Algal Research 7 (2015) 78–85

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Algal Research

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Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer productionby the diazotrophic cyanobacterium Nostoc muscorum Agardh: Processoptimization and polymer characterization

Ranjana Bhati 1, Nirupama Mallick ⁎Agricultural and Food Engineering Department, Indian Institute of Technology Kharagpur, Kharagpur 721302, West Bengal, India

⁎ Corresponding author.E-mail address: nm@agfe.iitkgp.ernet.in (N. Mallick).

1 Present address: Department of Microbiology, Bu284128, Uttar Pradesh, India.

http://dx.doi.org/10.1016/j.algal.2014.12.0032211-9264/© 2014 Published by Elsevier B.V.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 21 August 2014Received in revised form 4 November 2014Accepted 2 December 2014Available online xxxx

Keywords:Nostoc muscorum AgardhPHBP(3HB-co-3HV) copolymerDifferential scanning calorimetry (DSC)Thermogravimetric analysis (TGA)

Polyhydroxyalkanoate (PHA) homo- and copolymers are accumulated as cytoplasmic inclusions in awide variety ofbacteria. Cyanobacteria are emerging as a novel source for production of PHA polymers. In this report, response sur-face methodology (RSM) was used to evaluate the relationships between the critical variables that en-hanced the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer accumulation in a N2-fixingcyanobacterium, Nostoc muscorum Agardh. Using multifactor optimization strategy, a yield of 69% ofdry cell weight (dcw) ( 61 mg L−1 day−1) was achieved at reduced level of nutrients for an incubation periodof 7 days. The polymer productivitywas increased up to 98.3mg L-1day-1 (71% dcw) and 109.7mg L−1 day−1 (78% dcw),respectively under P- and N-deficiencies. Moreover, the poly-β-hydroxybutyrate (PHB) homopolymer and P(3HB-co-3HV) copolymer films produced by N. muscorum, were analyzed for their material (thermal andmechanical) proper-ties, and were compared with the PHAs obtained from other cyanobacterial and bacterial sources. Structural detailswere investigated by wide angle X-ray diffraction (WAXRD), which showed semicrystalline nature for PHB as wellas P(3HB-co-3HV) co-polymers. Thermal andmechanical properties of the polymer films produced by the test cyano-bacteriumare comparablewith the polymers obtained fromother cyanobacterial (Aulosira fertilissima CCC 444), bacte-rial (Cupriavidus necator) and the commercial polypropylene, and thus could be ensued for large-scale production.

© 2014 Published by Elsevier B.V.

1. Introduction

Over the years, substantial research efforts are being directed to de-velop biodegradable polymers that could substitute the conventionalpetrochemical-based plastics. In polyhydroxyalkanoate (PHA) family,poly-β-hydroxybutyrate (PHB) is themost common andwell character-ized member, and received much attention as a source for a novel bio-degradable plastic material. However, studies demonstrate that theproperties of PHB such as brittleness, low extension-to-break, and lackof flexibility limit its application [1]. To overcome these, production ofPHA copolymers is of current research interest. Byrom [1] reportedthat the copolymer P(3HB-co-3HV), i.e. poly(3-hydroxybutyrate-co-3-hydroxyvalerate) has two major advantages over PHB: (i) the meltingpoint, and (ii) the level of crystallinity, which are lower than that ofPHB. In addition, copolymers of P(3HB-co-3HV)with higher HV fractiontend to be softer and tougher [2].

Cyanobacteria are emerging as a novel source for production of PHApolymers [3–8]. Anabaena cylindrica 10 C was the first cyanobacterium

ndelkhand University, Jhansi

reported to accumulate P(3HB-co-3HV) copolymer under propionate-supplemented condition,with amaximumvalue of 2%of dry cellweight(dcw) only [9]. Subsequent studies conducted in our laboratory withNostoc muscorum with a few randomly selected variables revealed anaccumulation of the copolymer up to 31% (dcw) under propionate-supplemented condition [10]. Most recently, Samantaray and Mallick[8] reported an accumulation of 77% (dcw) in Aulosira fertilissima CCC444.

In our previous report [6], we have evaluated the suitability of vari-ous carbon sources, viz. glucose, acetate, maltose, fructose, sucrose, pro-pionate and valerate to enhance the production of P(3HB-co-3HV)copolymer inN.muscorumAgardh. Analysis of the results demonstratedthat the key factors affecting the performance in the terms of polymeraccumulation are concentrations of acetate, glucose and valerate.Incubation period was also found to affect the polymer accumulationsignificantly. This warrants further research on the interaction of multi-variables on polymer yield using appropriate method(s). One of themost widely used tools for studying the interaction of process variablesis multifactor optimization using response surface methodology (RSM).

