation feedstocks studied for the production of PHA, vegetable oils gave the best yield because of the high carbon content in fatty acids [8]. Recently, because of the booming biofuel industry, glycerol is also becoming an attractive feedstock for fermentation. Bacteria that have the ability to use triglycerides and glycerol for cell growth and PHA production would be very useful. In previous studies, glycerol and triacylglycerol have been the subject of PHA production by Pseudomonas oleovorans NRRL B-14682, Pseudomonas corrugata 388, Pseudomonas resinovorans [10–12] and Burkholderia cepacia ATCC 17759 [13]. Here we report the identification of a new potential bacterium, Burkholderia sp. USM (JCM15050) with the ability to use vegetable oils, fatty acids and glycerol as the sole carbon sources for growth and PHA production. Previous studies reported the application of ultracentrifugation to study the biological macromolecules such as proteins [14], carbohydrates [15–17], and nucleic acids [18, 19]. Kalscheuer et al. [20] reported the isolation of lipid inclusions containing different specific densities using glycerol density gradient centrifugation. This method was also applied by Matsumoto et al. [21] as well as Loo and Sudesh [5] in separating different types of PHA granules. This idea has inspired the application of ultracentrifugation in separating the mixture of PHA-producing microorganisms. Conventional bacterial isolation involved several steps of serial dilutions prior to selecting pure colonies. This method has made the isolation procedure time-consuming. The idea of applying ultracentrifugation to partially separate the microorganisms from the mixed cultures arose from the basic concepts of the PHA densities that is dependent on existence of the type of monomers and thus affects the total densities of the cell biomass. However, to date, no study was reported about the application of ultracentrifugation in separating a mixture of microorganisms. In this study, the isolation procedure involved discrete sucrose density gradient ultracentrifugation. The resulting fractionation of microorganisms appeared as distinct bands in the sucrose gradient with denser microorganisms migrating furthest to the bottom of the tube. This is because the SCL monomers contain not only shorter side chain but also has been packed compactly in the PHA granules [5]. Conversely, MCL monomers possess longer side chain and exhibit steric effects, thus causing the polymer chain to arrange loosely in the PHA granules [5]. The difference in the packing of SCL and MCL polymer chains in the PHA granules is thought to contribute to their density difference. By using this method, we have successfully isolated a bacterium with the ability to convert various palm oil derivatives into PHA.
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Materials and Methods Cultivation of Mixed Microbial Culture Samples Oil polluted wastewater samples were collected from the drainage system of Seberang Prai industrial area, Penang, Malaysia. The natural microbial consortium in the samples was directly inoculated into nitrogen-limiting mineral salts medium with 1% (v/v) oleic acid as the sole carbon source. The mineral salts medium was prepared according to the method described previously [22]. A 1 mL of filter sterilized trace element and 0.25 g/L of MgSO47H2O were added aseptically into the mineral salts medium. The trace element solution consists of (per liter) 0.22 g CoCl26H2O, 9.7 g FeCl3, 7.8 g CaCl2, 0.12 g NiCl26H2O, 0.11 g CrCl36H2O, 0.16 g CuSO45H2O in 0.1 N HCl [9]. The pH of the medium was adjusted to 7.0. The culture was incubated at 30 °C, 200 rpm for 72 h to promote PHA accumulation. The wastewater sample was collected from the open environment during daytime where temperature was around 30 °C. Therefore, this temperature was applied during screening of PHA-producing microorganisms. After obtaining pure isolate, a series of bacterial identification tests were done to characterize the isolate, and at that time 37 °C was found to be the most optimum temperature for the growth of this isolate. Therefore, subsequent experiments were carried out at this temperature. Concentration of PHA-Containing Bacterial Strains Using Sucrose Density Gradient Ultracentrifugation Initially, the