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International Journal of Biological Macromolecules 76 (2015) 254–261

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

j ourna l ho me pa g e: www.elsev ier .com/ locate / i jb iomac

olyhydroxyalkanoate from marine Bacillus megaterium usingSMCRI’s Dry Sea Mix as a novel growth medium

aykishan H. Dhangdhariyaa, Sonam Dubeyb, Hiral B. Trivedia, Imran Panchab,c,walant K. Bhatta, Bharti P. Davea,∗∗, Sandhya Mishrab,c,∗

Department of Life Sciences, Maharaja Krishnakumarsinhji Bhavnagar University, Bhavnagar 364002, IndiaDiscipline of Salt & Marine Chemicals, CSIR-Central Salt and Marine Chemicals Research Institute, Bhavnagar 364002, IndiaAcademy of Scientific & Innovative Research (AcSIR), CSIR-Central Salt and Marine Chemicals Research Institute, Bhavnagar 364002, India

r t i c l e i n f o

rticle history:eceived 4 November 2014eceived in revised form 30 January 2015ccepted 9 February 2015vailable online 16 February 2015

eywords:ry sea mixolyhydroxyalkanoate

a b s t r a c t

Oceans have significant potential to empower mankind and thus marine organisms are believed to be anenormous source for useful biomolecules. Polyhydroxyalkanoates (PHAs) are biological macromoleculesthat can be applied in nearly all fields. In the present study, Bacillus megaterium strain JK4h has beenexploited for maximum PHB production using novel Dry Sea Mix (DSM) via Central Composite Design(CCD) of Response Surface Methodology (RSM) approach. The isolate was found to be producing 56.77%Cell Dry Weight (CDW) of PHAs within 24 h, with optimized combinations of peptone, yeast extractand glucose. The PHB yield had been increased 2.61 fold compared to un-optimized experiments. Theobtained PHA/PHB had been chemically characterized through Nuclear Magnetic Resonance (NMR),

entral Composite Design Fourier Transform Infrared Spectroscopy (FTIR), Thermo Gravimetric Analysis (TGA) and DifferentialScanning Calorimetry (DSC). The results indicate the successful optimization for maximum production ofbiological macromolecule and it was found to be highly pure polyhydroxybutyrate (PHB). Thus, DSM canbe served as a novel and cost effective medium for PHA production offering the use of marine resourcesas a “green” sustainable alternative.

© 2015 Elsevier B.V. All rights reserved.

. Introduction

The major cause of environmental pollution and economic dis-urbance is the excessive use of petroleum products, like syntheticlastic. One of the ways to attain pollution free environment isreen products through microbes where research is being directedowards finding an alternative to the conventional petroleum basedlastics [1].

Polyhydroxyalkanoates (PHAs) are eco-friendly polymers pro-uced by various types of bacteria with thermostability and

lasticity comparable to synthetic polyesters. To date more than00 different types of bacteria are known to produce various types

∗ Corresponding author at: Discipline of Salt & Marine Chemicals, CSIR-Centralalt and Marine Chemicals Research Institute, Bhavnagar 364002, India.el.: +91 278 256 5801/3805x6160; fax: +91 278 256 6970/7562.∗∗ Corresponding author at: Department of Life Sciences, Maharaja Krishnaku-

arsinhji Bhavnagar University, Bhavnagar - 364002, India. Tel.: +91 278 2521545.E-mail addresses: bpd8256@gmail.com (B.P. Dave), smishra@csmcri.org

S. Mishra).

ttp://dx.doi.org/10.1016/j.ijbiomac.2015.02.009141-8130/© 2015 Elsevier B.V. All rights reserved.

of biopolymers (PHA) under nutrient starvation or in presence ofexcess carbon source in the growth medium [2].

