Synthesis kinetics of poly(3-hydroxybutyrate) by using a Pseudomonas aeruginosa mutant strain grown on hexadecane

International Biodeterioration & Biodegradation 115 (2016) 171e178

Contents lists available at ScienceDirect

International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod

Synthesis kinetics of poly(3-hydroxybutyrate) by using a Pseudomonas aeruginosa mutant strain grown on hexadecane Zulfiqar Ali Raza a, *, Sharjeel Abid a, Asma Rehman b, Tanveer Hussain a a b

Chemistry Research Laboratory, National Textile University, Faisalabad, 37610, Pakistan Industrial Biotechnology Division, National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan

a r t i c l e i n f o

a b s t r a c t

Article history: Received 4 July 2016 Received in revised form 8 August 2016 Accepted 18 August 2016

Polyhydroxyalkanoates (PHAs) are eco-friendly bio-polyesters which are produced in many microorganisms and plants. In this study, hexadecane was utilized as sole carbon source for the biosynthesis and optimization of PHAs in a gamma ray mutant of Pseudomonas aeruginosa in minimal salts media. Up to six days, various process aspects were checked to achieve the high possible yield of PHAs on hexadecane in minimal salts media. The batch fermentation produced 1.5 g l
1 cell dry mass (CDM) and upto 40.66% (w w
1) PHAs accumulation. The different production yield values of YX/S, YP/S and YP/X were 0.242, 0.098 and 0.41 g g
1, respectively. Fourier transform infrared spectra were used to confirm and characterize the produced PHA. The LC-MS analysis of the PHA showed that produced copolymer had major molecular mass of m z
1 448.5 representing the presence of polyhydroxybutyrate. © 2016 Elsevier Ltd. All rights reserved.

Keywords: Hexadecane Polyhydroxyalkanoates Pseudomonas aeruginosa Shake flask

1. Introduction Polyhydroxyalkanoates (PHAs) are gaining interest over typical oil-based plastics due to their extraordinary properties (Verlinden et al., 2007). Conventional plastics have bad impact on the environment regarding their unsafe disposal (Chee et al., 2010; Verlinden et al., 2007). Biopolymers, specifically PHAs, have emerged as the answer to these problems (Lee et al., 2008). When certain prokaryotic microorganisms face unbalanced nutrition conditions, they assimilate carbon source and convert it into hydroxyalkanoates which are then polymerized in vivo to high molecular weight polymers known as PHAs (Loo and Sudesh, 2007). The PHAs can have short or medium lengths depending on the length of carbon chain (Hazer and Steinbuchel, 2007). The composition on the monomeric level of PHAs depends on many factors like; metabolic route for PHA synthesis, carbon source adaptation and degradation efficiency of microorganism, the specificity of enzyme system which synthesize PHA (Ward and O'Connor, 2005) and prokaryotic microorganism (Jaeger et al., 1995; Koller et al., 2010). More than 300 prokaryotic microorganisms have been reported to accumulate PHAs (Chee et al., 2010; Choi et al., 1998).

* Corresponding author. E-mail address: [email protected] (Z.A. Raza). http://dx.doi.org/10.1016/j.ibiod.2016.08.005 0964-8305/© 2016 Elsevier Ltd. All rights reserved.

Microorganisms can biosynthesize various complex polymers by biodeterioration and biodegradation of both natural and synthetic sources (Gu, 2003). Pseudomonas species is mainly known to produce PHAs from alkanes, alkenes, fatty acids, sugars, waste frying oils, carbohydrates and other waste materials (Muhr et al., 2013a, 2013b; Nitschke et al., 2011; Solaiman et al., 2001). For the growth of bacterial cell, easy access to carbon source and adaptation is very important. The synthesis of biosurfactant, during fermentation, increases the uptake of hydrocarbon by hydrophobic biological cells. A linear relationship between the concentration of produced biosurfactant and degradation rate of hexadecane by P. aeruginosa had been reported (Zhong et al., 2014). Pseudomonas species can easily degrade and use hexadecane as preferred growth substrate (Stoimenova et al., 2009); though majorly, it has been studied for the synthesis of rhamnolipids employing different microorganisms (Abouseoud et al., 2007; Cameotra and Singh, 2009; Christova et al., 2004; Liu et al., 2012; Raza et al., 2006a). The gamma ray mutant EBN-8 of P. aeruginosa has been successfully reported for the enhanced production of biosurfactant (Iqbal et al., 1995; Raza et al., 2006a, 2006b, 2014), but no work has been reported for the biosynthesis of PHAs when grown on hexadecane. The purpose of present study was the investigation of hexadecane biotransformation for the production of PHA from the P. aeruginosa gamma ray mutant.

172

Z.A. Raza et al. / International Biodeterioration & Biodegradation 115 (2016) 171e178

value (W1) and after centrifugation, the supernatant was separated and the ependrof including pellet was oven dried at 60  C to a constant mass value (W2) and then the cell dry mass (CDM) was measured by using Eq. (2):

2. Materials and methods 2.1. Materials Analytical grade chemicals were used in the present study. KH2PO4, K2HPO4, FeSO4$7H2O, NaNO3, CHCl3, C3H6O, CH3OH, C16H34 and poly(3-hydroxybutyrate) (PHB) were sourced from SigmaAldrich. CaCl2.2H2O, MgSO4$7H2O and CH3(CH2)4CH3 were obtained from Riedel-de Haen.