environmental sample was grown under PHAaccumulation conditions to promote the biosynthesis of PHA by the bacterial consortium in the sample. Subsequently, the cultivated cell suspension was centrifuged (4 °C, 92009g, 10 min) and the supernatant discarded. The cell pellet was then resuspended in 1 mL of mineral salts medium. The suspension was then subjected to ultracentrifugation by placing the suspension on sucrose density gradients. For this, an ultracentrifugation tube with 4 different sucrose concentrations, 1.0, 1.33, 1.67 and 2.0 M was prepared (Fig. 1). The resulting layers were subjected to ultracentrifugation at 4 °C, 280,0009g for 2 h [5]. After 2 h of ultracentrifugation, the fractionated bands of cell suspension were carefully withdrawn by pipetting out the layers of sucrose densities and cell suspension. The cell suspensions were then serially diluted and spread onto nutrient rich (NR) agar plate, containing (per liter) 10 g meat extract, 10 g peptone, 2 g of yeast extract and 15% of agar powder. The pH of the medium was adjusted to 7.0. The agar plates were incubated at 30 °C for 24 h. The resulting single colonies were picked and further purified
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Before
After
Cell suspension 1.0 M
1.33 M
1.66 M
2.0 M
Fig. 1 Ultracentrifugation tube with discrete sucrose gradient and cell suspension before and after ultracentrifugation. Sucrose density gradient ultracentrifugation was used to fractionate different types of microorganisms
by streaking on fresh NR plates until pure isolates were obtained. Identification of the Isolate Identification of the isolate was performed based on morphological observation, biochemical characterization, API 20NE and 16S rDNA analysis. Genomic DNA was extracted using G-spinTM Genomic DNA Extraction Kit (for Bacteria) (iNtRON Biotechnology, Inc., South Korea). Amplification of the 16S rDNA fragment was performed by PCR using universal primers 68F (50 -TNANACATGCAAG TCGAKCG-30 ) and 1392R (50 -ACGGGCGGTGTGTRC-30 ) [23]. The following PCR parameters were used: 94 °C for 2 min, 30 thermal cycles of 94 °C for 30 s, 58 °C for 30 s, 72 °C for 2 min; followed by 58 °C for 1 min and a final extension step at 72 °C for 2 min. Plasmids extraction, digestion of DNA with restriction endonucleases and transformation of Escherichia coli JM109 were carried out by standard procedures [24]. DNA sequencing analysis was performed using ABI 3730xl DNA Analyzer (Applied Biosystem Co., USA). The similarity and identity of the sequence obtained were compared to other sequences in the GenBank database using nucleotide-nucleotide BLAST command [25] in National Center for Biotechnology Information (NCBI). Clustal X was used for alignment purposes [26].
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Phylogenetic Analysis Nineteen 16S rDNA genes were utilized for phylogenetic analysis. These bacteria are PHA producing strains except for Burkholderia sp. isolate CRE 7 and Burkholderia cepacia strain B03. The PHA synthase protein sequences of these two strains were not found in the NCBI database. The 16S rDNA sequences (partial/full) were obtained from NCBI. Alignments and Bootstrap consensus tree using neighbor-joining program based on 500 replicates were generated using MEGA version 4 [27]. Carbon Sources Acidchem International Ltd. (Penang, Malaysia) and Unitata Ltd. (Perak, Malaysia) kindly provided crude palm kernel oil (CPKO), crude palm oil (CPO), palm olein (PO), palm kernel acid oil (PKAO), palm acid oil (PAO), palm stearin (PS), crude glycerol, glycerin pitch (GP), pure glycerol and palm fatty acid distillate (PFAD). Crude glycerol contained impurities including wastewater, spent catalysts, salts after neutralization, fat soap, free fatty acids (oleic acid and linoleic acid) as well as methanol from transesterification of biodiesel refineries [28–30]. The purity of glycerol in crude glycerol reportedly range from 65–85% [28]. Glycerine pitch is a waste generated from glycerol refining process, which contains
