Production and characterization of poly-β-hydroxybutyrate(PHB) polymer from Aulosira fertilissima

Shilalipi Samantaray & Nirupama Mallick

Received: 22 April 2011 /Revised and accepted: 29 June 2011 /Published online: 27 July 2011# Springer Science+Business Media B.V. 2011

Abstract Biopolymers such as polyhydroxyalkanoates(PHAs) are a class of secondary metabolites with promisingimportance in the field of environmental, agricultural, andbiomedical sciences. To date, high-cost commercial productionof PHAs is being carried out with heterotrophic bacterialspecies. In this study, a photoautotrophic N2-fixing cyanobac-terium, Aulosira fertilissima, has been identified as a potentialsource for the production of poly-β-hydroxybutyrate (PHB).An accumulation up to 66% dry cell weight (dcw) wasrecorded when the cyanobacterium was cultured in acetate(0.3%) + citrate (0.3%)-supplemented medium against 6%control. Aulosira culture supplemented with 0.5% citrateunder P deficiency followed by 5 days of dark incubationalso depicted a PHB accumulation of 51% (dcw). PHBcontent of A. fertilissima reached up to 77% (dcw) under Pdeficiency with 0.5% acetate supplementation. Optimizationof process parameters by response surface methodologyresulted into polymer accumulation up to 85% (dcw) at0.26% citrate, 0.28% acetate, and 5.58 mg L−1 K2HPO4 foran incubation period of 5 days. In the A. fertilissima culturespre-grown in fructose (1.0%)-supplemented BG 11 medium,when subjected to the optimized condition, the PHB poolboosted up to 1.59 g L−1, a value ∼50-fold higher than thecontrol. A. fertilissima is the first cyanobacterium where PHBaccumulation reached up to 85% (dcw) by manipulating thenutrient status of the culture medium. The polymer extractedfrom A. fertilissima exhibited comparable material properties

with the commercial polymer. As compared with heterotro-phic bacteria, carbon requirement in A. fertilissima for PHBproduction is lower by one order magnitude; thus, low-costPHB production can be envisaged.

Keywords Aulosira fertilissima . DSC .Mixotrophy .

P deficiency . Poly-β-hydroxybutyrate . RSM

Introduction

Polyhydroxyalkanoates (PHAs), long known in microbiologyas natural storage compounds, have tremendous potential asbiomaterials. Poly-β-hydroxybutyrate (PHB) is the bestcharacterized member of the PHA family and is widespreadin prokaryotic organisms (Liebergesell et al. 1994). PHB hasvarious properties such as natural origin, biodegradability,biocompatibility, streospecificity, piezoelectricity, opticalpurity, and thermoplasticity, which make it suitable for avariety of applications in the field of environmental,agricultural, and biomedical sciences (Fukada and Ando1986; Duvernoy et al. 1995).

Nowadays, commercial production of PHB is being carriedout with Wautersia eutropha (now called Cupriavidusnecator) by Metabolix, Massachusetts, USA, under fermen-tation condition. However, the use of PHB produced bybacterial fermentation is limited due to its high productioncost contributed by the expensive carbon sources and richoxygen supply during the fermentation process (Lee 1996).Synthesis of PHB in agricultural crops has also beentried. Expression of the enzymes for PHB synthesis fromC. necator into the plastid of Arabidopsis thaliana wasfound to produce PHB up to 14% of shoot dry weight(Nawrath et al. 1994). In an effort to bring this technology tothe field, a group of scientists at Monsanto has shown a

S. Samantaray :N. Mallick (*)Agricultural and Food Engineering Department,Indian Institute of Technology Kharagpur,Kharagpur 721302, West Bengal, Indiae-mail: nm@agfe.iitkgp.ernet.in

J Appl Phycol (2012) 24:803–814DOI 10.1007/s10811-011-9699-7

PHB biosynthetic pathway in the chloroplasts of stalks andleaves of corn and reported a level of PHB only up to 6%(Mitsky et al. 2000). But it was observed that plantsaccumulating PHB higher than 5% of dry weight werechlorotic; growth reduction and a negative impact onchloroplast function were also observed (Rezzonico et al.2002). Moreover, low expression level, long growthperiod, and difficulties in isolating the PHB from othercellular components are the major disadvantages in plant-based PHB production.