Derivatives of polyhydroxyalkanoates are frequently encoun-tered in nature and agglomerate in various bacteria as waterinsoluble, light reflecting and electrotranslucent inclusion bodies(in the form of mobile, amorphous and liquid granules) which couldbe utilized as carbon and energy sources under nutrient starvationconditions by the bacteria itself [3]. As the weight of the polymerranges from 200 to 3000 kDa, it can cause a striking alteration inthe bacterial shape [4]. The number and size of these granules,monomer composition, molecular structure and physico-chemicalproperties vary depending on the producer microorganisms, thesubstrate used as carbon source and on its harvesting time [5]. PHAsare synthesized using renewable carbon sources having plausiblerole in agriculture, waste disposal systems, biomedical applicationsetc., contributing to the conservation of finite fossil resources with-out any harm [4,6].

PHAs mainly belong to two groups: short chain length (scl)

PHAs with 3–5 carbon chain length hydroxyl fatty acid repeats,and medium chain length (mcl) PHAs with >6 carbon chain repeatsof hydroxyl fatty acids [7]. As the length of the side chain on the�-carbon of the PHA increases, the physical property of polymer

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J.H. Dhangdhariya et al. / International Journa

s changed from glassy state to more soft and sticky material [6].epending on the physico chemical properties of the polymer,

ts applicability could be decided e.g. scl-PHAs are brittle, stiff,rystalline thermoplastics [8] whereas mcl-PHAs and their copoly-ers have low tensile strength and glass transition temperature

nd high elongation to break ratios, with flexible elastomeric anddhesive properties which could be employed for packaging andoating materials or as biodegradable carriers for slow delivery ofrugs, agrochemicals, preparation of medical materials like surgi-al sutures, pins, swabs etc. (which requires flexible biocompatibleaterial). Extremely diverse properties and characteristics can be

chieved with combination of more than 150 different known typesf these monomer units [9].

Although it is environment friendly and biocompatible, its pro-uction cost is much higher than synthetic alternatives, which is theajor cause for struggle of polyhydroxyalkanoates in global mar-

ets. It is unfair to compare the price of green plastics to syntheticlastics as the latter is non-biodegradable [10]. The researchers stillope to achieve the price comparable to synthetic ones by pro-ucing it through the naturally isolated halophilic bacteria (a wildtrain) utilizing cheap resources and the highest possible yieldsn relatively short time. Scientists have now focused on reduc-ng the production costs, using waste materials as carbon sourcesike biodiesel derived crude glycerol and other algal/agro-residual

aste. Many of the halophilic bacteria are explored for its potentialo produce high amount of PHA [11,12]. This offers an advantagever other bacterial strains like Alcaligenes with remarkable reduc-ion in the cost of the raw materials resulting in the prohibitivelyigh cost of PHA production [13].

Dry Sea Mix (DSM) is a Central Salt and Marine Chemicalsesearch Institute (CSMCRI) – a CSIR laboratory – patented product,ade from the three different fractions obtained from evaporated

ea water in the crystallizers. By combining the three fractionsn suitable concentration mixed with fresh water/tap water giveseawater in its pristine form. This salt is an excellent seawater sub-titute for the growth of various marine and halophilic organisms14]. DSM mainly contains all the essential major and trace metalons required for growth of microorganisms, which are normallyresent in sea water and artificial sea water nutrient media.

The present study has been aimed to produce and characterizeHA using wild strain of halophilic bacterium Bacillus megateriumtrain JK4h isolated from Ghogha coast near Bhavnagar, Gujarat,ndia, utilizing an inexpensive DSM based bacterial growth mediumor the production of biopolymer in a shorter incubation time thanur previous reports [12].

. Materials and methods

.1. Chemicals

Nile Red and standard polyhydroxybutyrate (PHB) werebtained from Sigma, USA. Sodium hypochlorite 6% (v/v), methanolnd chloroform were procured from Rankem (India). DSM [14]as used as cultivation media for marine bacteria throughout theresent study.