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55–65% of glycerol and other impurities such as diglycerol, fatty acids and inorganic salts [31, 32]. PFAD is obtained as a by-product of palm oil refining, which contains mainly unesterified fatty acids such as palmitic acid, oleic acid, linoleic acid and stearic acid [33]. PHA Biosynthesis of the Isolate The isolate was grown in NR medium for 24 h. Then, 1.5 mL of the resulting cell culture was transferred to nitrogen-limiting mineral salts medium containing 0.5% (v/v or w/v) of various carbon sources. The incubation was carried out in a temperature-controlled shaker for 72 h at 37 °C and 200 rpm to enable cell growth and PHA accumulation. Later the cells were harvested by centrifugation (92009g, 10 min, 4 °C), washed once with hexane and finally with distilled water. The cells were then lyophilized before subjecting to methanolysis and gas chromatography (GC) analysis. Analytical Methods Determination of PHA content and composition by GC were carried out as described by Braunegg et al. [34]. Lipase Activity Assay Lipase activity in the supernatant of the culture was determined using p-nitrophenol laurate (pNPL) as the substrate. Approximately 5 mmol/L of pNPL dissolved in 10 mL of dimethyl sulphoxide (DMSO) was emulsified in 90 mL of 100 mM phosphate buffer (pH 7.0) containing mixtures of 0.1% polyvinyl alcohol (PVA) and 0.4% Triton-X 100. Approximately 25 lL of the cell-free supernatant was mixed with the emulsion solution to make 2 mL of mixture and incubated for 10 min at 40 °C. The absorbance was measured by spectrophotometer at 410 nm. One unit of lipase activity was defined as the amount of enzyme required to release 1 lmol of p-nitrophenol per min at 40 °C.
Results and Discussion Isolation and Identification of the Bacterial Strains Oil polluted wastewater samples were chosen as the source of microorganisms in this study. We hypothesized that the presence of oil would favor the growth and multiplication of microbes that have the ability to metabolize oil, fatty acids and/or glycerol. The wastewater samples were inoculated directly into mineral salts medium containing oleic acid as the sole carbon source for growth and biosynthesis of PHA. The isolate described here was enriched and purified from cultures grown on oleic acid. Initially Nile Blue A was applied to detect the accumulation of PHA under fluorescence microscope. Sucrose density gradient ultracentrifugation is a method that is widely used to isolate PHA granules based on their density [6, 21]. It is known that the increase in the sidechain of PHA monomer from SCL to MCL would correspond to a decrease in the PHA granules densities, thus contributed to the differences in microbial cell densities. Microbial cells with different types of PHA granules may be fractionated and concentrated by this method. Figure 1 shows the result that was obtained. Bacterial cells containing P(3HB) granules were concentrated in the band between 1.67 and 2.0 M sucrose solutions. While other bands contained MCL-PHA-producing bacterial cells. The concentrated bacterial cells in each band were gently withdrawn and further purified to obtain pure cultures. A total of four different strains were observed frequently and isolated from the four bands. Upon obtaining pure cultures, each bacterium was grown separately in oleic acid as the sole carbon source under conditions that favor PHA accumulation. GC analysis revealed that the isolates from band one to band three produced MCL-PHA while the isolate obtained from band four produced P(3HB) (Table 1). The latter isolate was the subject of this study due to its ability to convert palm oil derivatives into high PHA content. The morphological and physiological characteristics of the isolate are summarized in Table 2. The
Table 1 PHA contents and compositions produced by bacteria from band 1 to band 4 using oleic acid as the sole carbon source Samples
DCW
PHA content (wt%)
(g/L)
PHA compositions (mol%) SCL 3HB
Band 1
1.3 ± 0.3
8±5
Band 2
1.4 ± 0.2
4±2
6
Band 3
1.0 ± 0.1
10 ± 4
38
Band 4
0.7 ± 0.1
48 ± 5
100
MCL 3HV
3HHx
3
25
5 10
3HO
3HD
3HDD: cis
3HDD
3HTD
10
47
4
9
2
60
22
2
4
1
37
12
1
2
3HB 3-hydroxybutyrate, 3HV 3-hydroxyvalerate, 3HHx 3-hydroxyhexanoate, 3HO 3-hydroxyoctanote, 3HD 3-hydroxydecanoate, 3HDD:cis 3-hydroxy-5-cis-dodecanoate, 3HDD 3-hydroxydodecanoate, 3HTD 3-hydroxytetradecanoate
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Table 2 Taxonomic characteristics of the isolate Characteristics Morphological characteristics Shape
Rod
Size (lm)
(0.5–0.9) 9 (1.0–2.0)
Motility
?