The aim of the present study is therefore, to optimize the four mostcritical variables, viz. concentrations of glucose, acetate and valeratewith days of incubation to maximize P(3HB-co-3HV) copolymeraccumulation in N. muscorum Agardh. The mechanical and thermal

Table 2Central composite design matrix with actual and predicted responses of P(3HB-co-3HV)co-polymer accumulation.

Run Process variable Y = polymercontent (% dcw)

A B C D Actual Predicted

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properties bear immense importance for recommending the polymerfor any application. Hence, the PHB homopolymer and P(3HB-co-3HV)copolymer films produced by N. muscorum Agardh were characterizedand compared with the PHA polymers obtained from othercyanobacterial (A. fertilissima CCC 444) and bacterial (Cupriavidusnecator) sources, and also with the commercial polypropylene.

1 0.4 (0) 0.4 (0) 0.4 (0) 10 (0) 67.2 67.92 0.55 (+1) 0.55 (+1) 0.5 (+1) 7 (−1) 63.1 62.23 0.55 (+1) 0.25 (−1) 0.3 (−1) 13 (+1) 44.5 44.84 0.25 (−1) 0.55 (+1) 0.3 (−1) 7 (−1) 67.3 69.35 0.4 (0) 0.4 (0) 0.6 (+2) 10 (0) 41.0 44.16 0.25 (−1) 0.55 (+1) 0.5 (+1) 7 (−1) 61.0 59.47 0.55 (+1) 0.25 (−1) 0.3 (−1) 7 (−1) 54.4 54.78 0.4 (0) 0.4 (0) 0.4 (0) 10 (0) 67.2 67.99 0.25 (−1) 0.25 (−1) 0.5 (+1) 7 (−1) 53.0 53.910 0.7 (+2) 0.4 (0) 0.4 (0) 10 (0) 55.8 57.011 0.4 (0) 0.4 (0) 0.4 (0) 10 (0) 69.6 67.912 0.25 (−1) 0.25 (−1) 0.5 (+1) 13 (+1) 37.3 35.113 0.1 (−2) 0.4 (0) 0.4 (0) 10 (0) 59.3 58.314 0.4 (0) 0.7 (+2) 0.4 (0) 10 (0) 58.7 58.615 0.55 (+1) 0.55 (+1) 0.3 (−1) 13 (+1) 58.8 58.316 0.4 (0) 0.4 (0) 0.4 (0) 10 (0) 67.5 67.917 0.55 (+1) 0.25 (−1) 0.5 (+1) 7 (−1) 48.7 47.518 0.4 (0) 0.1 (−2) 0.4 (0) 10 (0) 39.2 39.619 0.25 (−1) 0.55 (+1) 0.5 (+1) 13 (+1) 38.5 39.120 0.4 (0) 0.4 (0) 0.4 (0) 10 (0) 66.8 67.921 0.4 (0) 0.4 (0) 0.4 (0) 10 (0) 69.1 67.922 0.55 (+1) 0.55 (+1) 0.5 (+1) 13 (+1) 47.9 46.523 0.25 (−1) 0.55 (+1) 0.3 (−1) 13 (+1) 53.2 53.224 0.25 (−1) 0.25 (−1) 0.3 (−1) 13 (+1) 47.0 48.925 0.55 (+1) 0.55 (+1) 0.3 (−1) 7 (−1) 68.8 69.826 0.4 (0) 0.4 (0) 0.4 (0) 4 (−2) 63.9 63.427 0.25 (−1) 0.25 (−1) 0.3 (−1) 7 (−1) 63.3 63.428 0.4 (0) 0.4 (0) 0.4 (0) 16 (+2) 32.5 33.229 0.55 (+1) 0.25 (−1) 0.5 (+1) 13 (+1) 34.3 33.330 0.4 (0) 0.4 (0) 0.2 (−2) 10 (0) 68.4 65.5

2. Materials and methods

2.1. Growth conditions of the test organism, and extraction and assay ofpolymers

Axenic cultures of the filamentous nitrogen-fixing cyanobacterium,N. muscorum Agardh were grown in 250 mL Erlenmeyer flasks contain-ing 100mL nitrate-free BG-11medium [11]. Cultures were incubated ina temperature-controlled culture room at 25 ± 2 °C, pH 8.0, under aphotoperiod of 14:10 h andat light intensity of 75 μmol photonm−2 s−1