.2. Isolation of marine bacteria and growth conditions

Sea water samples were collected in sterile BOD bottles [15]rom Ghogha coast near Bhavnagar (21◦39′35.8′′N, 72◦19′13.4′′E),ujarat, India. These samples had been serially diluted up to 10−6

nd 0.1 mL aliquot from this was withdrawn and inoculated in00 mL of DSM broth in 250 mL-Erlenmeyer flasks. The flasks hadeen placed on an environmental shaker (MaxQ 400, Thermo, USA)t 37 ◦C at 120 rpm for 24 h. From this, 0.1 mL had been inoculated

ological Macromolecules 76 (2015) 254–261 255

onto DSM medium and incubated for 24 h. After incubation the DSMplate has been spread with 0.1 mL of 24 h old DSM broth.

2.3. ICP analysis of DSM

DSM was analyzed chemically for its chemical constituents. Itis extremely cheaper, convenient and showing a very good micro-bial diversity against conventional growth media for cultivation ofmarine bacteria. For ICP sample preparation, 1 g of DSM has beenplaced in silica dish and 10 mL of concentrated HNO3 was addedand left overnight. Next day, silica dish had been incinerated overa burner until no white smoke was emitted. Further, the samplewith silica dish was kept in muffle furnace at 550–600 ◦C for 5 h.Sample was removed from furnace and allowed to cool at roomtemperature. The digested sample was dissolved in 100 mL of 4%HCl and filtered through 0.22 �M filter (Millipore, USA) and analysiswas carried out using Inductively Coupled Plasma atomic emissionspectroscopy (ICP) (Perkin-Elmer, Optima 2000, USA) using induc-tively coupled plasma multi-element reference standard solution[16].

2.4. Screening the isolates for PHAs production

2.4.1. Primary screeningNile Red dye was amended in DSM broth in the final concen-

tration of 0.5 �g/mL and heat fixed smears had been prepared after24 h and observed under fluorescence microscope (Zeiss, AxiovisonA1, Germany) [17].

2.4.2. Secondary screeningFor this experiment, 10 mL (10% v/v) of the 18 h old bacterial

suspension was inoculated into 90 mL of sterilized DSM taken in250 mL capacity Erlenmeyer flask, which was incubated at 37 ◦C onan environmental shaker at 120 rpm for 24 h. After 24 h of incuba-tion, the bacterial culture was centrifuged at 10,000 rpm for 10 min.The cell pellets were oven dried at 60 ◦C overnight. Cellular diges-tion of dried cell pellet was carried out by re-suspending it in 6%(v/v) sodium hypochlorite solution followed by centrifugation at10,000 rpm for 5 min. Further, the digested cell pellets were washedtwice with methanol followed by distilled water to remove thetraces of impurities resulting in a purified product, which was re-dissolved in chloroform and weighed after air drying [7,18,19].

2.5. Identification of the isolate

The isolate showing maximum fluorescence was primarilyidentified by examining the colony characteristics and cellularmorphology. Physiological and biochemical characteristics wereexamined by standard methods [20]. The identification was furtherconfirmed using Biolog bacterial identification system and molec-ular characterization using 16s rDNA partial sequencing [21].

2.5.1. Biochemical characterizationVarious biochemical tests such as nitrate reduction, oxidase,

catalase, indole production, casein and starch hydrolysis, gelatineliquefaction, acid and H2S production were examined [20].

2.5.2. Biolog assayBiolog GPMicroPlatesTM was inoculated with 100 �L of inocu-

lation fluid (IF) containing bacterial strain and incubated for 24 hat 37 ◦C. The isolate was identified using Biolog MicrostationTM

system and software Microlog version 5.2.01 [21].