Gram staining
Negative
Features of colonies Shape
Circular
Opacity
Opaque
Elevation
Convex
Surface
Smooth, glistening
Edge Emulsifiability
Entire Forms uniformly turbid suspension in water
Color
Beige
Physiological characteristics Catalase
?
Oxidase
Oxidative
O/F test
?
Urease test
?
Gelatin liquefaction
-
Simmons citrate test
?
Arginine dihydrolase
-
Reduction of nitrate Growth temperatures
? 9–44 °C
Aerobic conditions
?
Nutritional characteristics Glucose
?
Fructose
?
Sucrose
?
Lactose
?
D-Arabinose
?
Raffinose
-
D-Xylose
?
Glycerol
?
Inositol
?
Mannitol
?
Sorbitol
?
D-Gluconate
?
Starch Vegetable oil
?
Morphological characteristics and features of the isolate were monitored on NR agar plate. Growth on carbohydrates and triglyceride was monitored on mineral salts medium. Other physiological characteristics were determined according to Mac Faddin [35]
isolate was grown in various types of carbohydrates and alcohols as the carbon source. It was found that this isolate was able to utilize most of the carbon sources, with better growth noticed in vegetable oils and glycerol.
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A series of biochemical test and also API 20NE were utilized to identify the isolate. The identification was then further performed with 16S rDNA analysis. A partial 16S rDNA sequence of 1349 bp was obtained by PCR. The BLASTX analysis revealed a 99% identity to partial sequence of 16S rRNA gene of Burkholderia sp. isolate CRE 7 (1347/1349) (accession no. U37340); followed by also a 99% identity to partial sequence of 16S rRNA gene of Burkholderia cepacia strain B03 (1346/1349) (accession no. DQ387437). The third closest identity was showed by 16S rRNA gene of Burkholderia vietnamiensis G4 with 99% identity (1346/1349) (accession no. CP000614). The sequence was deposited in the GenBank database with the accession number FJ667272. The isolate was found to be in the Burkholderia sp. Linage according to the phylogenetic tree generated (Fig. 2). The isolate was identified as a strain of Burkholderia cepacia according to all of the identification results. The isolate was then deposited in the Japanese Culture of Microorganisms under the code name Burkholderia sp. USM (JCM15050). Study of Growth Profile and P(3HB) Accumulation The ability of Burkholderia sp. USM (JCM15050) to utilize triglyceride, glycerol and fatty acids to produce PHA was investigated. As shown in Fig. 3, the growth profile of the isolate was studied in mineral medium containing CPKO as the sole carbon source. The cells grew rapidly after 12 h of adaptation and the cell biomass increased until about 36 h of cultivation. During the period of exponential growth, accumulation of P(3HB) increased gradually, amounting to 47 wt% of the dry cell weight at 24 h, 63 wt% at 36 h until it reached a maximum of 70 wt% at 72 h. Conversion of readily available carbon sources such as vegetable oil and its by-products into PHA has been the subject of studies in recent years. Burkholderia sp. is known as one of the most metabolically versatile bacterium [36], is able to secrete lipase to break down triglycerides [37] into fatty acids and glycerol. Therefore, the production of lipase by the isolate was also determined during the growth study. In the first 12 h, the lipase was induced and secreted into the culture medium. During the first 48 h, there was a steep increase in lipase activity until it reached a maximum of 117 U/mL, even though the residual oil concentration showed that CPKO was completely hydrolyzed at 24 h. Although the depletion of triglyceride was detected at 24 h, the accumulation of P(3HB) in the cells continued to increase until 36 h. Biosynthesis of PHA from Fatty Acids and Glycerol Derivatives by Burkholderia sp Table 3 shows the relative % (w/w) of fatty acids compositions in the samples used in this study. Palm kernel