1 PARwithout sparging with air or CO2. This was referred to as the con-trol culture. Biomass was harvested at stipulated time intervals near tothe endof the light cycle, andwas dried under vacuumat 60 °C. Polymerwas extracted from the dried biomass in hot chloroform following theprotocol of Yellore and Desia [12]. Detection and quantification ofP(3HB-co-3HV) copolymer was done by gas chromatography (Clarus500, Perkin Elmer, Shelton, CT, USA) following Bhati and Mallick [6].The polymer sample was thoroughly mixed with KBr in a ratio of1:100, and a thin pellet (12 mm diameter with 1 mm thickness) wasmade using hydraulic press. The functional groups present in the poly-mers were analyzed by a FTIR spectrophotometer (NEXUS 870, ThermoNicolet Co., USA). The spectra were recorded within the range of500–4000 cm−1.

Values in the parentheses denote coded level of the variables.

2.2. Optimization study

A central composite rotary design (CCRD) with five levels of fourvariables requiring 30 experiments (‘design-Expert®’, version 7.1.1,Stat-Ease Inc., Minneapolis, USA)was used in this study. Concentrationsof acetate (A), glucose (B) and valerate (C) with incubation period(D) were the independent variables selected to optimize the P(3HB-co-3HV) copolymer accumulation in the test cyanobacterium. Freshlyprepared inoculum of N. muscorum Agardh was transferred to BG-11medium with varying concentrations of acetate, glucose and valeratefor stipulated periods as given in Table 1. Experimental data obtainedfrom CCRD were analyzed with the help of RSM. ‘Point optimization’technique was used to find out the level of each variable for maximumresponse.

2.3. Effects of N-/P-deficiency on polymer accumulation

As deficiencies of nitrogen and phosphorus are known to triggerpolymer accumulation, the interaction of the optimized conditionwith the above deficiencies was also elucidated. These conditionswere achieved following Samantaray and Mallick [8].

Table 1Variables and levels of experimental design for response surface.

Independent variable Codedsymbol

Level

−2(−α)

−1 0 1 2(+α)

Acetate (% w/v) A 0.1 0.25 0.4 0.55 0.7Glucose (% w/v) B 0.1 0.25 0.4 0.55 0.7Valerate (% w/v) C 0.2 0.3 0.4 0.5 0.6Incubation period (days) D 4 7 10 13 16

2.4. Characterization of the homo- and co-polymers

2.4.1. Casting of the filmsThick films of PHB and P(3HB-co-3HV) polymers were prepared at

room temperature by chloroform solvent casting technique with 1% so-lution using glass Petri dishes as casting surface [13]. The resulting filmswere further dried at 50 °C for 3 h to remove any residual solvent andmoisture, and were taken for further analysis.

2.4.2. Differential scanning calorimetry (DSC)For analyzing the thermal properties, DSC was performed with the

help of a Pyris DiamondDifferential ScanningCalorimeter (PerkinElmer,USA). 5 mg of the sample was exposed to−50 to 200 °C at a rate of 10°Cmin−1. The melting temperature (Tm) was determined from the DSCendotherm. For measurement of the glass-transition temperature (Tg),the sample was maintained at 200 °C for 1 min, and then rapidlyquenched to −150 °C. The sample was again heated to 200 °C at arate of 20 °C min−1. Tg was taken as the midpoint of the heat capacitychange [14].

Table 3ANOVA analysis of the response surface quadratic model.

Source Sum of squares Df Mean squares F-value Probability N F

Model 3994.80 14 285.34 84.46 b0.0001Residual 50.67 15 3.37Lack of fit 44.21 10 4.42 3.42 0.0934Pure error 6.45 5 1.29Corrected Total 4045.48 29

Coefficient of determination (R2) = 0.9874, adjusted R2 = 0.9757.Adequate precision = 28.16, coefficient of variation (CV) = 3.3%.

(AA)

((C)

(BB)

Fig. 1. 3D response surface: interactive effects of (A) varied glucose and acetate concentrations at zero level of valerate concentration and days of incubation, (B) varied days of incubationand acetate concentration at zero level of glucose and valerate concentrations, and (C) varied days of incubation and valerate concentration at zero level of glucose and acetateconcentrations.

80 R. Bhati, N. Mallick / Algal Research 7 (2015) 78–85

The degree of crystallinity (Xc) was calculated as follows:

Xc ¼ ΔHm=ΔH0PHB

� �� 100

where, ΔHm is the measured enthalpy of melting of the given sample,and ΔH0

PHB is the enthalpy of melting of PHB [15].

