UTILIZATION OF MUSTARD OIL FOR THE PRODUCTION OF POLYHYDROXYALKANOATES BY Pseudomonas

aeruginosa

Hasnain Javed*1, Nazia Jamil

Address(es): Mr. Hasnain Javed (Corresponding author)
1
Universty of the Punjab, faculty of Life sciences, Department of Microbiology and Molecular Genetics, Quaid-e-Azam Campus Lahore-54590, Pakistan.

*Corresponding author: hasnain_javed@hotmail.com doi: 10.15414/jmbfs.2015.4.5.412-414

ARTICLE INFO ABSTRACT

Received 10. 12. 2014 With the unnecessary use of plastics and cumulative pressure being placed on capacities available for plastic waste disposal, the need for
Revised 15. 1. 2015 biodegradable plastics and biodegradation of plastic wastes has assumed increasing importance in the last few years. Bioplastic
Accepted 16. 2. 2015 production from mustard oil was considered relatively cheap, easily available, included in vegetable oil and don’t having much volatile
Published 1. 4. 2015 characteristics. Total of 67 bacterial strains were isolated and purified from different regions of the Pakistan, and were checked for
Polyhydroxyalkanoates (PHA) production by Sudan black and Nile blue staining. Quantitative analysis for biodegradable plastic
produced by different bacterial species was performed by Modified surfactant hypochlorite method. High PHA production was detected
Regular article
in 35 strains belonging to different genera including Pseudomonas, Staphylococcus, Escherichia and Enterobacter. Fermentation and
PHA production was done in batch culture. The PHA production of P. aeruginosa by mustered oil cultivation was studied under six
experimental conditions, such as air flow rates, pH, Temperature, optical density, substrates concentration and cell dry weight. PHA
production of Pseudomonas species were subsequently authenticated at molecular level by PCR amplifications and sequence analysis.
PHA polymerase 1 (PhaC1) and PHA polymerase 2 (PhaC2) from Pseudomonas aeruginosa were amplified, sequenced and submitted
to gene bank.

Keywords: Polyhydroxyalkanoates, Pseudomonas aeruginosa, Mustard oil, batch culture fermentation

INTRODUCTION on production cost (Lau et al., 2014). So to reduce the bioplastic production cost
as compared to the production cost of synthetic plastics, there is an urgent need to
PHAs are recognized as biodegradable plastics as they possess material find the cheaper media with necessary requirements and cheaper carbon source
properties similar to synthetic thermoplastics and are easily degraded to water for the PHA (Allen et al., 2010). More than 59 PHA synthase genes have been
and carbon dioxide (and methane under anaerobic conditions) by enzymatic cloned from 45 bacterial species and broadly characterized into four different
action of microbes in various environments such as soil, sea water, lake water, classes, based on their in vivo substrate specificities, amino acid sequences, and
and sewage (Kung et al., 2007; Chaudhry et al., 2011). Synthetic plastics, subunit composition (Tajima et al., 2012). Class I and class II PHA synthases
although are non-degradable and pose one of the major causes of environmental comprise enzymes consisting of only one type of subunit phaC (Yang et al.,
pollution (Bhuwal et al., 2013), are used because the use of plastics cannot be 2013). Pseudomonas is highly stress resistant bacteria and often isolated from
eradicated from our daily life at all. To solve the current problem of different minimal nutrient environments (Caton et al., 2014). The class II PHA
environmental pollution, interests have been shifted to the development of synthase genes phaC1 and phaC2 that favor mcl 3PHA have been identified and
bioplastics because they provide dual benefits of utilizing the waste and cost- characterized in Pseudomonas sp. including Pseudomonas aeruginosa),
effective production of biodegradable plastic (Ramezani et al., 2014). Pseudomonas oleovorans (Tan et al,. 2010). The different studies showed that
For the commercialization of PHA, inexpensive carbon sources are required. both of them exhibit very similar properties (Rehman et al., 2007).
Therefore, research efforts are abound to exploit inexpensive raw materials as Objective of our study was to find out cost effective substrate for PHA
substrates in order to reduce the high cost of PHAs production (Iles and Martin, production. For this purpose, soil and waste water isolates were characterized,
2013). Different efforts are in process to reduce the PHA production cost (Wang their PHA production ability with different carbon sources was optimized and
et al., 2014). A good candidate for PHAs production would be a bacteria that can molecular characterization of phaC, phaC1 and phaC2 genes were done. After
store high level of PHAs using inexpensive substrate. Numerous inexpensive that Mustard oil was used as carbon source in lab scale fermenter and PHA
carbon substrates such as molasses, whey, plant oils etc. can be excellent production was compared with Glucose.
substrates for bacteria to produce PHAs, which could lead to substantial
economic benefits (Ciesielski et al., 2014). In an attempt to reduce production MATERIAL AND METHODS
costs in the recent years, focus has shifted to the use of less valuable, renewable
materials as substrates for PHA production, as soy molasses (Solaiman et al., Sampling and growth of PHA producing bacteria
2006), wheat-based co-products (Koutinas et al., 2007), refined and crude
glycerol (Cavalheiro et al., 2009; Zhu et al., 2010; Ashby et al., 2011). Soil and waste water samples were collected from different areas of Pakistan and
The isolation and the purification of bacterial PHAs are the key step of the were plated on PHA detection media (PDA) containing 2 g/l (NH4)2SO4, 13.3g/l
process profitability in the fermentation system (Khan et al., 2013). Detailed KH2PO4, 1.2 g/l MgSO4.7H2O, 1.7g/l citric acid, trace element solution 10ml/l,
studies have been carried out to investigate that which fermentation system i.e. 5µg/ml Nile blue, 2% carbon source (glucose or mustard oil), incubation was
batch, fed-batch or continuous fermentations is efficient for the desired PHA. The given at 37oC for 24 to 48 hours.
ideal method should lead to a high purity and recovery level at a low production
cost. To achieve the volumetric productivity and to develop a comparatively Microscopy for PHA detection
economically competitive method for the production of PHAs, various factors are
very important like choice of bacterial strain, optimized conditions and most PHA granules or PHA inclusion bodies were detected in the cells using Sudan
importantly the choice of media and carbon sources which have direct influence Black staining method in which heat fixed bacterial smear (from bacterial strain