CDM ¼ ðW2
W1 Þ  666:67

(2)

2.6. Extraction of PHA from cells 2.2. Microorganism The mutant strain of Pseudomonas aeruginosa designated as EBN-8 (Iqbal et al., 1995) was provided by National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan. The strain was maintained on nutrient agar plate and refreshed, weekly. In normal saline (0.89%, w v
1, NaCl), the bacterial cells were suspended and at 660 nm, the optical density of the suspended bacterial cells was adjusted to 0.7. This suspension was used as inoculum for all the experiments. 2.3. Shake flask experiments The minimal salts medium (g l
1) was prepared in distilled water in the following recipe: KH2PO4 (0.07), K2HPO4 (0.13), MgSO4$7H2O (0.03), NaNO3 (0.2), CaCl2$2H2O (0.01) and FeSO4$7H2O (0.0001). The pH-value of solution was adjusted at 7.0 with K2HPO4 when required. In this study, 250 ml Erlenmeyer flasks were used having 100 ml of minimal salts media. The sterilization was done in an autoclave (WiseClave®; WAC-60, Korea) at 121  C for 15 min. Pre-sterilized hexadecane (autoclaved in a separate glass container) was added from 1 to 3% (v v
1) as carbon source followed by the addition of 1% (v v
1) inoculum. Then, the flasks were incubated on an orbital shaker (WiseShake®; SHO-2D, Daihan, Korea) at (37 ± 1  C), which is an optimum temperature for bacterial growth (Haba et al., 2007), and 150 rpm. The experiments were carried out up to six days and the whole flasks were removed from the orbital shaker each day for analyzing degradation of hexadecane and biosynthesis of PHAs. 2.4. Carbon source utilization The utilization percentage of hexadecane was determined by calculating the residual hexadecane present in cell free cultural broth (CFCB). For estimation of residual hexadecane, 100 ml of CFCB was washed thoroughly with 1:2 (v v
1) hexane using a separating funnel (Raza et al., 2006b). The organic layer was separated on a rotary evaporation machine (Strike® 202, STEROGLASS, Italy) at 60  C to a constant mass. The carbon source utilization (w w
1%) was measured by Eq. (1) as followed:

  Hexadecane utilization ww
1 %   Initial carbon source e Residual carbon source  100 ¼ Initial carbon source (1)

2.5. Biomass estimation To collect cell pellet, an aliquot (1.5 ml) of cultural medium was centrifuged at 10,000 rcf and 4  C with the help of a refrigerated centrifuge machine (MSE, Harrier 18/80R, England). The ependorfs used for sampling were first heated at 60  C upto constant mass

Selection of PHA extraction method is crucial with respect to the purity as some methods may damage the polymer (Koller et al., 2013). Among the methods, solvent extraction provide no harm to the bio-polymer (Jacquel et al., 2008). The centrifuged cells were washed with acetone to remove cell membrane components like phospholipids. Then the cells were washed with Triton X-100 to make them permeable (Koley and Bard, 2010). The cells were lyophilized overnight, re-suspended in chloroform and shifted in a flask on the orbital shaker at 180 rpm at 30  C for 24 h. The cell debris was removed by filtering the contents with a Whatman filter paper and the filtrate was concentrated. Then, chilled methanol was added (1:3) dropwise into concentrated polymer containing solvent to precipitate PHAs (Tan et al., 2014). This solution was then concentrated on a rotary evaporator to a constant mass at 60  C (Jiang et al., 2008). 2.7. Production kinetics of fermentation The fermentation production kinetics were studied by finding the product yields with respect to substrate consumption (YP/S in g g
1), the product yield with respect to biomass (YP/X in g g
1), the biomass yield related to substrate consumption (YX/S in g g
1) and the volumetric productivity (PV in g l
1 h
1) of the cultural media (Aiba et al., 1973). 2.8. Fourier transform infrared spectroscopy (FTIR) FTIR (Bruker, Tensor 27) was used to characterize the produced bio-polymer. 2.9. Liquid chromatography-mass spectroscopy (LC-MS) A matrix of PHB (or PHA) was prepared by dissolving in chloroform and its ESI chemical analysis was performed on a double focusing mass spectrometer (LTQ XL, Thermo electron Corporation USA) in both negative and positive ion modes equipped with a liquid chromatograph (Finnigan Surveyor, Thermo electron Corporation USA). The temperature of capillary was 280  C, the flow rate of sheath gas was 25, the flow rate of auxiliary gas was 5, tube lenz was 70 V, source voltage was 3.5 KV and the flow rate was 10 ml min
1. 3. Results and discussion The selected bacterial strain of P. aeruginosa gamma ray mutant showed good growth and PHAs accumulation when grown on different concentrations of hexadecane under shake flask experiments. Selection of appropriate carbon source, concentration of carbon source and bacterial strain are significant factors in the PHAs production. Utilization of carbon source, biomass formation and PHAs production are important factors for consideration as these attributes contribute in the final cost of produced polymer.