2.4.3. Thermogravimetric analysis (TGA)TG analyses of PHB and P(3HB-co-3HV) polymers were done with

the help of Pyris Dimond TG–DTA machine (PerkinElmer, USA) at aheating rate of 10 °C min−1 over a temperature range of 50–350 °Cunder nitrogen atmosphere, as detailed in Samantaray and Mallick [8].

Table 4Co-polymer content before and after optimization of critical variables.

Variable Before optimization After optimization

Acetate (% w/v) 0.4 0.28Glucose (% w/v) 0 0.38Valerate (% w/v) 0.4 0.30Incubation period (days) 10 7

2.4.4. Mechanical propertiesFor mechanical test, films were cut to rectangular shape (length:

60 ± 0.5 mm, width: 20 ± 0.2 mm), with thickness 0.35 ± 0.03 mm,and were measured by a dial gauge. The ‘elongation-to-break’ value,‘Young's modulus’ and ‘tensile strength’ were determined using auniversal testing machine (ElectroPuls E1000, Instron, UK) at roomtemperature. The extension rate was 10 mmmin−1. Mechanical ten-sile data were calculated from such curves on an average of threereplicates.

2.4.5. Crystalline structureThe crystalline structure of the polymer samples was studied by X-

ray diffraction (XRD), andmore precisely bywide angle X-ray scattering

P(3HB-co-3HV) co-polymer content (% dcw)

Before optimization After optimization

Predicted Experimental

50.2 71.4 69.0 ± 1.7

(ΑΑ)

((Β)

Fig. 2. (A) Normal % probability of internally studentized residuals. (B) Plot of internallystudentized residuals vs. predicted response.

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(WAXS) using an X-ray diffractometer (model PW 1710, Philips, Hol-land). A Philips-model PW 1729 X-ray generator with Fe filter was op-erated at 40 kV and 20 mA which provides a Co Kα radiation (λ =1.79 Å). The scattering angel (2θ) covered was from 10 to 80° at a stepof 0.05° and sampling interval of 1 s.

3. Results

3.1. Response surface optimization

The results at each point based on the experimental design demon-strated a variation in polymer content between 32.5 and 69.6% (dcw) at

Table 5Interactive effects of the optimized condition with N- and P-deficiencies on P(3HB-co-3HV) co

Culture condition Biomass (mg L−1) Polymer content

(mg L−1) (% dc

aOptimized condition 619.3 ± 2.5b 427.3 ± 1.1a 69.0 ±Optimized condition + N-deficiency 561.2 ± 3.3a 438.9 ± 1.0a 78.2 ±Optimized condition + P-deficiency 553.7 ± 4.0a 393.1 ± 0.9b 71.0 ±

All values are mean ± SE, n = 3.Values in the column superscripted by different letters are significantly (P b 0.05) different froSeparate analysis was done for each column.

a Polymer was extracted after 7 days as per the optimized condition.

different combinations of the variables (Table 2). The predicted values,calculated using the model were within the range of 33.2–69.8%(dcw). Regression analysis of the actual response demonstrated thatthe linear model terms (B, C and D), quadratic model terms (A2, B2, C2

and D2) and the interactive model terms (AB, AD and CD) were signifi-cant (Pb 0.05). However, the interactivemodel termsAC, BC, and BDdidnot depict significant effects on the polymer accumulation (P N 0.05).

Results were fitted to a second-order polynomial equation by apply-ingmultiple regression analysis and the followingmathematical regres-sion model for polymer accumulation was obtained in terms of codedfactors:

Y P 3HB−co−3HVð Þpolymer content % dcwð Þ½ � ¼ þ67:88–0:32Aþ4:76B–5:32C–7:56Dþ 2:29ABþ 0:58ACþ 1:15AD–0:07BC–0:38BD–1:04CD2:55A2

–4:69B2–3:26C2

–4:90D2:

The analysis of variance (ANOVA) report is presented in Table 3. Themodel was found to be highly reliable with R2 value of 0.9874 and ‘ad-justed R2’ value of 0.9757. The F-value of 84.46 implied the significanceof the model (probability N F= 0.0001). A very low value of coefficientof the variation (CV: 3.3%) again demonstrated a very high degree ofprecision and reliability of the experimental data.