2.5.3. 16s rDNA analysisIndividual colonies of the above isolate were scrapped off from

the plates into sterile deionized water. The genomic DNA was

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56 J.H. Dhangdhariya et al. / International Journa

solated by boiling the above solution for 15 min in a water bathaintained at 95 ◦C and collecting the supernatant following cen-

rifugation at 4500 rpm for 10 min [22]. The supernatant wasirectly used for polymerase chain reaction (PCR) using univer-al bacterial primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and492R (5′-ACGGCTACCTTGTTACGACTT-3′). The initial PCR reactionas carried out in a total reaction volume of 20 �L. The reactionixture contained 1× PCR buffer (Sigma, USA), 1 mM MgCl2, 0.5 �M

f each primer, 0.5 �M dNTPs, and 1 U Taq DNA polymerase (Sigma,SA) and 15 ng template. PCR amplification was performed with

nitial denaturation for 5 min at 95 ◦C followed by 35 cycles withenaturation for 5 min at 95 ◦C, annealing for 1 min at 62 ◦C, andxtension for 1 min at 72 ◦C, with final extension for 5 min at 72 ◦C.fter completion of PCR, the mixture was loaded on 0.8% agaroseel and run under voltage of 80 mV. The bands of DNA, if any, wereisualized under the gel documentation apparatus (BioRad, Gel-ocXR+, USA). The amplified product was eluted using HiPurAgelurification kit (HiMedia Pvt. Ltd., India) as per the manufacturer’srotocol. The final eluted product was quantified spectrophotomet-ically using nano-photometer (Implen, 2192, Germany) as well asy visualizing on agarose gel. The purified PCR products of approxi-ately 1400 bp were sequenced by using two primers as described

bove. Sequencing was performed using Big Dye terminator cycleequencing kit (Applied BioSystems, USA). Sequencing productsere resolved on automated DNA sequencing system (3730XL,pplied BioSystems, USA). Contig and phylogenetic tree was gen-rated using the top 10 best matching 16S rDNA using the MEGA 6oftware [21].

.6. Optimization of DSM medium for PHAs production

Using 2k + 2k + n0, design indicating dependent variables (k) andumber of repeated experiments (n0) at central point were stud-

ed. Three media components viz. peptone (x1), yeast extract (x2)nd glucose (x3) were studied at five values ranging from −2o +2 values, where the ranges of independent variables were asollows: peptone 0.1–0.5 g/100 mL, yeast extract 0.05–0.1 g/100 mLnd glucose 1–5 g/100 mL.

Experimental design, regression and graphical analysis wereonducted throughout using Minitab v16. The results were sub-ected to ANOVA and dependent variables were coded as:

i = xi − x0

�xi

here xi represents variable value, x0 is the mid value of xi, and �xis the step change in xi. PHA production was explained as a secondrder model with four independent variables:

= ˇ0 +∑

ˇixi +∑

ˇijx2 +

∑ˇijxixj

here ˇ0 represents the constant process between xi and xj on PHAroduction. The significance of the above equation was strength-ned by Fisher’s test, the proportion of ANOVA and R2 values1,21,23].

.7. Validation experiment

Based on the combinations of independent variables executedy CCD design matrix, a validation experiment having same set ofonditions have been performed [23].

.8. Extraction and chemical characterization of the biopolymer

Based on the optimum concentrations of peptone, yeast extractnd glucose achieved, DSM had been modified so as to achieve max-mum production of PHAs. The isolate was inoculated in 100 mL of

ological Macromolecules 76 (2015) 254–261

DSM and incubated at 120 rpm for 24 h. Extraction and purifica-tion of polymer was carried out as mentioned in Section 2.4.2. Thepurified PHA has been subjected to the following analyses.

2.8.1. FTIR analysisPurified PHA was mixed with KBr. FTIR spectrum was

recorded with PerkinElmer spectrum GX FTIR spectrometer in4000–400 cm−1 [24].

2.8.2. Differential scanning calorimetric analysis (DSC)Purified PHA sample was run in a differential scanning calorime-

ter (DSC) using DSC 204 F1 Phoenix instrument with Netzschsoftware at a temperature range from −20 to 450 ◦C with the heat-ing rate of 10 ◦C min−1. Melting temperature was compared withstandard PHB (Sigma, USA) [25].