In this context, cyanobacteria are emerging as analternative host system due to their minimal nutrientrequirements and photoautotrophic nature. These organismswith a short generation time need some simple inorganicnutrients such as phosphate, nitrate (not in case of nitrogenfixers), magnesium, sodium, potassium, and calcium asmacronutrients and Fe, Mn, Zn, Mo, Co, B, and Cu asmicronutrients for their growth and multiplication. To date,about 50 cyanobacterial species, belonging to more than 14different genera, have been analyzed for the presence ofPHAs, but the contents, in general, are found to be low andamounted to <10% of dry cell weight (dcw) underphotoautotrophic growth condition (Vincenzini and DePhilippis 1999). A major breakthrough in cyanobacterialPHAs research was, however, obtained by Miyake and hisgroup with a thermophilic cyanobacterium, SynechococcusMA19, isolated from the surface of a wet volcanic rock inJapan, reported to accumulate PHB up to 55% (dcw;Nishioka et al. 2001). Subsequently, Sharma and Mallick(2005) and Panda and Mallick (2007) demonstrated PHBaccumulation up to 43% and 38% (dcw), respectively, inNostoc muscorum and Synechocystis sp. PCC 6803 undervarious specific conditions. Mallick and her group alsoobserved the accumulation of P(3HB-co-3HV) copolymerin these test cyanobacteria under propionate/valerate-supplemented conditions (Mallick et al. 2007; Panda2008). In this report, an unexplored diazotrophic cyano-bacterium, Aulosira fertilissima, biomass was examined as apossible potent source for the production of PHB byidentifying and evaluating the interrelationships betweenthe critical variables that influence PHB accumulation withthe help of a multifactor optimization study, i.e., responsesurface methodology (RSM). The PHB film was furthercharacterized for future applications.

Materials and methods

The filamentous nitrogen-fixing cyanobacterium, Aulosirafertilissima CCC 444 (National Centre for Conservationand Utilization of Blue-Green Algae, Indian Agricultural

Research Institute, New Delhi, India), was used andmaintained in 150-mL Erlenmeyer flasks containing50 mL of nitrate-free BG-11 medium (Rippkaet al. 1979). The cultures were incubated in atemperature-controlled culture room at 28±2°C, pH 8.5,under a photoperiod of 14/10 h and at light intensity of75 μmol photon m−2 s−1 PAR without sparging with air orCO2. This is referred to as control culture.

Cyanobacterial biomass was harvested by centrifugationat 3,500×g for 10 min. The harvested biomass was driedunder vacuum at 60°C until a constant weight wasobtained, and dcw was determined gravimetrically follow-ing Rai et al. (1991). Polymer extraction was carried outfollowing the protocol of Yellore and Desia (1998) withcertain modifications. Detection of PHB was done using thegas chromatography (GC) method of Riis and Mai (1988)with a GC (Clarus 500, Perkin-Elmer, USA), as detailed inBhati et al. (2010). Chemical proof for the presence of PHBwas obtained from UV spectrophotometry (Law andSlepecky 1961), GC-MS, and 1H-NMR study as detailedin Bhati et al. (2010).