To investigate the interactive effects of the four critical variables oncopolymer accumulation, the response surface and contour plots(Fig. 1) were generated as graphical representations of the regressionequation. Mutual interactions between the independent variableswhether significant or not are indicated by the shape of the correspond-ing contour plot. In the 3D response surface graphs (Fig. 1A–C), ellipticalnature of contours depicted that the mutual interactions between thevariables were highly significant. The plot based on varying the concen-tration of glucose and acetate is shown in Fig. 1A, where the other twovariables, i.e. concentration of valerate and incubation period werekept at a constant (zero) level. In this plot, the copolymer accumulationwas found to increase with increase in the level of glucose and acetateabove the zero level. The interaction of acetate and incubation periodat zero level of glucose and valerate concentration is shown in Fig. 1B,where the copolymer content increased with the decrease in the levelof incubation period at zero level of acetate. Similarly, Fig. 1C depictsthe interaction of incubation period and valerate concentration at zerolevel of acetate and glucose, where an increase in valerate concentrationtill certain point contributes for increasing polymer accumulation atzero level of incubation period. On the other hand, the interactivemodel terms AC (varying acetate and valerate concentrations at zerolevel of glucose and incubation period), BC (varying glucose and valer-ate concentrations at zero level of acetate and incubation period) andBD (varying glucose concentrations and incubation period at zerolevel of acetate and valerate) are not shown graphically since they didnot illustrate significant effects on the copolymer accumulation, whichwas reflected from the flat response surface with parallel contourlines. Optimization was carried on with the help of ‘point optimization’technique. The optimum condition of the selected variables for maxi-mum copolymer accumulation is presented in Table 4; a maximum co-polymer content of 71.4% (dcw) was predicted at 0.28% (w/v) acetate,

polymer accumulation in N. muscorum Agardh after 4 days incubation.

Composition (mol %) Polymer productivity (mg L−1 day−1)

w) 3HB 3HV

0.6a 83.9 ± 0.4b 16.1 ± 0.3a 61.0 ± 1.3a

0.7bc 73.2 ± 0.5a 26.8 ± 0.2b 109.7 ± 1.1c

0.6b 72.8 ± 0.5a 27.2 ± 0.2b 98.3 ± 0.93b

m each other (Duncan's new multiple range test).

Fig. 3. FTIR spectra of A) PHB homopolymer, B) P(3HB-co-8% 3HV), C) P(3HB-co-16%3HV), and D) P(3HB-co-27% 3HV) co-polymers films obtained from N. muscorum Agardh.

82 R. Bhati, N. Mallick / Algal Research 7 (2015) 78–85

0.38% (w/v) glucose, and 0.3% (w/v) valerate for an incubation period of7 days.

Validation of the model was done with experiments performed intriplicate, and repeated thrice under the aforementioned optimal condi-tion. Predicted (71.4%) and experimental (69.0%) values, after optimiza-tion are not varied significantly and hence, the model was successfullyvalidated. In order to judge the model adequacy, residuals from theleast square fit play an important role in judging the model adequacy[16]. Normality assumption (Fig. 2A)was checked by constructing a nor-mal probability plot of the residuals. The normality assumption was sat-isfied as the residual plot approximated along a straight line. A plot ofresiduals versus the predicted response is presented in Fig. 2B. Both theplots (Fig. 2A and B) were found highly satisfactory, thus depictinggood reliability of the model. Copolymer content of 427.3 mg L−1 (69%dcw) and biomass concentration of 619.3 mg L−1 was recorded withnet PHA productivity of 61.0 mg L−1 day−1. The polymer productivitywas increasedup to 98.3mgL-1 day-1 (71%dcw)and109.7mgL−1 day−1

(78% dcw), respectively under P- and N-deficiency (Table 5).

3.2. Properties of the polymer

3.2.1. Chemical structureThe FTIR spectra of PHB homopolymer and P(3HB-co-3HV) copoly-

mer films with different mol% of HV depicted characteristic bands(Fig. 3), showing a strong transmittance at 1724 and 1280 cm−1, corre-sponding to C_O and C\O stretching groups, respectively. Transmit-tance region from 2800 to 3100 cm−1 corresponds to stretching

Table 6Thermal and mechanical properties of PHA polymers with varying HV fractions produced by N

Monomercomposition(mol %)

Tg(°C)

Tm(°C)

ΔHm

(J g−1)Xc

(%)Td(5%)(°C)

HB HV

100 0 0.8 178 91.7 62.8 25292 8 −1.2 165 82.6 56.6 26284 16 −2.2 154 65.2 44.7 26973 27 −4.7 145 57.7 39.5 275

Values are means of three samples.

vibration of C\H bonds of methyl (CH3) and methylene (CH2) groups.Although the functional groups were found to be similar in the homo-and the copolymer samples, the relative band intensity was found tovary significantly; the relative intensity of the band at 1720 cm−1 wasbetter resolved than other bands and increased significantly with in-creasing HV content.