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6
140
5
120 100
4
80 3 60 2
40
1
Lipase activity (U/mL)
Dry cell weight (g/L) P(3HB) concentration (g/L) Residual oil concentration (g/L)
Fig. 2 Neighbour-joining tree of 16S rDNA gene sequence similarity showing phylogenetic positions of Burkholderia sp. USM (JCM15050) (FJ667272), Burkholderia sp. isolate CRE 7 (U37340), B. cepacia strain B03 (DQ387437), PHA-producing bacteria: B. vietnamiensis G4 (CP000614), B. cenocepacia MC0-3 (CP000958), B. ambifaria MC406 (CP001025), B. multivorans ATCC 17616 (AP009385), B. pseudomallei K96243 (BX571966), B. mallei NCTC 10229 (CP000546), R. eutropha H16 (AM260479), A. vinosum DSM 180 (FM178268), A. vinelandii (AB175657), P. aeruginosa PAO1 (AE004091), P. mendocina ymp (CP000680), P. oleovorans (D84018), P. corrugata (AF348508), P. putida KT2440 (AE015451), P. fluorescens Pf0-1 (CP000094) and B. megaterium (AB334764). Numbers at nodes represent the levels of bootstrap support based on analyses on 500 replicates. Bar: 0.01 substitutions per site
20
0 0
12
24
36
48
60
72
84
0 96
Time (h)
Fig. 3 Time profiles of growth and lipase activity for growthassociated P(3HB) synthesis by Burkholderia sp. grown in nitrogenlimiting MM containing 0.5% (v/v) of CPKO and 0.5 g/L of NH4Cl at 37 °C. (filled diamond) Dried cell weight (DCW); (filled triangle) P(3HB) concentration; (cross) Residual oil concentration; (filled square) Lipase activity
extracts consisted of higher concentrations of lauric acid (C12) and myristic acid (C14), whereas high concentration of palmitic acid (C16) was found in mesocarp extracts. The effect of different fatty acids on cell growth and PHA production were determined by feeding the cells with individual fatty acid as the sole carbon source. We also determined the various concentrations of fatty acids to investigate the level of tolerance of the fatty acids by the
isolate. Table 4 shows that neither caproic acid (C6) nor caprylic acid (C8) was able to support the growth of the isolate at the concentrations of more than 0.1% (v/v). Capric acid (C10) could not at all support the cell growth even at 0.1% (v/v). In contrast, with lauric acid and myristic acid, Burkholderia sp. USM (JCM15050) produced relatively high amount of P(3HB) whereby 69 and 38 wt% respectively were produced at concentrations of 0.5% (w/v). Therefore, higher concentrations of lauric acid and myristic acid in PKO, CPKO and PKAO were believed to contribute to better cell growth and P(3HB) production. Unsaturated fatty acid such as oleic acid (C18:1) contributed to the production of P(3HB) but had an adverse effect on the cell biomass. Saturated stearic acid (C18:0) and unsaturated oleic acid produced significantly different amount of P(3HB), whereby the former resulted in the production of less than 1 wt% while the latter produced 48 wt% P(3HB). It is reported that the toxicity level of the fatty acid is related to the concentration of the unionized form of the fatty acid and the size of the carbon chain [38]. Indeed the fatty acids with shorter n-alkyl chains exhibit higher toxicity than the longer chains [39]. On the other hand, longchain fatty acids were preferentially acted as the substrates for phospholipids acylation reactions [40, 41] during high rate of b-oxidation metabolism at exponential growth phase. Another lipase-hydrolyzed compound, glycerol could also support both the cell growth and P(3HB) accumulation. Glycerol derivatives which varied in the degree of purities were used as the sole carbon source. The results showed that the P(3HB) content was 54 wt% in 2.5 g/L of dry cell weight when fed with pure glycerol (Table 5). However, other by-products such as crude glycerol and glycerine pitch (GP), only achieved 31 and 22 wt% of P(3HB) content respectively. P(3HB) concentration obtained from glycerol (1.35 g/L) was almost similar to those obtained from CPKO (1.54 g/L) and lauric acid (1.17 g/L). The P(3HB) contents obtained from crude glycerol, glycerine pitch and PFAD were lower than that obtained from pure glycerol. This is attributed to the fact that these by-products contain different impurities of approximately 12–15% methanol and 23–25% soap as a product from transesterification of biodiesel refineries [28]. Glycerol is metabolized via glycolysis pathway. Initially, glycerol is first converted into glycerol-3-phosphate with the aid of ATP molecule to activate the reaction. Then it is followed by the conversion to dihydroxyacetone phosphate before entering the glycolysis pathway to generate acetyl-CoA and followed by three step P(3HB) synthesis pathway. Besides Burkholderia sp., glycerol derivatives were also tested independently for their ability to support cell growth and PHA production by Cupriavidus necator H16. Table 5
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Table 3 Relative % (w/w) fatty acid compositions of crude palm oil, palm stearin, palm olein, palm acid oil, crude palm kernel oil, palm kernel oil and palm kernel acid oil Fatty acids
Saturated
Unsaturated
Caproic (6:0)
Caprylic (8:0)
Capric (10:0)
CPO
–
–
–
PS
–
–
–
PO
–
–
–
PAO
–
Lauric (12:0) 0.1 0.2
Myristic (14:0)
Palmitic (16:0)
Stearic (18:0)
Arachidic (20:0)
Palmitoleic (16:1)
Oleic (18:1)
Linoleic (18:2)
Linolenic (18:3)
0.9
43.8
4.0
0.4
–
42.1
8.9
0.2
1.1
58.9
4.4
–
–
27.7
8.1
–
1.0
35.8
4.1
0.4
0.1
43.8
14.3
0.2
–
–
0.8
1.1
44.7
3.7
0.4
–
40.3
9.4
–
CPKO –
3.0
3.5
48.5
16.2
7.5
2.6
–
–
15.7
2.1
–
PKO
4.0
3.6
48.3
15.5
8.0
2.1
–
–
15.4
2.6
–
2.0
2.5
44.1
17.8
10.8
3.1
–
–
17.3
2.3
–
0.5
PKAO –
Source: Unitata Ltd CPO crude palm oil, PS palm stearin, PO palm olein, PAO palm acid oil, CPKO crude palm kernel oil, PKO palm kernel oil, PKAO palm kernel acid oil
Table 4 Biosynthesis of P(3HB) from various fatty acids as the sole carbon sources using one-stage cultivation nitrogen-limiting MM by the isolate Burkholderia spa Concentrations of fatty acids
0.1% (v/v) or (w/v) Dry cell weight (g/L)
P(3HB) content (wt%)
Caproic (6:0)
0.7 ± 0.3
Caprylic (8:0) Capric (10:0) Lauric (12:0)
1.4 ± 0.2
0.3% (v/v) or (w/v) b
0.5% (v/v) or (w/v) Dry cell weight (g/L)
P(3HB) contentb (wt%)
ND
NG
ND
NG
ND
NG
ND
NG
ND
NG
ND
2.1 ± 0.1
49 ± 3
1.7 ± 0.1
69 ± 7
Dry cell weight (g/L)
P(3HB) content (wt%)
Tr
NG
0.8 ± 0.1
Tr
NG
ND 8±1
b
Myristic (14:0)
1.1 ± 0.3
1 ± 0.2
1.6 ± 0.1
49 ± 2
1.9 ± 0.2
38 ± 4
Palmitic (16:0)
0.6 ± 0.2
Tr
1.2 ± 0.1
3±1
1.5 ± 0.2
9±6
Stearic (18:0)
0.5 ± 0.1
1 ± 0.1
1.0 ± 0.3
1 ± 0.8
0.9 ± 0.3