Impact of pH, temperature, and deficiencies of phosphorusand nitrogen on PHB accumulation

Fifty milliliters of the medium was taken in 150-mLErlenmeyer flasks. The pH was adjusted to different values,ranging from 5.5 to 10.5 (MES buffer, 4 mM for pH 5.5 and6.5, and Tris buffer, 4 mM for pH 7.5–10.5) beforeintroducing A. fertilissima inoculum. Cultures were grownfor the fixed time period. PHB accumulation was alsostudied in A. fertilissima cultures grown at various temper-atures, ranging from 16 to 36°C, with an interval of 4°C. Tos t udy the impac t o f phospho ru s de f i c i encyon PHB accumulation, A. fertilissima was grown inphosphate-deficient medium, where K2HPO4 of themedium was substituted by equimolar concentrations ofKCl. For nitrogen deficiency, the medium was substitutedby equimolar concentrations of ferric citrate and CoCl2⋅6H2O,respectively, in place of ferrous ammonium citrate and Co(NO3)2⋅6H2O. To achieve phosphorus and nitrogen deficien-cies, cultures grown in BG-11 medium were centrifuged, thebiomass was washed with the medium without the specificnutrient for two to three times, and finally transferred to thedeficient medium.

PHB accumulation under mixotrophyand chemoheterotrophy

Mixotrophic growth conditions were achieved by supple-menting BG-11 medium with various concentrations (0.2–

804 J Appl Phycol (2012) 24:803–814

1.5%, w/v) of organic carbon sources such as acetate,sucrose, fructose, glucose, maltose, and citrate at the timeof inoculation. Samples were withdrawn at an interval of7 days and were subjected to analysis. The interactiveeffects of acetate with glucose, fructose, and citrate on PHBaccumulation of the test cyanobacterium were studied bysupplementing carbon sources at the time of inoculation.These carbon sources were selected based upon theirperformance on biomass and PHB yield in earlier experi-ments. For the chemoheterotrophic condition, the stationaryphase photoautotrophic cultures supplemented with variouscarbon doses (0.3, 0.5, and 1.0%) were incubated undercomplete darkness.

To study the interaction of mixotrophy with N/Pdeficiency, logarithmic phase Aulosira cultures were sub-jected to N/P deficiency with exogenous carbon, and thepolymer content was analyzed at an interval of 2 days. Tostudy the interactive effects of chemoheterotrophy and N/Pdeficiency on PHB accumulation, the stationary phasephotoautotrophic cultures were subjected to N or Pdeficiency in the presence of appropriate carbon doses andwere kept under complete darkness. These doses wereselected based on their performance on growth and PHByield in earlier experiments.

Optimization study for PHB yield

A five-level, four-factor central composite rotary design(CCRD) obtained using the commercial statistical package,Design Expert version 7.1.1 (Stat-Ease, USA), wasemployed to find out the interactive effects of fourvariables, viz., concentrations of citrate (A), acetate (B),K2HPO4 (C), and incubation period (D) on PHB productionin 30 runs (Montgomery 2004). Stationary phase cultures ofA. fertilissima were transferred into BG-11 medium withvarying concentrations of citrate, acetate, and phosphate, asgiven in Table 1. Duration of culture was as per theexperimental design. The experimental data obtained fromCCRD were analyzed by RSM. To optimize the level ofeach factor for maximum response, “point optimization”technique was employed.

Characterization of the polymer

The thermal properties of the polymer were studiedwith the help of a Pyris Diamond Differential ScanningCalorimeter (Perkin-Elmer) equipped with a liquidnitrogen cooling accessory. Samples (5 mg) were ex-posed to −50–200°C at a heating rate of 10°C min−1 forthe first run. The samples were maintained at 200°C for 1 minand then rapidly quenched in liquid nitrogen at −150°C. Then,the samples were again analyzed during a second heating scanfrom −50°C to 200°C at a heating rate of 10°C min−1. Themelting temperature (Tm) was determined from the DSCendotherms. The glass transition temperature (Tg) was takenas the midpoint of the heat capacity change. The degree ofcrystallinity (Xc) was calculated following Lupke et al.(1998).

The isolated polymer film was cut to a rectangular shape(length, 50±0.5 mm; width, 10±0.2 mm) and the thicknessmeasured by a Dial gauge before using it for mechanicaltests. The elongation-to-break, Young’s modulus, andtensile strength of the film were determined using aUniversal testing machine (H-10KS, Hounsfield, UK) atroom temperature. The extension rate was 10 mm min−1

(Bibers and Kalnins 1999). All the experiments wererepeated three times to check the reproducibility, and theresults were statistically analyzed by Duncan’s new multiplerange test.