Z.A. Raza et al. / International Biodeterioration & Biodegradation 115 (2016) 171e178

3.1. Carbon source utilization

173

1.8 1.6 1.4 1.2

CDM g l-1

At either concentration of hexadecane, low consumption of hexadecane was observed upto 3rd day of incubation as shown in Fig. 1. This might be attributed to the lag phase of the bacterial culture. When the bacterial culture was in the lag phase it showed low metabolic activity due to carbon source adaptation and cell density was also very low in the cultures. An abrupt increase in the consumption of hexadecane was observed on 3rd day which could be attributed to various aspects like; the exponential growth phase, adaptation of hexadecane and production of biosurfactant (which helps in solubilizing the hydrocarbon) (Raza et al., 2006a). The production of biosurfactant starts within 24 h of incubation which reduces the cultural media's surface tension (El-Sheshtawy and Doheim, 2014). As hydrophobic carbon source is not miscible with aqueous minimal media, so during early days of incubation access to carbon source is less. Hence, the formation of biosurfactant plays major role in interaction of bacterial cultures with the carbon source (Ratledge, 2012). The maximum hydrocarbon utilization of upto 73.97% (w w
1) was achieved from the cultures with 1% (v v
1) hexadecane. At 2 % (v v
1) hexadecane concentration, the cultures showed maximum utilization upto 61.90% (w w
1). The result were close with the finding of a study performed on 2% hexadecane as carbon source using Pseudomonas species where upto 6th day of incubation about 52e53% (w w
1) carbon source was utilized (Cameotra and Singh, 2009). The cultures of 3% (v v
1) hexadecane flasks consumed only 47.33% (w w
1) hydrocarbon. This low consumption of carbon source in 3% (v v
1) hexadecane experiments might be due to the more carbon source supplied at start of experiment which had a negative impact on biomass formation and on 6th day of incubation only 0.95 g l
1 biomass was achieved.

1 1% Hexadecane 0.8

2% Hexadecance

0.6

3% Hexadecane

0.4 0.2 0 1

2

3

4

5

6

IncubaƟon Ɵme (d) Fig. 2. Cell dry mass (CDM) formation during incubation under shake flask conditions.

hexadecane. Further increasing hexadecane from 2 to 3% (v v
1), the CDM reduced from 1.5 to 0.95 g l
1. This concentration decrease trend could be attributed to the fact that excessive carbon source might had an inhibitory effect on cell growth, so less growth was observed. This finding is in accordance to the finding where increased concentration of carbon source reduced the growth (Khandpur et al., 2012). So, the optimum value of 1.5 g l
1 of CDM was achieved on 2% (v v
1) hexadecane cultures under the experimental conditions. Different studies reports represent different biomass formation on hexadecane where 1% (v v
1) carbon source using Pseudomonas sp. showed 2 g l
1 (El-Sheshtawy and Doheim, 2014) and 4.23 g l
1 of CDM (Joice and Parthasarathi, 2014).

3.3. Production of PHAs 3.2. Biomass formation The biomass values calculated from the experiments on 1, 2 and 3% (v v
1) hexadecane are shown in Fig. 2. At all three used concentrations of hexadecane, first 2e3 days showed very less growth due to the under adaptation of hexadecane as carbon source during lag phase and less access to hexadecane. On 3rd day, at all three concentrations of hexadecane, better biomass yield was achieved because bacteria entered the exponential phase. Using 1% (v v
1) hexadecane maximum CDM of 1.3 g l
1 was achieved at 6th day on incubation while 1.5 g l
1 CDM was achieved on utilizing 2% (v v
1)

90

The cells collected by centrifugation were subjected to acetone washing because cell membrane is composed of lipids, proteins and sugars. These lipids are soluble in acetone and other organic solvents (Brown, 1996). The lipids are permeability barriers of cell membranes (Cronan, 2003), so removal of lipids is necessary to make them permeable. After extraction and precipitation of polymer from cell, it was heated to get a constant mass value against each different cultural flasks. The yield of PHA is shown in Fig. 3. First two days showed no detection of PHAs through solvent extraction irrespective of the used hexadecane concentrations. This might be due to the lower biomass formation, as cells showed very less growth during the first two days of incubation and they were in lag phase hence accumulation of PHA was not detectable. On the

70

0.7

60

0.6

50

0.5

1% Hexadecane 40

2% Hexadecane 3% Hexadecane

30

PHA yiled (g l-1)

Substrate uƟlizaƟon (%)

80

0.4 1% Hexadecane 0.3

20

0.2

10

0.1

0

2% Hexadecane 3% Hexadecane

0

1

2

3

4

5

6

IncubaƟon Ɵme (d) Fig. 1. Carbon source utilization during incubation under shake flask conditions.

1

2

3

4

5

6

IncubaƟon Ɵme (d) Fig. 3. PHAs production during incubation under shake flask conditions.

174

Z.A. Raza et al. / International Biodeterioration & Biodegradation 115 (2016) 171e178