Tr
Oleic (18:1)
1.0 ± 0.1
1 ± 0.2
1.4 ± 0.2
40 ± 2
0.7 ± 0.1
48 ± 5
a
Cultivation at 37 °C for 72 h, pH 7.0, 200 rpm
b
P(3HB) content in freeze-dried cells Tr PHA content less than 1 wt%, NG no growth, ND not determined
shows that palm kernel and palm mesocarp derivatives could readily support the growth and P(3HB) accumulation of C. necator H16. Approximately, 71 wt% P(3HB) of the dry cell weight was obtained. Glycerol, which supported the growth and P(3HB) production of Burkholderia sp. USM (JCM15050), however, was less preferred by C. necator H16, whereby, only 30 wt% of P(3HB) was attained (Table 5). Biosynthesis of PHA from Various Palm Oil Products by Burkholderia sp A total of nine types of palm oil products were tested for the synthesis of PHA by this isolate. This strain was capable of incorporating P(3HB) homopolymer regardless
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of the type of triglycerides fed. Among the various triglycerides tested, CPKO supported the best synthesis of P(3HB), amounting to 70 wt% of the dry cell weight using one-stage cultivation. Table 5 shows that products derived from palm kernel such as CPKO and PKAO contributed to higher P(3HB) accumulation rather than PO, PS, CPO and PAO, which products derived from palm mesocarp. Overall, the dry cell weight from all the carbon sources screened was in the range of 1.7–2.6 g/L with P(3HB) content ranging from 41–70 wt%. In this study, although the P(3HB) contents gained from glycerol derivatives were not as high as that from CPKO, Burkholderia sp. USM (JCM15050) demonstrated good versatility in the choice of palm oil derived carbon sources for both cell growth and P(3HB) production.
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Table 5 Biosynthesis of P(3HB) from various types of palm oil and glycerol as the sole carbon source using one-stage cultivation of nitrogenlimiting MM by Burkholderia sp. USM (JCM15050)a and C. necator H16 Carbon sources (0.5%) (v/v) or (w/v)
Burkholderia sp. USM (JCM15050)
Cupriavidus necator H16 b
Dry cell weight (g/L)
P(3HB) contentb (% wt)
Source
Dry cell weight (g/L)
P(3HB) content (% wt)
CPKOc
2.2 ± 0.4
70 ± 3
5.0
67
[42]
PKAOc
1.9 ± 0.2
58 ± 4
4.7 ± 0.3
42 ± 2
[43]
POc
2.4 ± 0.1
46 ± 6
5.2
70
[42]
PSc
2.4 ± 0.3
52 ± 3
–
–
–
PSd
1.9 ± 0.1
41 ± 1
–
–
–
CPOc
1.8 ± 0.2
63 ± 6
4.6
75
[42]
c
1.8 ± 0.1
57 ± 1
4.5 ± 0.2
31 ± 2
[43]
Glycerolc
2.0 ± 0.4
60 ± 4
1.3 ± 0.3
33 ± 3
This study
Glycerold
2.5 ± 0.4
54 ± 4
1.8 ± 0.1
31 ± 3
This study
Crude glycerol
1.9 ± 0.2
31 ± 3
1.2 ± 0.2
34 ± 4
This study
1.9 ± 0.1
22 ± 3
1.3 ± 0.2
18 ± 1
This study
1.3 ± 0.3
43 ± 3
1.9 ± 0.5
47 ± 1
This study
Palm kernel
Palm mesocarp
PAO
Glycerol derivatives
GP Fatty acid derivative PFAD a
Cultivation at 37 °C for 72 h, pH 7.0, 200 rpm
b
P(3HB) content in freeze-dried cells
c
Source from Unitata Ltd
d
Source from Acidchem Ltd
CPKO crude palm kernel oil, PO palm olein, PKAO palm kernel acid oil, PS palm stearin, CPO crude palm oil, PAO palm acid oil, Glycerol pure glycerol, GP glycerine pitch, PFAD palm fatty acid distillate Acknowledgements This material is based upon work supported by the Malaysia Toray Science Foundation. The authors would like to thank Teoh Chai Sin and Chan Chin Keong for assisting with the experimental works. Generous supply of palm oil products by Acidchem International Ltd. and Unitata Ltd. is gratefully acknowledged.
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