412
 J Microbiol Biotech Food Sci / Javed and Jamil 2015 : 4 (5) 412-414

grown in PHA accumulating conditions) was treated with 0.3% Sudan Black B PHA production using different carbon sources
(in ethylene glycol) for 5- 15 minutes. Then washed with several times dipping in
xylene and counter stained with safranine and rinsed. Using the batch culture conditions PHA producing strain was grown in 5 liter lab
scale fermenter of Bioengineering Company using glucose and mustard oil as the
Time profiling and PHA extraction carbon source in two different reactions. Comparisons of PHA production and
growth and survival conditions were noted by taking samples out of the fermenter
Time profiling of PHA production was also done by taking the sample out in with the regular intervals of 2 hours for 3 consecutive days.
regular intervals of 2 hours (0-72 hours) and total biomass weight was measured
by taking optical density of sample at 600nm. PHA producer Bacterial cells RESULTS AND DISCUSSION
grown in PDA medium (liquid culture) for 72 hours were collected by
centrifugation and biomass was re-suspended in 1:1 ratio of 0.4 % sodium Isolation and characterization of PHA producing bacterial strains:
hypochlorite for one hour and then dissolved in chloroform. PHA granules were
collected by centrifugation and the pellet was washed with alcohol and A total of 67 bacterial strains were isolated from 15 different samples collected
chloroform. The percentage of PHA in bacterial strain was noted with the from various areas of Pakistan. Out of these 27 samples showed positive results
formula: for PHA production in PDA media contacting Nile blue (Figure 1A) and
PHA% = weight of PHA/ weight of biomass × 100 confirmed production by microscopic analysis with Sudan Black staining (Figure
1B).
Isolation and molecular characterization of DNA