3rd day of incubation, small quantities of PHAs were observed and then the PHA accumulation of CDM increased in all cultures. Maximum product yield of 0.610 g l
1 was observed on the 2% (v v
1) hexadecane cultures. While, with 1 and 3% (v v
1) hexadecane cultures, the product yield of 0.426 and 0.320 g l
1 was observed, respectively. Moving from 1 to 2% (v v
1) hexadecane, the product yield increased but on further increasing concentration from 2 to 3% (v v
1) hexadecane led to decrease product yield of PHA. This decrease could be due to the undesirable conditions for growth. The 1% (v v
1) hexadecane cultures showed PHA accumulation of 32.78% (w w
1) of CDM at 6th day of incubation. Moreover, 2 and 3% (v v
1) hexadecane fed flasks produced maximum PHAs accumulation of 40.67% and 33.68% (w w
1) of CDM, respectively. In literature, 55 different bacterial strains of Pseudomonas sp. had been reported to accumulate PHAs ranging from less than 5% upto 78% (w w
1) of CDM (Timm and Steinbüchel, 1990). Another study reported that P. aeruginosa could accumulate PHAs from 0 to 23% (w w
1) of CDM (Haywood et al., 1989). In a study, only 7.5% (w w
1) PHAs accumulation was observed in P. aeruginosa strain grown on 8% (w w
1)hexadecane at 34  C (Chayabutra and Ju, 2001) while using only upto 2% (w w
1) concentration of hexadecane, we achieved higher production (40.67% w w
1) PHAs accumulation of CDM. The increased PHAs accumulation could be attributed to the much suitable fermentation temperature (37  C) provided as well as the consequences of genetic mutation. The PHAs accumulation from 15 to 57% (w w
1) of CDM on different alkanoic acids as carbon source has been reported using P. putida strains (Ward and O'Connor, 2005). The lower accumulation of PHAs by P. aeruginosa gamma ray mutant when compared with other carbon sources, could be attributed to the longer chain length of hexadecane or to the particular strain used as it had been already reported that the amount of PHAs produced and composition of produced PHAs differs on changing the carbon source from same bacterial strain (Lageveen et al., 1988).

3.4. Production kinetics of fermentation Kinetic parameters of formation of cell biomass and production of PHA on hexadecane are shown in Table 1. The bacteria successfully adapted hydrocarbon for biotransformation and produced PHAs. The best growth yield coefficient, PX/S, of 0.242 g biomass per g of carbon source was observed on 2% (v v
1) hexadecane concentration followed by 0.201 g g
1 on 3% (v v
1) cultures. The experiments showed an increase in PX/S value as the concentration of hexadecane was gradually increased from 1 to 3% (v v
1). The values of YP/S was different for different amount of hydrocarbon fed and maximum value of YP/S was 0.098 g product per gram of substrate achieved on 2% hexadecane cultures. YP/S of 1 and 3% (v v
1) hexadecane cultures was 0.058 and 0.068 g g
1, respectively. P. putida strain has been reported to give YP/S values of 0.11, 0.17 and 0.22 g g
1 on different carbon sources (Ward et al., 2005) and YP/S values from 0.10 to 0.41 g g
1on different alkanoic acids (Ward and O'Connor, 2005). In the present study, maximum YP/X was achieved on 2% (v v
1) hexadecane as 0.41 g g
1 then by 3% (v v
1) hexadecane as 0.34 g g
1 whereas 0.33 g g
1 value was observed on 1%

hexadecane . The C/N ratio is known to perform an important role in PHAs accumulation (Lee et al., 2008; Singh Saharan et al., 2014). Increasing the concentration of hexadecane from 1 to 2% (v v
1), the value of C/N ratio also enhanced which favored more PHAs accumulation thus, YP/X yield increased but shifting from 2 to 3% carbon source reduced YP/X yield as less quantity of biomass formation was observed in that case. Maximum value of volumetric productivity (PV) achieved was 0.0042 g l
1 h
1 on 2% (v v
1) hexadecane medium followed by 0.0029 g l
1 h
1 on 1% (v v
1) hexadecane medium. While 3% (v v
1) hexadecane cultures showed only 0.0022 g l
1 h
1 volumetric productivity which was lower as compared to other media and that might be due to the unfavorable conditions like excessive hexadecane which reduced biomass formation hence the chances of the PHA accumulation reduced as well. The shake flask experiments are preliminary procedure before continuing on bioreactor scale for the production. To optimize the fermentation conditions, improved medium and proper selection of bacterial strain for desired purposes is important before bioreactor trials. According to the literature, shake flask experiments is an practical approach for these experiments (Kennedy et al., 1994). 3.5. Chemical analysis of PHA The produced PHA polymer was analyzed against the standard PHB of Sigma-Aldrich using FTIR. The standard PHB absorption spectrum and the spectrum of produced PHA from P. aeruginosa gamma ray mutant has been shown in Fig. 4. The presence of peak from 1700 to 1750 cm
1 was due to ester group (C]O) in the main chain of the standard PHB polymer. The peak at 1278.25 cm
1 represented the presence of eCH group. The overall spectra of standard PHB was very close to already published spectra (Sindhu et al., 2011). The PHAs produced by P. aeruginosa showed peaks at 1740.45 and 1458.97 cm
1 that confirmed the existence of ester group (C] O) and bacterial protein amide (NC]O), respectively (Randriamahefa et al., 2003). Peaks near 1364.11 and 1376.87 cm
1 could be due to the CeH bond's asymmetric deformation (Srivastava and Tripathi, 2013). The peaks in the region from 1000 to 1300 cm
1 was due to the CeO stretching vibration of ester. The region from 2800 to 3100 cm
1 represented the vibrational stretching of CeH bonds of methyl and methylene groups (Shamala et al., 2009). The main difference in biosynthesized PHAs from the standard PHB was the region of peaks near 2900 cm
1 is a confirmation of longer chain length polymer than PHB (mcl-PHA) (Khare et al., 2014). The mass spectra of the PHAs structures that could be observed in Fig. 5 which showed that PHA co-polymers were produced having molecular mass of m z
1 448.5 corresponding to polyhydroxybutyrate. Terminal group's molecular mass was 104.5 and the monomer unit possessed 86 molecular mass as per the following structure:

Table 1 Kinetic parameters of PHA production by P. aeruginosa EBN-8 grown on hexadecane. hexadecane conc. (w v
1%)

YX/S (g g
1)

YP/S (g g
1)

YP/X (g g
1)

PV (g l
1 h
1)

1 2 3

0.176 0.242 0.201

0.058 0.098 0.068

0.33 0.41 0.34

0.0029 0.0042 0.0022

175

3500

3000

3500

3000

2000 1500 Wavenumber cm-1

2500

2000 1500 Wavenumber cm-1

1000

677.98 626.74 619.74 578.30 530.82 514.03

1453.06 1402.57 1379.24 1357.99 1278.25 1261.27 1228.25 1181.35 1130.93 1100.38 1055.28 1044.67 979.36 954.03 929.90 910.82 895.67 838.79 826.40

1720.33 1686.66

1996.82

2165.96

2308.76

2500

500

b

1000

758.01 720.42 667.25 648.52 580.91 544.33 526.73

827.22

887.94

1119.75 1097.74 1081.47 1021.71 964.61

1246.11

1184.79

1376.87 1364.11

1510.62 1458.97

1647.94 1609.94

1740.35

3007.99 2953.56 2921.85 2852.87

1987.90

40

50

Transmittance [%] 60 80 70

90

100

a

2995.81 2976.05 2933.36

3434.93

20

40

Transmittance [%] 80 60

100

Z.A. Raza et al. / International Biodeterioration & Biodegradation 115 (2016) 171e178

500

Fig. 4. FTIR spectra of (a) PHB and (b) PHA produced by P. aeruginosa grown on hexadecane as carbon source.

According to the calculations (Alarfaj et al., 2015), 4 monotonous units (i.e., degree of polymerization equals 4) were coupled with the primary monomer unit, as given Eq. (3):

consist of 4 basic monomers linked in a chain. Pseudomonas sp. has been reported to accumulate PHB (Catone et al., 2014; Jiang et al., 2008). While, the other present peaks require characterization in future through proper analysis with either GCMS or GC-FID.

Mol: mass of PHA ¼ 104:5 þ 86:0  4 ¼ 104:5 þ 344 ¼ 448:5amu

(3)

The biosynthesized PHAs on hexadecane showed molecular mass peak at 448.5, due to positive ionization phenomenon of mass spectra proton in the case of both the standard PHB and produced PHAs molecules yielded [MþH]þ cations at m z
1 449.5 (Fig. 5a, b). Whereas, the negative mode LC-ESI-MS of both standard PHB and produced PHA molecules yielded [M
H]
anions at m z
1 447.5 which can be seen from the spectra giving corroboration of the compound (Fig. 5c, d). Repetitive units were used to estimate the final molecular mass of biosynthesized PHAs. Each repetitious unit were consonants with the molecular mass of 86.0 KD. At last, the other peak at 447.5 dovetail with the value perceived after deprotonation are approximately the same hence reaffirmed the PHAs to

4. Conclusion Pseudomonas aeruginosa gamma ray mutant successfully biotransformed hexadecane into PHAs. From the selected concentrations, 2% (v v
1) hexadecane showed maximum product yield of 0.61 g l
1, PHAs accumulation of CDM 40.67% (w w
1), 0.41 g g
1 product yield with respect to biomass (YP/X) and volumetric productivity (PV) as 0.0042 g l
1 h
1. Thus, it could be asserted that from the selected concentrations, 2% (v v
1) hexadecane was the optimum condition for PHAs production. The concentration of fed hydrocarbon proved to be one of the crucial parameters for proper growth and production kinetics of PHAs.

176

Z.A. Raza et al. / International Biodeterioration & Biodegradation 115 (2016) 171e178 337.08

100 90 80 Relative Abundance

70 60 50 40 30 199.08

20 10

449.25 215.08 243.17 232.17 274.17 291.17

155.17 171.08

0 150

a

200

250

319.25

377.25 391.08 405.33

346.25

300

350

481.58 498.58

400

450

500

550

m/z

449.42

100 90 318.42

Relative Abundance

80 70

274.33

60

289.25

50

362.42

40

405.42

245.25

30

413.33

20 10

391.42

301.25 218.25

240.17

220

240

0

260

280

346.42

321.33

255.17 300

320

340 m/z

437.42

383.33 360

380

400

420

440

471.42 460

480

b 447.25

100 90 80 Relative Abundance

70 60 50 40

653.42

30

285.17

20 10 0

205.00 229.25 267.25 200

250

311.42 347.08 385.25 300

350

417.17

400

491.08 485.25 511.42 450

c

500 m/z

623.25

563.17 550

600

670.67 650

700

750

80

447.17

100 90

Relative Abundance

80 70 60 50 653.33

40 30 285.17

20 10

355.25

277.25

0 200

300

417.17 400

491.00 500

563.08 623.33 671.00 600

700 m/z

800

900

1000

1100

1200

d Fig. 5. Representative LC-ESI-MS positive mode spectra of (a) PHB and (b) produced PHA; and respective negative mode spectra of (c) PHB and (d) produced PHA.