Results

The time course of growth and PHB accumulation in A.fertilissima are presented in Fig. 1. Growth of A. fertil-issima increased steadily with a lag phase of 6 daysfollowed by the logarithmic phase and reached thestationary phase on day 20. The accumulation of PHB,though started at the early phase, maximum accumulationof 6.4% dcw, was observed on the 14th day. A decliningtrend in the polymer content was observed after day 20.PHB accumulation was found to be maximum at pH 8.5(6.4% dcw) followed by pH 7.5 (4.2% dcw) on the 14th

Independent variable Coded symbol Level

−2 (α) −1 0 1 2 (+α)

Citrate (%, w/v) A 0.2 0.25 0.3 0.35 0.4

Acetate (%, w/v) B 0.2 0.25 0.3 0.35 0.4

KH2PO4 (mg L−1) C 0 5 10 15 20

Incubation period (days) D 2 5 8 11 14

Table 1 Variables andexperimental design levelsfor response surface

J Appl Phycol (2012) 24:803–814 805

day of incubation. Acidic and high alkaline pH values werenot suitable for PHB accumulation. A temperature range of28–32°C was optimum for PHB accumulation (data notshown). A marginal rise in PHB content was observedunder nitrogen deficiency on day 4 (9.8% dcw) against6.4% of control. Under phosphate deficiency, PHB contentalso reached up to 10.5% (dcw, Fig. 2).

The impact of exogenous carbon supplementation onPHB accumulation was studied by supplementing differ-ent concentrations of acetate, sucrose, fructose, glucose,maltose, and citrate to the growth medium. The mostsignificant rise in PHB content up to 34.2% (dcw) wasobserved in 0.3% citrate-supplemented cultures onday 28 of incubation, followed by 27.1% in 0.3% acetateon the 14th day and 24% in 1% sucrose- and 19.3% in0.3% glucose-supplemented cultures on day 28 ofincubation (Table 2). The total polymer content pervolume of medium was increased up to 381.4 mg L−1 in1% fructose-supplemented culture, which was 12-foldhigher against 31.7 mg L−1 under the control condition.It is noteworthy here that the total polymer content alsoincreased up to 277.5 mg L−1 in 1% maltose- and261.3 mg L−1 in 1% sucrose-supplemented vessels onday 28 of incubation. No further rise in PHB contentwas observed at increasing concentrations of theabove carbon compounds. The interactive effects ofacetate with glucose, fructose, and citrate were alsoinvestigated (Fig. 3). The most significant enhancement inPHB yield was observed under acetate–citrate interaction.The total PHB yield was boosted to 65.9% (dcw) withacetate (0.3%) + citrate (0.3%) supplementation on day 14 ofincubation (Fig. 3c).

The cultures (14 days old) incubated under darkness for5 days showed a PHB accumulation up to 13.5% (dcw). Amaximum accumulation up to 24.4% (dcw) was recorded in0.3% acetate-supplemented culture after 5 days of darkincubation. However, the productivity per volume ofmedium was increased up to 211.3 mg L−1 in 0.5%fructose-supplemented culture after 5 days dark incubation,which was ∼6-fold higher than the 32.3 mg L−1 control(Table 3). Prolonged incubation, i.e., incubation for morethan 7 days under darkness, however, had a negative impacton PHB accumulation.

PHB accumulation was also studied under the interactiveconditions of P and N deficiencies in the presence of carbonsources as these variables were found to stimulate PHBaccumulation significantly (Table 4). The PHB pool reached77.2% (dcw) in the presence of 0.5% acetate under Pdeficiency on day 4 of incubation. PHB accumulationreached up to 70.1% (dcw) under the interactive conditionof N deficiency with 0.1% citrate. The interactive effects ofchemoheterotrophy with N/P deficiencies on PHB yieldwere also found to be stimulatory. After 5 days of darkincubation, an accumulation up to 50.7% (dcw) wasrecorded in 0.5% citrate-supplemented culture under Pdeficiency (data not shown).