2.8.3. Thermogravimetric analysis (TGA)Purified PHA sample was subjected to thermogravimetric anal-

ysis (TGA) with help of TG–DTA system in TG 209 F1 instrument. Allanalyses were performed with 5 mg sample in an aluminium panover a temperature range of 27–450 ◦C under nitrogen atmosphere,at a heating rate of 10 ◦C min−1 [25].

2.8.4. NMR analysis1H NMR analysis of purified PHA was done using BrukerAvance-

II 500 (Ultra shield) spectrometer, Switzerland, at 500 MHz, inCDCl3 [9,26,27].

3. Results and discussion

3.1. Isolation and screening of marine bacteria

After incubation, the growth of marine bacteria was observed,and a total 109 morphologically different colonies were obtainedfrom triplicates of the DSM agar plates. The isolates were repeat-edly sub-cultured to obtain them in pure form. The isolates weremaintained on DSM slants and stored at 4 ◦C until further use.

3.1.1. Primary screeningA fluorescent dye Nile Red (0.5 �g/mL) amended DSM plates

were observed under UV transilluminator for fluorescent colonies.Amongst 109 isolates, 26 isolates (designated as JK1 to JK26) exhib-ited fluorescence as observed under GelDoc XR+ (BioRad, USA).Based on maximum fluorescence, 8 isolates (JK1 to JK8) were fur-ther selected for secondary screening of PHA production. Thesecolonies were picked up and again streaked on DSM plates con-taining Nile Red. From these plates, cells were heat fixed and slideswere observed under fluorescence microscope. Isolate JK4h exhib-ited maximum fluorescence (Fig. 1).

3.1.2. Secondary screeningA total of eight potent primarily screened isolates with max-

imum fluorescence intensity were further examined for PHAproduction. Amongst these eight isolates, JK4h showed maximumPHA producing capacity (21.72% yield) (data not shown) and thushas been selected for optimization experiment.

3.2. ICP analysis of DSM

The results obtained through the present study (Table 1) haveestablished the use of minerals present naturally in DSM which

could replace the use of synthetic marine media for bacterialgrowth. This would surely help to fabricate a growth medium forPHA production making a process more economic and easy to scaleup.

J.H. Dhangdhariya et al. / International Journal of Biological Macromolecules 76 (2015) 254–261 257

3

3

r

3

iM

TI

TB

+

Fig. 1. Fluorescent PHA granules in isolate JK4h.

.3. Identification of the isolate JK4h

.3.1. Biochemical characterizationThe biochemical characterization of isolate JK4h has been car-

ied out and the results are as shown in Table 2.

.3.2. Biolog identification

On the basis of utilization of 96 different carbon sources,

solate JK4h had been identified as B. megaterium using BiologicrostationTM system and Microlog version 5.2.01 software.

able 1CP analysis of DSM.

Sr. no. Element (%)

1 Ca 06.292 Cd 00.003 Co 00.004 Cr 00.015 Cu 00.016 Fe 00.017 K 00.278 Mg 02.379 Mn 00.00

10 Mo 00.0411 Na 25.9712 Ni 00.0313 Pb 00.0214 Zn 00.59

able 2iochemical characterization of isolate JK4h.

Sr. no. Test Result

1 Hydrolysis of starch +2 Hydrolysis of casein +3 Hydrolysis of gelatine +4 Hydrolysis of urea −5 Catalase +6 Oxidase −7 Voges-Proskauer −8 Nitrate reduction −9 Indole production −

10 Sugar fermentationGlucose +Fructose +Lactose +Sucrose +Xylose +

= positive; − = negative.

Fig. 2. Lane 1 showing 1500 bp amplified DNA of isolate JK4h, Lane 4 showing 100 bpladder.