Z.A. Raza et al. / International Biodeterioration & Biodegradation 115 (2016) 171e178

5. Future work Cost effective production of PHAs depends on finding improved biosynthetic strategies and downstream processing needed to obtain the level of product purity of PHAs desirable for particular applications. Crude PHAs may find applications in textile industry such as meshes and fibers however, a host of interesting features of purified PHAs has a lot of potential in many biomedical applications. Moreover, investigating the effects of carbon sources on the mixed PHAs species produced, and the distinct roles and characteristics of each homolog produced are of great importance to be better elucidated, both in individual and mutual properties. Acknowledgement The authors acknowledge Higher Education Commission, Islamabad, Pakistan for research grant (NRPU-3154). References Abouseoud, M., Maachi, R., Amrane, A., 2007. Biosurfactant production from olive oil by pseudomonas fluorescens. Commun. Curr. Res. Educ. Top. Trends Appl. Microbiol. 340e347. Aiba, S., Humphrey, A.E., Millis, N.F., 1973. Biochemical Engineering. Academic Press. Alarfaj, A.A., Arshad, M., Sholkamy, E.N., Munusamy, M.A., 2015. Extraction and characterization of polyhydroxybutyrates (PHB) from Bacillus thuringiensis KSADL127 isolated from mangrove environments of Saudi Arabia. Braz. Arch. Biol. Technol. 58, 781e788. http://dx.doi.org/10.1590/S1516-891320150500003. Brown, B.S., 1996. Biological Membranes. Biochemical Society, London, p. 44. Cameotra, S.S., Singh, P., 2009. Synthesis of rhamnolipid biosurfactant and mode of hexadecane uptake by Pseudomonas species. Microb. Cell Fact. 8, 16. http:// dx.doi.org/10.1186/1475-2859-8-16. Catone, M.V., Ruiz, J.A., Castellanos, M., Segura, D., Espin, G., Lopez, N.I., 2014. High polyhydroxybutyrate production in Pseudomonas extremaustralis is associated with differential expression of horizontally acquired and core genome polyhydroxyalkanoate synthase genes. PLoS One 9. http://dx.doi.org/10.1371/ journal.pone.0098873. Chayabutra, C., Ju, L.K., 2001. Polyhydroxyalkanoic acids and rhamnolipids are synthesized sequentially in hexadecane fermentation by Pseudomonas aeruginosa ATCC 10145. Biotechnol. Prog. 17, 419e423. http://dx.doi.org/10.1021/ bp010036a. Chee, J., Yoga, S., Lau, N., Ling, S., Abed, R.M.M., 2010. Bacterially produced Polyhydroxyalkanoate (PHA): converting renewable resources into bioplastics. In: Mendez-Vilas, A. (Ed.), Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology. Formatex research center, pp. 1395e1404. Choi, J.I., Lee, S.Y., Han, K., 1998. Cloning of the Alcaligenes latus polyhydroxyalkanoate biosynthesis genes and use of these genes for enhanced production of Poly(3-hydroxybutyrate) in Escherichia coli. Appl. Environ. Microbiol. 64, 4897e4903. Christova, N., Tuleva, B., Lalchev, Z., Jordanova, A., Jordanov, B., 2004. Rhamnolipid biosurfactants produced by Renibacterium salmoninarum 27BN during Growth on n-hexadecane. Z. fur Naturforsch. e Sect. C J. Biosci. 59, 70e74. Cronan, J.E., 2003. Bacterial membrane lipids: where do we stand? Annu. Rev. Microbiol. 57, 203e224. http://dx.doi.org/10.1146/ annurev.micro.57.030502.090851. El-Sheshtawy, H.S., Doheim, M.M., 2014. Selection of Pseudomonas aeruginosa for biosurfactant production and studies of its antimicrobial activity. Egypt. J. Pet. 23, 1e6. http://dx.doi.org/10.1016/j.ejpe.2014.02.001. Gu, J.D., 2003. Microbiological deterioration and degradation of synthetic polymeric materials: recent research advances. Int. Biodeterior. Biodegr. 52, 69e91. http:// dx.doi.org/10.1016/S0964-8305(02)00177-4. Haba, E., Vidal Mas, J., Bassas, M., Espuny, M.J., Llorens, J., Manresa, A., 2007. Poly 3(hydroxyalkanoates) produced from oily substrates by Pseudomonas aeruginosa 47T2 (NCBIM 40044): effect of nutrients and incubation temperature on polymer composition. Biochem. Eng. J. 35, 99e106. http://dx.doi.org/10.1016/ j.bej.2006.11.021. Haywood, G.W., Anderson, A.J., Dawes, E.A., 1989. A survey of the accumulation of novel polyhydroxyalkanoates by bacteria. Biotechnol. Lett. 11, 471e476. http:// dx.doi.org/10.1007/BF01026644. Hazer, B., Steinbuchel, A., 2007. Increased diversification of polyhydroxyalkanoates by modification reactions for industrial and medical applications. Appl. Microbiol. Biotechnol. http://dx.doi.org/10.1007/s00253-006-0732-8. Iqbal, S., Khalid, Z.M., Malik, K.A., 1995. Enhanced biodegradation and emulsification of crude oil and hyperproduction of biosurfactants by a gamma rayinduced mutant of Pseudomonas aeruginosa. Lett. Appl. Microbiol. 21, 176e179. Jacquel, N., Lo, C.W., Wei, Y.H., Wu, H.S., Wang, S.S., 2008. Isolation and purification of bacterial poly(3-hydroxyalkanoates). Biochem. Eng. J. http://dx.doi.org/ 10.1016/j.bej.2007.11.029.