For molecular characterization genomic and plasmid DNA was extracted using
phenol chloroform method and was subjected to 16s rRNA sequencing. DNA
was subjected to amplification with forward primer 16S-3,
CCCGGGAACGTATTCACCG and reverse primer 16S-5,
GCYTAAYACATGCAAGTCGA (Chaudhry et al., 2011) under the conditions
like denaturation at 95oC, annealing at 52oC and extension at 72oC. Gene
sequencing was done with chain termination method and sequence alignment was
checked using NCBI BLAST. A B
Figure 1 Characterization of PHA producing bacterial strains. 1A: Nile blue
Identification and amplification of phaC gene from PHA producers staining. 1B: Strains positive for PHA production showing granules on sudan
black staining.
The 16S rRNA gene was sequenced at the Center for Excellence in Molecular
Biology, (Pakistan) by Sanger sequencing method. The phaC polymerase gene Biochemical characterization of all PHA producing stains was done and genus
was amplified using forward primer 179–L and the reverse primer 179–R identification was done as Staphylococci (07), Enterobacter (04), Klebsiella (02),
(Chaudhry et al., 2011). PCR was carried out by denaturing DNA at 110°C for Pseudomonas (11), and Bacillus (05) (Table 1). Pseudomonas strains showed
10 min followed by 30 cycles of amplification (95°C for 2 min, 56°C for 1 min high percent PHA production and better growth compared to all other strains and
and 72°C for 2 min). The phaC1 and phaC2 gene extensions of phaC were also their 16s rRNA ribotyping was done. Different bacterial strains have been noted
amplified using the ORF primers for Pseudomonas denaturation at 95oC, to form these biopolymers but there is need to minimize the production and
annealing temperature 58oC and extension at 72oC for 2 minutes each. bacterial cultivation cost to get more advantage from these polymeric substances.
Strain GS1 showed homology to P. auregenosa on the basis of ribotyping. The
16S rRNA gene partial sequence was submitted to the NCBI database under the
accession number HM598296.

Table 1 Genus identification of strains found positive for biopolymers production

Sample Strain Sample Strain
Area Genus Area Genus
type code type code
LW2 Staphylococcus LS2 Staphylococcus
Lahore LW4 Escherichia Lahore LS3 Bacillus
LW5 Pseudomonas LS6 Bacillus
GW1 Staphylococcus GS1 Pseudomonas
Gujrat GW3 Pseudomonas Gujrat GS4 Klebsiella
GW5 Staphylococcus GS6 Pseudomonas
KW2 Klebsiella KS1 Enterobacter
Water Kasoor Soil Kasoor
KW5 Pseudomonas KS3 Pseudomonas
JW2 Pseudomonas Jehlum JS1 Pseudomonas
Jehlum
JW3 Bacillus BS2 Pseudomonas
Bhakkar
BW1 Escherichia BS4 Staphylococcus
Bhakkar
BW2 Pseudomonas SS1 Bacillus
Sheikhupura SW1 Pseudomonas Sheikhupura SS2 Staphylococcus
- - - SS4 Bacillus

Optimization of PHA production in different carbon sources

Our aim was to get the production of PHA using cost-effective sources of carbon.
Using glucose and mustard oil as carbon sources, the PHA production was
checked and growth conditions were optimized in separate reactions. Figure 2
shows the results for the PHA production and its percent in the cell dry weight at
different time intervals. This is in agreement with (Ng KS et al., 2010) and
(Rehman et al., 2007) as linear polyesters- polyhydroxyalkanoates are produced
naturally by bacteria as the reserve materials for carbon and energy; along with
this these are used as the source of bio-plastics. High carbon to nitrogen ratio,
lack of certain macro and trace elements during growth, and excess of carbon
cause certain microorganisms to store energy in this form. As these biopolymers
are biodegradable so they are getting important in agriculture, economy and
health sciences. Plant oils are desirable feedstock for PHAs production because
they are relatively cheap as compared to most sugars (Le Meur et al., 2012). In
search of inexpensive carbon source, mustard oil was chosen in this study as it is Figure 2 Comparison of PHA percentage (PHA %) of two carbon sources
cheap, easily available and less volatile. (Glucose and Mustard oil) from GS1 strain