3.2.2. Differential scanning calorimetric (DSC) analysisThe thermal characteristics of the homo- and the copolymer films

from N. muscorum Agardh were inferred from DSC thermograms (datanot shown). Double melting peaks were observed for P(3HB-co-3HV)copolymer samples with different HV contents. The Tm of P(3HB-co-3HV) copolymers decreased from 178 to 145 °C with an increase inthe 3HVunits from0 to 27mol% (Table 6). Similarly, the Tg and enthalpyof fusion (ΔHm) values were also decreased from 0.8 to −4.7 °C and91.7 to 57.7 J g−1, respectively. The degree of crystallinity (Xc) can becalculated from enthalpy of fusion. The crystallinity of the copolymersthus showed lower values as compared to PHB homopolymer (de-creased from 62.8 to 39.5%with increase in 3HV content from 0 to 27%).

3.2.3. Thermogravimetric analysis (TGA)TGA analysis was carried out to determine the degradation temper-

ature of the polymers produced fromN. muscorumAgardh. The temper-ature at 5% weight loss (Td(5%)) was evaluated to check the thermalstability of the polymers. The initial decomposition temperature ofPHB was observed to be 223 °C, and 5% weight loss of the polymerwas recorded at 252 °C. The 5% weight loss for P(3HB-co-3HV) copoly-mer with 8, 16 and 27 mol% HV was observed at 262, 269 and 275 °C,respectively (data not shown). Maximum degradation temperature,i.e. Tmaxwas found to be 274 °C for PHB as obtained fromderivative ther-mo gravimetric (DTG) analysis. The Tmax for P(3HB-co-3HV) copolymerwith 8 mol% HV was 278 °C, whereas the maximum degradation tem-perature for copolymer with 16 and 27 mol% HV units was found tobe 285 and 291 °C, respectively.

3.2.4. Mechanical propertiesP(3HB-co-3HV) copolymers with different 3HV fractions were char-

acterized for their mechanical properties (tensile strength, Young'smodulus and elongation-to-break), and were compared with the PHBhomopolymer (Table 6). Young's modulus of the copolymer showed adecreasing trend with increasing 3HV mol%. The tensile strength ofthe polymers also showed a decreasing trend with an increase in 3HVfraction and varied from 20.9 to 32.4 MPa. Elongation-to-break valuesshowed a profound rise with incorporation of 3HV monomers and thecopolymer with 27 mol% 3HV fraction depicted a rise by 19-fold.

Fig. 4 shows the WAXRD patterns of the P(3HB-co-3HV) samplescontaining 0 to 27 mol% HV. WAXRD diffractograms of P(3HB-co-3HV) polymers containing 0 to 27 mol% HV units showed that thesepolymers were semicrystalline in nature, and the presence of only onecrystalline phase even with different HV mol%. PHB homopolymershowed characteristic reflection patterns at 2θ = 15.6°, 19.9°, 26.2°and 29.8°. Similar diffraction peaks were also observed for P(3HB-co-

. muscorum Agardh.

Tmax

(°C)Tensile strength(MPa)

Young's modulus(GPa)

Elongation- to-break(%)

274 32.4 3.8 4.9278 29.3 2.8 61.2285 26.5 1.2 72.1291 20.9 0.6 92.3

Fig. 4. X-ray diffractograms of A) PHB, B) P(3HB-co-8% 3HV), C) P(3HB-co-16% 3HV), andD) P(3HB-co-27% 3HV) films from N. muscorum Agardh.

83R. Bhati, N. Mallick / Algal Research 7 (2015) 78–85

3HV) copolymers with different mol% of HV produced by the testcyanobacterium.

4. Discussion

Optimization of critical variables affecting P(3HB-co-3HV) copoly-mer accumulation in any organism is important for maximizing poly-mer production. Analysis of variance of the predicted model (Table 3)showed the adequacy of the model with a very high ‘F’ value (84.46)and low ‘P’ value (b0.0001). The R2 value of 0.987 shows highly signifi-cant correlation between the experimental and predicted values. Thecloser the R2 is to 1, the stronger the model and the better it predictsthe response [16]. In addition, the high value of adjusted R2, higher de-gree of adequate precision and insignificant ‘lack of fit’ also advocatedfor the significance of the model. Reliability and better precision of theexperiments carried out was indicated by a relatively lower value of CV.