Optimization of PHB yield

The results of CCRD for studying the interactive effectsof four independent variables, i.e., citrate, acetate,

0

2

4

6

8

10

12

0 2 4 6 8 10 12 14

PH

B c

on

ten

t (%

dcw

)

Incubation period (days)

Fig. 2 Effects of nitrogen and phosphorus deficiencies on PHBaccumulation potential of A. fertilissima. Control (white circle), Ndeficiency (black square), and P deficiency (black diamond)

0

1

2

3

4

5

6

7

0

100

200

300

400

500

600

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0 6 12 18 24 30 36 42

PH

B c

on

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t (%

dcw

)

Bio

mas

sco

nce

ntr

atio

n (

mg

L-1

)

Incubation period (days)

Biomass

PHB

Fig. 1 Accumulation of PHB in A. fertilissima with reference to growth

806 J Appl Phycol (2012) 24:803–814

K2HPO4, and incubation period on the production ofPHB, as constituents of A. fertilissima, are presented inTable 5. The yield varied between 17.0% and 85.5%(dcw) at different combinations of the variables. Thepredicted values, calculated using the model, were in therange of 20.0–87.9% (dcw). The regression analysis of

the experimental design demonstrated that the linearmodel terms (A, B, C, and D); quadratic model terms(A2, B2, and C2); and the interactive model terms (AB, AC,AD, and BC) were significant (P<0.05). However, thequadratic model term D2 and the interactive model termsBD and CD were found to be insignificant (P>0.05).

Table 2 Effects of exogenous carbon supplementation on biomass concentration and PHB content of A. fertilissima on days 14 and 28 ofincubation

Carbon concentration(%, w/v)

Incubation period (days)

14 28

Biomass concentration(mg L−1)

PHB content (mg L−1) Biomass concentration(mg L−1)

PHB content

(mg L−1) (% dcw) (mg L−1) (% dcw)