413
 J Microbiol Biotech Food Sci / Javed and Jamil 2015 : 4 (5) 412-414

Characterization of phaC gene from strain GS1 CHAUDHRY, W.N., JAMIL, N., ALI, I., AYAZ, M.H., HASNAIN, S. 2011.
Screening for polyhydroxyalkanoate (PHA)-producing bacterial strains and
From PHA producing strain GS1 which was Pseudomonas aeruginosa, phaC comparison of PHA production from various inexpensive carbon sources. Annals
gene was amplified and 540 bp product was successfully sequenced and of microbiology, 61(3), 623-629. http://dx.doi.org/10.1007/s13213-010-0181-6
submitted to NCBI gene bank under the accession number HM598297. Results ILES, A., MARTIN, A.N.2013. Expanding bioplastics production: sustainable
indicated the homology with the Pseudomonas aeruginosa gene for PHA business innovation in the chemical industry. Journal of cleaner production, 45,
production. Along with this using ORF primers for Pseudomonas region adjacent 38-49. http://dx.doi.org/10.1007/s13213-010-0181-6
to phaC was amplified named phaC1 and phaC2 (Figure. 3) and submitted to LE, M., SYLVAINE, M.Z., THOMAS, E., LINDA, T-M, QUN, R. 2012.
NCBI gene bank under the accession number HM598298. Sequencing results Production of medium-chain-length polyhydroxyalkanoates by sequential feeding
indicated the homology of this region with the phaC flanking regions of the of xylose and octanoic acid in engineered Pseudomonas putida KT2440. BMC
operon. biotechnology, 12(1), 53. http://dx.doi.org/10.1186/1472-6750-12-53
KHAN, A.B., MEHMOOD, I.K., OMER, M.T., FARZANA, H., KHALID, J.,
ARFA, Y., SHOUKAT, P. 2013. Separation of polyhydroxyalkanoates-
producing bacterial strains using PHA synthase gene and their evaluation for
PHA deposition. Brazilian Archives of Biology and Technology, 56(4), 645-652.
http://dx.doi.org/10.1590/s1516-89132013000400015
KOUTINAS, A.A., XU Y, W. R., WEBB, C. 2007. Polyhydroxybutyrate
production from a novel feedstock derived from a wheat-based biorefinery.
Enzyme and microbial technology, 40(5), 1035-
1044. http://dx.doi.org/10.1016/j.enzmictec.2006.08.002
KUNG, S.S., CHUANG, Y.C., CHEN, C.H., CHIEN, C.C. 2007. Isolation of
polyhydroxyalkanoates‐producing bacteria using a combination of phenotypic
and genotypic approach. Letters of Applied Microbiology, 44(4), 364-
371. http://dx.doi.org/10.1111/j.1472-765x.2006.02090.x
LAU, N. S., CH'NG, D. H. E., CHIA, K. H., WONG, Y. M., & SUDESH, K.
2014. Advances in Polyhydroxyalkanoate (PHA): Unraveling the Development
and New Perspectives. Journal of Biobased Materials and Bioenergy, 8(2), 118-
129. http://dx.doi.org/10.1166/jbmb.2014.1418
NG, K.S., OOI, W.Y., GOH, L.K., SHENBAGARATHAI, R., SUDESH, K.
2010. Evaluation of jatropha oil to produce poly (3-hydroxybutyrate) by
Cupriavidus necator H16. Polymer Degradation and Stability, 95(8), 1365-1369.
http://dx.doi.org/10.1016/j.polymdegradstab.2010.01.021
REHMAN, S., JAMIL, N., HUSNAIN, S. 2007. Screening of different
Figure 3 PCR amplification of genes. (a) Lane 1 is indicating gene ruler (1kb) contaminated environments for polyhydroxyalkanoates-producing bacterial
while lane 2,3 and 4 showing phaC (540bp), phaC1 (1700bp) and 16s rRNA strains. Biologia, 62(6), 650-656. http://dx.doi.org/10.2478/s11756-007-0144-y
(1500bp) gene respectively (b) Lane 1 is showing phaC 2 gene and lane 2 is RAMEZANI, M., AMOOZEGAR, M.A., VENTOSA, A. 2014. Screening and
showing gene ruler. comparative assay of poly-hydroxyalkanoates produced by bacteria isolated from
the Gavkhooni Wetland in Iran and evaluation of poly-β-hydroxybutyrate
CONCLUSION production by halotolerant bacterium Oceanimonas sp. GK1. Annals of
Microbiology, 1-10. http://dx.doi.org/10.1007/s13213-014-0887-y
Our strain GS1 gave the maximum yield of PHA in lab scale fermenter when all SAMBROOK, J., RUSSELL, D.W. 2001. Molecular Cloning: A Laboratory
the conditions like as air flow rates, pH, Temperature, Optical density, substrates Manual. Cold Spring Harbor Laboratory Press.