Fig. 5. Proposed biosynthetic pathway forAdapted from [18,19,35].

All the variables considered in this study, viz. concentrations ofacetate, glucose and valerate with incubation period contributedsignificantly to the accumulation of P(3HB-co-3HV) copolymer inN. muscorum Agardh. P(3HB-co-3HV) copolymer biosynthesis requires3-hydroxybutyryl-CoA (3HB-CoA) and 3-hydroxyvaleryl-CoA (3HV-CoA) as substrates for the polymerization reaction catalyzed by the en-zyme, PHA synthase (Fig. 5). Condensation of propionyl-CoA and acetyl-CoA to 3-ketovaleryl-CoA which further by the action of the enzymeacetoacetyl-CoA reductase resulted to form 3HV-CoA [17,18]. Therefore,to promote the formation of 3HV-CoA, the addition of 3HV precursors isneeded. Valerate was reported to act as precursors for incorporation of3-hydroxyvalerate (3HV) units into PHB backbone [19]. Enhanced poly-mer accumulation with acetate supplementation could be ascribed tothe increased availability precursor, i.e. acetyl-CoA [20,21]. The positiveeffect of glucose could be due to the increased supply of reduced cofac-tor NADPH along with acetyl-CoA, which are prerequisites for the en-zyme acetoacetyl-CoA reductase activity [22]. The molar ratio of 3HBand 3HV units in the copolymer could be regulated by controlling theconcentration of 3HV to 3HB precursors [20].

The contour plots in 3D response surface depicted the variation inthe copolymer content of cells as a function of interaction of variables(Fig. 1). In Fig. 1A–C it could be concluded that decreasing the concen-trations of acetate and valerate and shortening the incubation periodwith increased glucose addition resulted into higher polymer accumula-tionwithin the surface. It could be visualized in Table 4 that the productaccumulationwas increased up to 69% (dcw) after optimization. The re-quirement of acetate and valerate was also reduced by 30 and 22%, re-spectively. This is imperative from large-scale production point ofview as acetate and valerate are more expensive substrates.

Assessment of the thermal and mechanical properties of the poly-mers carries great importance for their applications. Melting tempera-ture is an important feature for polymer processing [13]. The meltingtemperature of P(3HB-co-3HV) copolymer with various 3HV fractions

PHB and P(3HB-co-3HV) co-polymer.

Table 7A comparison on the properties of PHAfilms produced byN. muscorumAgardhwith the PHA polymers obtained from Aulosira fertilissima CCC 444, Cupriavidus necator and the commercialpolypropylene.

Property Commercial Polymers Alcaligenes eutrophus(Cupriavidus necator)

Aulosira fertilissima CCC 444 Nostoc muscorum Agardh

Polypropylene PHB P(3HB-co-3HV)(mol fraction)

PHB P(3HB-co-3HV) (molfraction)

PHB P(3HB-co-3HV) (molfraction)

100:0 90:10 80:20 100:0 88:12 75:25 100:0 92:8 84:16 73:27

Tm (°C) 176 177 140 130 174 168 155 178 165 154 145Tg (°C) −10 nr nr nr 0.6 −3.8 −5.5 0.8 −1.2 −2.2 −4.7Xc (%) 50–70 80 60 35 60.7 52.6 46.1 62.8 56.6 44.7 39.5Young's modulus (GPa) 1.7 3.5 1.2 0.8 3.4 1.2 0.6 3.8 2.8 1.2 0.6Tensile strength (MPa) 38 40 25 20 37.6 29.5 18.1 32.4 29.3 26.5 20.9Elongation to break (%) 400 8 20 50 4.9 75.6 87.2 4.9 61.2 72.1 92.3Reference Vincenzini and De Philippis [34] Luzier [33] Samantaray and Mallick [7,8] This study