3.3.3. 16s rDNA identificationQuantification of purified 1400 bp genomic DNA using nanopho-

tometer (Implen, 2196, Germany) was done taking 1 �L of sample.A highly purified 180 ng/�L of DNA with 1.9 purity ratio of260/280 nm was obtained. Agarose gel electrophoresis was doneusing ethidium bromide staining and visualized in BioRad gel doc-umentation system (Fig. 2). Based on the nucleotide sequencehomology and phylogenetic analysis (Fig. 3), sequenced fragmentof gene is of B. megaterium strain JK4h (GenBank Accession No. KP122748)

3.4. Optimization of DSM medium for maximum PHA production

The outcome of the optimization represents robust and repro-ducible model data. Maximum PHA production had been observedin Run no. 12 (55.97% yield) (Table 3) compared to un-optimizedconditions (21.72% yield). The independent variables were opti-mized at level 0 hence they need not be optimized further.Moreover, higher (+1 and +2) and lower (−1 and −2) coded valuesresulted in decrement in PHA production (Table 3).

The other striking observation is the dependence of PHB produc-tion on glucose amendment. There was no PHA production (Run no.2) (Table 3) in absence of glucose. This showed the prime impor-tance of carbon source for PHA production.

Also, both peptone and yeast extract significantly affected PHAproduction and the interaction which is eminent from the con-tour plots (Fig. 4). The design model showed highly significantinteractions between independent variables except yeast extract

(Tables 4 and 5). The model showed R2 value as high as 99.20%(0.9920) with adjusted R2 (0.9848) and predicted (0.9506) haveindicated its high accuracy and reproducibility (Table 5).

258 J.H. Dhangdhariya et al. / International Journal of Biological Macromolecules 76 (2015) 254–261

Fig. 3. Phylogenetic tree of isolate JK4h with its closest neighbours.

Table 3Optimization of medium components for biopolymer production.

Run no. Peptone (x1) Yeast extract (x2) Glucose (x3) Biopolymer(g/100 mL)

Biopolymer (g/100 mL)(predicted)

CorrespondingCDW (g/100 mL)

Correspondingyield (%)

Actual Coded Actual Coded Actual Coded

1 0.3 0 0.075 0 3 0 0.0340 0.0332 0.0610 55.732 0.3 0 0.075 0 −0.36358 −2 0.0000 0.0004 0.0383 0.0003 0.5 1 0.050 −1 5 1 0.0179 0.0162 0.0580 30.864 0.3 0 0.075 0 3 0 0.0320 0.0332 0.0650 49.235 0.1 −1 0.100 1 1 −1 0.0129 0.0135 0.0470 27.446 0.1 −1 0.050 −1 5 1 0.0225 0.0224 0.0610 36.887 0.3 0 0.075 0 3 0 0.0328 0.0332 0.0660 49.698 0.3 0 0.075 0 3 0 0.0330 0.0332 0.0620 53.229 −0.03635 −2 0.075 0 3 0 0.0247 0.0241 0.0630 39.20

10 0.3 0 0.11704 2 3 0 0.0200 0.0201 0.0580 34.4811 0.1 −1 0.100 1 5 1 0.0185 0.0184 0.0620 29.8312 0.3 0 0.075 0 3 0 0.0342 0.0332 0.0611 55.9713 0.5 1 0.050 −1 1 −1 0.0129 0.0119 0.0490 26.3214 0.5 1 0.010 1 5 1 0.0110 0.0104 0.0430 25.5815 0.3 0 0.075 0 6.36358 2 0.0073 0.0081 0.0380 19.2116 0.1 −1 0.050 −1 1 −1 0.0085 0.0078 0.0420 20.2317 0.5 1 0.100 1 1 −1 0.0168 0.0158 0.0590 28.4718 0.63635 2 0.075 0 3 0 0.0189 0.0208 0.0610 30.9819 0.3 0 0.075 0 3 0 0.0340 0.0332 0.0611 55.64

0

C

3

oabt

TA

x

20 0.3 0 0.03295 −2 3 0

DW = cell dry weight.