concentration and dry cell weight were under controlled. PHA production was at http://dx.doi.org/10.1002/jobm.3620300824
its peak at 30th hour of fermentation process and it produced 9.01 gram/liter of SOLAIMAN, D.Y., ASHBY, R., HOTCHKISS, A.J.R., FOGLIA, T. 2006.
PHA (20.1 %PHA). So it is concluded that Mustard oil is also cheap carbon Biosynthesis of Medium-chain-length Poly(hydroxyalkanoates) from Soy
source for bioplastic production as it is easily available in South Asia and its use Molasses. Biotechnology letters, 28, 157-162. http://dx.doi.org/10.1007/s10529-
can lead to bio-plastic production at commercial level. 005-5329-2
TAJIMA, K., HAN, X., SATOH, Y., ISHII, A., ARAKI, Y., MUNEKATA, M.,
Acknowledgement: It is acknowledged that this research work was supported by TAGUCHI, S. 2012. In vitro synthesis of polyhydroxyalkanoate (PHA)
International Foundation for Science (IFS) Sweden and University of the Punjab incorporating lactate (LA) with a block sequence by using a newly engineered
Lahore, Pakistan. thermostable PHA synthase from Pseudomonas sp. SG4502 with acquired LA-
polymerizing activity. Applied microbiology and biotechnology, 94(2), 365-376.
REFERENCES http://dx.doi.org/10.1007/s00253-011-3840-z
TAN, Y., NEO, P. C., NAJIMUDIN, N., SUDESH, K., MUHAMMAD, T. S. T.,
ALLEN, A. D., ANDERSON, W. A., AYORINDE, F. O., & ERIBO, B. E. 2010. OTHMAN, A. S., SAMIAN, R. 2010. Cloning and characterization of poly (3‐
Biosynthesis and characterization of copolymer poly (3HB-co-3HV) from hydroxybutyrate) biosynthesis genes from Pseudomonas sp. USM 4‐55. Journal
saponified Jatropha curcas oil by Pseudomonas oleovorans. Journal of industrial of basic microbiology, 50(2), 179-189.
microbiology & biotechnology, 37(8), 849-856. http://dx.doi.org/10.1002/jobm.200900138
http://dx.doi.org/10.1007/s10295-010-0732-7 WANG, Y., YIN, J., CHEN, G.Q. 2014. Polyhydroxyalkanoates, challenges and
ASHBY, R.D., SOLAIMAN, D.K., AND STRAHAN, G.D. 2011. Efficient opportunities. Current opinion in biotechnology, 30, 59-65.
utilization of crude glycerol as fermentation substrate in the synthesis of poly (3- http://dx.doi.org/10.1016/j.copbio.2014.06.001
hydroxybutyrate) biopolymers. Journal of American oil chemist’s society, 88(7) YANG, C., WEI, Z., RUIHUA, L., CHI, Z., TING, G., QIANG, L.I.,
949-959. http://dx.doi.org/10.1007/s11746-011-1755-6 SHUFANG, W., CUNJIANG, S. 2013. Analysis of polyhydroxyalkanoate (PHA)
BHUWAL, A.K., SINGH, G., AGGARWAL, N.K., GOYAL, V., YADAV, A. synthase gene and PHA‐producing bacteria in activated sludge that produces
2013. Isolation and Screening of Polyhydroxyalkanoates Producing Bacteria PHA containing 3‐hydroxydodecanoate. FEMS microbiology letters, 346(1), 56-
from Pulp, Paper, and Cardboard Industry Wastes. International journal of 64. http://dx.doi.org/10.1111/1574-6968.12201
biomaterials. http://dx.doi.org/10.1155/2013/752821 ZHU, C., NOMURA, C.T., PERROTTA, J.A., STIPANOVIC, A.J., NAKAS,
CATONE, M. V., RUIZ, J. A., CASTELLANOS, M., SEGURA, D., ESPIN, G., J.P. 2010. Production and characterization of poly-3-hydroxybutyrate from
& LÓPEZ, N. I. 2014. High Polyhydroxybutyrate Production in Pseudomonas biodiesel‐glycerol by Burkholderia cepacia ATCC 17759. Biotechnology
extremaustralis Is Associated with Differential Expression of Horizontally Progress, 26, 424-430. http://dx.doi.org/10.1002/btpr.355
Acquired and Core Genome Polyhydroxyalkanoate Synthase Genes. PloS
one,9(6), e98873. http://dx.doi.org/10.1371/journal.pone.0098873
CAVALHEIRO, J.M., DE ALMEIDA, M., GRANDFILS, C., DA FONSECA,
M. 2009. Poly (3-hydroxybutyrate) production by Cupriavidus necator using
waste glycerol. Process Biochemistry. 44(5), 509-515.
http://dx.doi.org/10.1016/j.procbio.2009.01.008
CIESIELSKI, S., MOŻEJKO, J., PISUTPAISAL, N. 2014. Plant oils as
promising substrates for polyhydroxyalkanoates production. Journal of Cleaner
Production. http://dx.doi.org/10.1016/j.jclepro.2014.09.040

414