177

Jaeger, K.E., Steinbuchel, A., Jendrossek, D., 1995. Substrate specificities of bacterial polyhydroxyalkanoate depolymerases and lipases: bacterial lipases hydrolyze poly(w-hydroxyalkanoates). Appl. Environ. Microbiol. 61, 3113e3118. Jiang, Y., Song, X., Gong, L., Li, P., Dai, C., Shao, W., 2008. High poly(hydroxybutyrate) production by Pseudomonas fluorescens A2a5 from inexpensive substrates. Enzyme Microb. Technol. 42, 167e172. http://dx.doi.org/10.1016/ j.enzmictec.2007.09.003. Joice, P.A., Parthasarathi, R., 2014. Optimization of biosurfactant production from Pseudomonas aeruginosa PBSC1. Int. J. Curr. Microbiol. Appl. Sci. 3, 140e151. Kennedy, M.J., Reader, S.L., Davies, R.J., Rhoades, D.A., Silby, H.W., 1994. The scale up of mycelial shake flask fermentations: a case study of gamma linolenic acid production by Mucor hiemalis IRL 51. J. Ind. Microbiol. 13, 212e216. http:// dx.doi.org/10.1007/BF01569750. Khandpur, P., Jabeen, E.T., Rohini, K.V.L., Varaprasad, Y., 2012. Study on production, extraction and analysis of polyhydroxyalkanoate ( PHA ) from bacterial isolates. IOSR J. Pharm. Biol. Sci. IOSRJBPS 1, 31e38. Khare, E., Chopra, J., Arora, N.K., 2014. Screening for mcl-PHA-producing fluorescent Pseudomonads and comparison of mcl-PHA production under iso-osmotic conditions induced by PEG and NaCl. Curr. Microbiol. 68, 457e462. http:// dx.doi.org/10.1007/s00284-013-0497-0. Koley, D., Bard, A.J., 2010. Triton X-100 concentration effects on membrane permeability of a single HeLa cell by scanning electrochemical microscopy (SECM). Proc. Natl. Acad. Sci. U. S. A. 107, 16783e16787. http://dx.doi.org/ 10.1073/pnas.1011614107. Koller, M., Niebelschutz, H., Braunegg, G., 2013. Strategies for recovery and purification of poly[(R)-3-hydroxyalkanoates] (PHA) biopolyesters from surrounding biomass. Eng. Life Sci. 13, 549e562. http://dx.doi.org/10.1002/elsc.201300021. Koller, M., Salerno, A., Dias, M., Reiterer, A., Braunegg, G., 2010. Modern biotechnological polymer synthesis: a review. Food Technol. Biotechnol. 48, 255e269. Lageveen, R.G., Huisman, G.W., Preusting, H., Ketelaar, P., Eggink, G., Witholt, B., 1988. Formation of polyesters by Pseudomonas oleovorans: effect of substrates on formation and composition of poly-(R)-3-hydroxyalkanoates and poly-(R)3-hydroxyalkenoates. Appl. Environ. Microbiol. 54, 2924e2932. Lee, W.H., Loo, C.Y., Nomura, C.T., Sudesh, K., 2008. Biosynthesis of polyhydroxyalkanoate copolymers from mixtures of plant oils and 3hydroxyvalerate precursors. Bioresour. Technol. 99, 6844e6851. http:// dx.doi.org/10.1016/j.biortech.2008.01.051. Liu, T., Wang, F., Guo, L., Li, X., Yang, X., Lin, A.J., 2012. Biodegradation of n-hexadecane by bacterial strains B1 and B2 isolated from petroleum-contaminated soil. Sci. China Chem. 55, 1968e1975. http://dx.doi.org/10.1007/s11426-0124618-6. Loo, C., Sudesh, K., 2007. Polyhydroxyalkanoates: bio-based microbial plastics and their properties. Malays. Polym. J. 2, 31e57. Muhr, A., Rechberger, E.M., Salerno, A., Reiterer, A., Malli, K., Strohmeier, K., Schober, S., Mittelbach, M., Koller, M., 2013a. Novel description of mcl-PHA biosynthesis by Pseudomonas chlororaphis from animal-derived waste. J. Biotechnol. 165, 45e51. http://dx.doi.org/10.1016/j.jbiotec.2013.02.003. Muhr, A., Rechberger, E.M., Salerno, A., Reiterer, A., Schiller, M., Kwiecien, M., Adamus, G., Kowalczuk, M., Strohmeier, K., Schober, S., Mittelbach, M., Koller, M., 2013b. Biodegradable latexes from animal-derived waste: biosynthesis and characterization of mcl-PHA accumulated by Ps. citronellolis. React. Funct. Polym. 73, 1391e1398. http://dx.doi.org/10.1016/ j.reactfunctpolym.2012.12.009. Nitschke, M., Costa, S.G.V.A.O., Contiero, J., 2011. Rhamnolipids and PHAs: recent reports on Pseudomonas-derived molecules of increasing industrial interest. Process Biochem. 46, 621e630. http://dx.doi.org/10.1016/j.procbio.2010.12.012. rin, P., Langlois, V., 2003. Fourier transform Randriamahefa, S., Renard, E., Gue infrared spectroscopy for screening and quantifying production of PHAs by Pseudomonas grown on sodium octanoate. Biomacromolecules 4, 1092e1097. http://dx.doi.org/10.1021/bm034104o. Ratledge, C., 2012. Physiology of Biodegradative Microorganisms. Springer Science & Business Media, Berlin e Heidelberg. Raza, Z.A., Khan, M.S., Khalid, Z.M., Rehman, A., 2006a. Production of biosurfactant using different hydrocarbons by Pseudomonas aeruginosa EBN-8 mutant. Z. fur Naturforsch. e Sect. C J. Biosci. 61, 87e94. Raza, Z.A., Khan, M.S., Khalid, Z.M., Rehman, A., 2006b. Production kinetics and tensioactive characteristics of biosurfactant from a Pseudomonas aeruginosa mutant grown on waste frying oils. Biotechnol. Lett. 28, 1623e1631. http:// dx.doi.org/10.1007/s10529-006-9134-3. Raza, Z.A., Rehman, A., Hussain, M.T., Masood, R., Haq, A., Saddique, M.T., Javid, A., Ahmad, N., 2014. Production of rhamnolipid surfactant and its application in bioscouring of cotton fabric. Carbohydr. Res. 391, 97e105. http://dx.doi.org/ 10.1016/j.carres.2014.03.009. Shamala, T.R., Divyashree, M.S., Davis, R., Kumari, K.S.L., Vijayendra, S.V.N., Raj, B., 2009. Production and characterization of bacterial polyhydroxyalkanoate copolymers and evaluation of their blends by fourier transform infrared spectroscopy and scanning electron microscopy. Indian J. Microbiol. 49, 251e258. http://dx.doi.org/10.1007/s12088-009-0031-z. Sindhu, R., Ammu, B., Binod, P., Deepthi, S.K., Ramachandran, K.B., Soccol, C.R., Pandey, A., 2011. Production and characterization of poly-3-hydroxybutyrate from crude glycerol by Bacillus sphaericus NII 0838 and improving its thermal properties by blending with other polymers. Braz. Arch. Biol. Technol. 54, 783e794. http://dx.doi.org/10.1590/S1516-89132011000400019. Singh Saharan, B., Grewal, A., Kumar, P., 2014. Biotechnological production of polyhydroxyalkanoates: a review on trends and latest developments. Chin. J. Biol.