.5. Validation experiment

A growth linked validation has resulted in 56.77% yield (Fig. 5)f PHA production, validating the reliability of the statistical

pproach. The maximum value was observed within 24 h of incu-ation and resulted in 2.61 fold increment in PHA yield comparedo un-optimized conditions (21.77% yield).

able 4NOVA for CCD of DSM media components.

Source DF Seq SS Adj SS Adj MS F P

Regression 9 0.001976 0.001976 0.000220 137.66 0.0000Linear 3 0.000085 0.000085 0.000028 17.71 0.0000

x1 1 0.000013 0.000013 0.000030 8.19 0.0170x2 1 0.000000 0.000000 0.000000 0.02 0.8810x3 1 0.000072 0.000072 0.000072 44.91 0.0000

Square 3 0.001790 0.001790 0.000597 374.03 0.0000x1 * x1 1 0.000090 0.000210 0.000210 131.71 0.0000x2 * x2 1 0.000190 0.000309 0.000309 193.79 0.0000x3 * x3 1 0.001510 0.001510 0.001510 946.47 0.0000

Interaction 3 0.000102 0.000102 0.000034 21.25 0.0000x1 * x2 1 0.000002 0.000002 0.000002 1.02 0.3370x1 * x3 1 0.000053 0.000053 0.000053 33.26 0.0000x2 * x3 1 0.000047 0.000047 0.000047 29.49 0.0000

Residual error 10 0.000016 0.000016 0.000002Lack of fit 5 0.000012 0.000012 0.000002 3.18Pure error 5 0.000004 0.000004 0.000001Total 19 0.001992

1 = peptone; x2 = yeast extract; x3 = glucose.

.0190 0.0202 0.0630 30.15

3.6. Characterization of PHA biopolymer

3.6.1. FTIR analysisFig. 6 shows FTIR spectra of standard PHB and obtained biopoly-

mer. The absorption bands at 1726 cm−1 indicate ester carbonylgroup of PHA. In addition, the peaks at 1283, 1227 cm−1 are due tothe C O C stretching modes present in the polymer. The band at2932 cm−1 is characteristic of the stretching and deformation vibra-

tions of the O H groups. The presence of the absorption bands at2978 cm−1 may be due to the C H· · ·O hydrogen bond. The peakat 3440 cm−1 is attributed to the hydroxyl end group present. The

Table 5Estimated regression coefficient of PHA production by CCD.

Sr. no. Term Coefficient SE coefficient T P

1 Constant 0.033294 0.000515 64.635 0.00002 x1 −0.000978 0.000342 −2.861 0.01703 x2 −0.000053 0.000342 −0.154 0.88104 x3 0.00229 0.000342 6.701 0.00005 x1 * x1 −0.003818 0.000333 −11.477 0.00006 x2 * x2 −0.004631 0.000333 −13.921 0.00007 x3 * x3 −0.010235 0.000333 −30.765 0.00008 x1 * x2 −0.00045 0.000447 −1.008 0.33709 x1 * x3 −0.002575 0.000447 −5.767 0.0000

10 x2 * x3 −0.002425 0.000447 −5.431 0.0000

R2 = 0.9920; R2(predicted) = 0.9506; R2(adjusted) = 0.9848.x1 = peptone; x2 = yeast extract; x3 = glucose.

J.H. Dhangdhariya et al. / International Journal of Biological Macromolecules 76 (2015) 254–261 259

variab

Fse

3t

gbT

Fb

Fig. 4. Contour plots showing interactions between process

TIR results obtained from PHA produced (Fig. 6) by B. megateriumtrain JK4h was found to be corroborated to the work of Kulkarnit al. [9], Maaloul et al. [4].