84 R. Bhati, N. Mallick / Algal Research 7 (2015) 78–85

showed a declining trend with increasing HV fraction (Table 6). Similarobservation was reported by Yoshie et al. [23]. This is advantageous aslow Tm widens the processing range of the polymer as compared toPHB for which themelting temperature is very close to the degradationtemperature [13]. The enthalpy of fusion was also found to decreasewith increasing HV% (Table 6). As the enthalpy of fusion decreased,the crystallization of PHBwasmore andmore hindered with increasingHVmol%, andmore imperfect crystals are formed in the copolymer [24].The degree of crystallinity of PHB was found to be highest (62.8%)than that of the P(3HB-co-3HV) copolymer with 8, 16 and 27 mol% HV(56.6, 44.7, 39.5%, respectively) (Table 5). These are well in accordwith the findings of Kulkarni et al. [25]. Furthermore, the Tg value de-creased from 0.8 °C to −4.7 °C with increasing 3HV fraction from 0 to27 mol%. This is known to be due to the presence of 3HV monomerswhich increased the mobility in the amorphous state, thus offering alarger free volume of molecular movement [26,27]. Thermal stabilityplays an important role in polymer melt processing. This characteristicwas determined by TGA. The temperature at 5% weight loss (Td(5%))was used to evaluate the polymer thermal stability [27]. Thermogravi-metric analysis showed alteration of thermal stability of copolymerswith the incorporation of 3HV units. The Td(5%) of the three isolated co-polymersfilms ranged from262 to 275 °C, higher than thePHB (252 °C),thus highlighting its improved thermal stability (Table 5).

In Table 6, it is also clear that the PHBfilms obtained from the test cy-anobacterium is a brittle material with low elongation-to-break value(4.9%), whereas the copolymers are less brittle in nature. Young's mod-ulus of the copolymers decreased significantly with increasing 3HVmol%. The low Young's modulus demonstrates high flexibility of the co-polymer produced by the test cyanobacterium. The decrease in Young'smodulus from3.8 to 0.6 GPa is well in accordwith the report of Doi [20],where the elastic modulus decreased with increasing HV fraction andthe copolymer with 25 mol% HV possesses a value of 0.7 GPa. Tensilestrength varied between 21 and 32 MPa, which is also in accord withthe earlier findings [13,14]. Elongation-to-break values showed a pro-found rise with incorporation of 3HV monomers, thus demonstratinghigh elasticity of the copolymers of N. muscorum.

WAXRD again describes the crystalline structure of P(3HB-co-3HV)copolymers produced by N. muscorum Agardh (Fig. 4). According toYamanea et al. [28], PHB is crystallized in orthorhombic unit cell withlattice parameter a = 0.576 nm, b = 1.320 nm, c = 0.596 nm.WAXRD patterns of the P(3HB-co-3HV) copolymer samples containing0 to 27mol% correspond to the patterns given by the PHB crystalline lat-tice as observed by earlier studies [29–31]. It has been reported thatP(HB-co-HV) crystallizes as PHB crystalline lattice for HV contentlower than 50 mol% [32,24]. Our result is consistent with these reports.

A comparison on the thermal and mechanical properties of PHApolymers obtained from N. muscorum Agardh with polymers of othercyanobacterial (A. fertilissima CCC 444), bacterial (Cupriavidus necator)sources and the commercial polypropylene is presented in Table 7. Poly-mers produced by N. muscorum Agardh exhibited crystallinity in the

range of 40–63%, which is quite in tune with the polymers ofA. fertilissima CCC 444. Interestingly, the copolymers obtained showedbetter flexibility than the commercial polypropylene (Table 6). Howev-er, a reduction in tensile strength was observed whichwas in tunewiththe report of Luzier [33]. Overall, the thermal andmechanical propertiesof the films obtained from both the filamentous cyanobacteria(N. muscorum Agardh and A. fertilissima CCC 444) were comparablewith the polymers obtained from C. necator, although the elongation-to-break valueswere significantly lower as compared to the polypropyl-ene. A profound rise in elongation-to-break value (4.9 to 92.3%)was ob-served with incorporation of HV monomers in to the PHB backbone,which could be improved further by increasing the mol% of 3HV units.

5. Conclusion

In conclusion,N. muscorumAgardh is capable of accumulating P(3HB-co-3HV) copolymerup to 69%of dry cellweight ( 61mg L−1 day−1) underthe optimized condition. Copolymer productivity reached up to110mg L−1 day−1 (78% dcw) under N-deficient condition, which is com-paratively higher than the reported value for A. fertilissima CCC 444 [8].This is the second photosynthetic organism, which has shown such highpolymer accumulating potential. It has been observed that the thermaland mechanical properties of the polymer produced varied significantlywith variation in the mol% of 3HV monomers. The variation in 3HV con-tent in P(3HB-co-3HV) co-polymer is highly desirable for industrial appli-cations, as it offers possibilities of producing a range of thermoplasticswith varying degrees of flexibility and toughness. Therefore, further stud-ies are needed at pilot-scale tomake ‘cyano-plastics’ a commercial reality.

Acknowledgment

Financial support from the Council of Scientific and Industrial Re-search (CSIR 38(1146)/07/EMRII), New Delhi, India is thankfullyacknowledged.

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