178

Z.A. Raza et al. / International Biodeterioration & Biodegradation 115 (2016) 171e178

2014, 1e18. http://dx.doi.org/10.1155/2014/802984. Solaiman, D.K.Y., Ashby, R.D., Foglia, T.A., 2001. Production of polyhydroxyalkanoates from intact triacylglycerols by genetically engineered Pseudomonas. Appl. Microbiol. Biotechnol. 56, 664e669. http://dx.doi.org/ 10.1007/s002530100692. Srivastava, S.K., Tripathi, A.D., 2013. Effect of saturated and unsaturated fatty acid supplementation on bio-plastic production under submerged fermentation. 3 Biotech. 3, 389e397. http://dx.doi.org/10.1007/s13205-012-0110-4. Stoimenova, E., Vasileva-Tonkova, E., Sotirova, A., Galabova, D., Lalchev, Z., 2009. Evaluation of different carbon sources for growth and biosurfactant production by Pseudomonas fluorescens isolated from wastewaters. Z. für Naturforsch. C. J. Biosci. 64, 96e102. Tan, G.Y., Chen, C.L., Li, L., Ge, L., Wang, L., Razaad, I., Li, Y., Zhao, L., Mo, Y., Wang, J.Y., 2014. Start a research on biopolymer polyhydroxyalkanoate (PHA): a review. Polym. (Basel) 6, 706e754. http://dx.doi.org/10.3390/polym6030706. Timm, A., Steinbüchel, A., 1990. Formation of polyesters consisting of medium-

chain-length 3-hydroxyalkanoic acids from gluconate by Pseudomonas aeruginosa and other fluorescent pseudomonads. Appl. Environ. Microbiol. 56, 3360e3367. Verlinden, R.A.J., Hill, D.J., Kenward, M.A., Williams, C.D., Radecka, I., 2007. Bacterial synthesis of biodegradable polyhydroxyalkanoates. J. Appl. Microbiol. 102, 1437e1449. http://dx.doi.org/10.1111/j.1365-2672.2007.03335.x. Ward, P.G., de Roo, G., O'Connor, K.E., 2005. Accumulation of polyhydroxyalkanoate from styrene and phenylacetic acid by Pseudomonas putida CA-3. Appl. Environ. Microbiol. 71, 2046e2052. http://dx.doi.org/10.1128/AEM.71.4.2046-2052.2005. Ward, P.G., O'Connor, K.E., 2005. Bacterial synthesis of polyhydroxyalkanoates containing aromatic and aliphatic monomers by Pseudomonas putida CA-3. Int. J. Biol. Macromol. 35, 127e133. http://dx.doi.org/10.1016/j.ijbiomac.2005.01.001. Zhong, H., Liu, Y., Liu, Z., Jiang, Y., Tan, F., Zeng, G., Yuan, X., Yan, M., Niu, Q., Liang, Y., 2014. Degradation of pseudo-solubilized and mass hexadecane by a Pseudomonas aeruginosa with treatment of rhamnolipid biosurfactant. Int. Biodeterior. Biodegr. 94, 152e159. http://dx.doi.org/10.1016/j.ibiod.2014.07.012.