.6.2. Differential scanning calorimetric analysis (DSC) andhermogravimetric analysis (TGA)

DSC analysis was done to find out the melting temperature,

lass transition temperature and heat associated in melting of PHAiopolymer. Fig. 7 shows DSC analysis of produced biopolymerm 176.6 ◦C which is similar to standard PHB (Sigma, USA), and

ig. 5. Validation experiment of growth linked biopolymer production as obtainedy CCD model.

les on biopolymer production by B. megaterium strain JK4h.

heat associated with melting is 2.627 J/(g K) and 16.211 J/(g K) forfirst and second peak respectively. TGA of biopolymer was doneto find out thermal stability of PHA. Fig. 7 revealed that degra-dation of PHA biopolymer is done mainly in two stages. In thefirst stage at around 180 ◦C, only 1.46% of the total mass was lostwhich is mainly due to low molecular size mass present in a sam-ple, in the second stage, at around 260–300 ◦C, 88.34% of the totalmass was lost (Fig. 7).

3.6.3. Nuclear magnetic resonance (NMR) analysis1H NMR resonances of the obtained polymer appeared at

ı = 1.6 ppm for methyl (CH3), ı = 2.4 ppm for methylene (CH2) and

Fig. 6. FTIR spectrum of extracted biopolymer of B. megaterium strain JK4h.

260 J.H. Dhangdhariya et al. / International Journal of Biological Macromolecules 76 (2015) 254–261

Fig. 7. Differential scanning calorimetric analysis of PHA (a) and thermogra

Ff

ım

4

bumommcisfTplsap

[[

[

[[[

[

ig. 8. 1H NMR spectra of standard PHA (a) and NMR spectra of biopolymer obtainedrom B. megaterium strain JK4h (b).

= 5.2 ppm for methane (CH) group confirming the produced poly-er was PHB when compared to standard PHB (Sigma, USA) (Fig. 8).

. Conclusion

Our previous study was focused on the use of Zobell marineroth for raising the inoculum and biodiesel wastes and crude lev-linic acid as production medium for the production of PHA througharine Halomonas hydrothermalis having optimum harvesting time

f 96 h, while the current study demonstrates the use of wild typearine B. megaterium strain JK4h using DSM as a novel growthedium for production of PHA in 24 h of incubation, minimizing the

hances of contamination, since, the optimum harvesting time is anmportant factor for the scaling up of the process. CCD optimizationtudies have revealed that maximum polymer productivity wasound to be 56.77%, exploiting marine B. megaterium strain JK4h.his piece of work enabled us to utilize coastal ecosystem in a welllanned manner through the wild strain of halophilic bacteria iso-

ated from the Arabian Sea and using DSM prepared in the solaralt pans for the culturing and further production of a biodegrad-ble polymer (PHB) in a short incubation time. This opens up theossibility of large scale production of biological macromolecules

[

[[

vimetric analysis (b) of PHA biopolymer of B. megaterium strain JK4h.

like PHA in cost-effective and environmental friendly manner infuture through the halophilic B. megaterium strain JK4h utilizingDSM of the present study.

This research would lead towards use of marine bioresources as“green” alternative for sustainable development of our oceans interms of keeping them free of toxic hazardous products.

Acknowledgements

CSIR – CSMCRI Ref no – 182/2014. Authors would like toacknowledge analytical discipline CSMCRI for the help in charac-terization of the sample and Mr. S.C. Upadhyay for providing usDSM. JD, HT, JB, BD would like to thank Earth Science and Technol-ogy Cell (ESTC), Ministry of Earth Sciences for financial support. Wewould also like to thank Dr. K.H. Mody, CSMCRI and Prof. S.R. Dave,Gujarat University, Ahmedabad for the help in Biolog. SD wouldlike acknowledge CSC 0203 for financial support. IP would like toacknowledge CSIR and AcSIR for fellowship and PhD registration.

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