Control 496.5±1.01d 31.7±0.23b 6.4±0.05 bc 631.4±1.05ef 18.1±0.33c 2.8±0.01 ab

Acetate

0.2 508.3±1.23e 43.3±0.55de 8.5±0.07 cd 657.4±1.7efg 21.4±0.32c 3.2±0.02 b

0.3 526.7±1.34e 143.2±0.33j 27.1±0.09j 682.7±2.9gh 47.1±0.43f 6.8±0.07 c

0.5 362.4±0.67c 47.6±0.91ef 14.5±0.08f 454.9±0.6cd 42.3±0.78e 9.2±0.09 d

1.0 262.3±0.3ab 33.5 ± 0.25bc 12.7±0.06cf 401.1±1.4c 12.7±0.19b 3.1±0.04 b

Glucose

0.2 456.4±1.05d 44.7±0.73e 9.7±0.05d 491.2±1.3e 52.7±0.5g 10.7±0.09de

0.3 524.7±2.36e 54.4±0.6fgh 10.3±0.08de 593.6±1.9e 114.9±0.3j 19.3±0.12gh

0.5 600.5±3.35f 36.3±0.27cd 6.0±0.04bc 695.5±1.8g 90.0±0.5i 12.9±0.12ef

1.0 656.4±1.22gh 33.6±0.24bc 5.1±0.05b 716.3±1.3h 84.1±0.7i 11.7±0.11e

Fructose

0.3 513.4±1.37e 28.8±0.31b 5.6±0.03b 712.8±1.1h 24.6±0.3d 3.4±0.03b

0.5 1763.8±5.48m 121.5±0.72i 6.8±0.04c 2,021.7±4.2m 53.9±0.7g 2.6±0.02ab

1.0 2386.5±6.23n 381.4±1.75k 15.9±0.09f 2,510.3±7.5n 244.1±1.9k 9.7±0.08d

1.5 281.2±9.58o 11.9±0.64i 4.2±0.03b 214.5±0.1b 6.1±0.7a 2.8±0.01ab

Sucrose

0.3 610.3±1.13fg 34.1±0.54c 5.6±0.03b 894.2±1.6i 64.4±0.9h 7.2±0.09cd

0.5 685.8±1.39h 43.5±0.33bc 6.3±0.04bc 930.5±2.7i 83.4±0.7i 8.9±0.07d

1.0 755.3±2.25i 58.8±0.72h 7.7±0.06cd 1087.3±4.0k 261.3±1.3l 24.0±0.19i

1.5 312.7±3.51bc 7.9±0.11a 2.5±0.01a 183.6±1.3ab 5.7±0.2a 3.1±0.05b

Maltose

0.3 825.3±1.22j 35.2±0.92c 4.2±0.03b 1,016.7±3.5j 61.8±0.8h 6.0±0.04c

0.5 915.9±3.17k 60.1±0.73efg 6.5±0.04bc 1,112.3±6.4k 80.7±0.4i 7.2±0.08cd

1.0 1066.2±6.63l 56.5±0.44gh 5.2±0.03b 1,683.2±6.3l 277.5±1.6m 16.4±0.12f

1.5 217.5±2.53a 6.0±0.1a 2.7±0.01a 131.4±0.2a 1.9±0.4a 1.4±0.01a

Citrate 0.1 0.3 0.5 1.0

0.1 236.2±0.89a 58.1±0.42h 24.5±0.19i 358.1±1.4cb 105.6±1.4j 29.4±0.19k

0.3 241.9±0.91a 61.9±0.81h 25.5±0.21ij 378.9±1.8c 129.7±2.8j 34.2±0.21l

0.5 252.3±1.13b 43.6±0.64e 17.2±0.15g 298.6±0.9b 61.2±0.6h 20.4±0.18h

1.0 270.3±1.34b 34.9±0.32c 12.9±0.18ef 286.4±1.3b 52.2±0.7g 18.2±0.16g

Values are the mean±SE, n=3

Values in the column followed by different letters are significantly (P<0.05) different from each other (Duncan’s new multiple range test).Separate analysis was done for each column

J Appl Phycol (2012) 24:803–814 807

Applying a multiple regression analysis, the results werefitted to a second-order polynomial equation. Thus, themathematical regression model for polymer production

fitted in terms of the coded factors was obtained asfollows:

Y PHB yield½ � ¼ þ73:72� 5:59Aþ 5:25B� 4:99C

� 7:59Dþ 5:89AB� 12:54AC � 3:74

ADþ 5:86 BC � 0:68 BDþ 1:63 CD� 6:41 A2

� 8:05 B2 � 8:30 C2 � 0:23 D2

The P value of the model was <0.0001, whichindicated that the model was highly significant. A lack-of-fit value of 3.15 was found insignificant relative to thepure error (0.109, P>0.05). The high values of thecoefficient of determination (R2=0.986) and also theadjusted R2 of 0.972 also showed that the model washighly significant.

The fitted polynomial equation was expressed as 3Dsurface plots to visualize the relationship between theresponse and the experimental levels of each factor usedin this design and also to demonstrate the interactionsamong the variables. An elliptical nature of the contour inthe 3D response surface graphs (Fig. 4a–d) depicted themutual interactions between the independent variables to besignificant. The positive interaction between varied acetateand citrate concentrations at zero level of K2HPO4 andincubation period demonstrated that a decrease in theconcentration of acetate and citrate contributed to anincreased PHB yield (Fig. 4a). A similar explanation couldalso be valid for Fig. 4b, where a simultaneous decrease inK2HPO4 and citrate concentration from zero level, keepingacetate and incubation period unchanged, increases thePHB yield to a certain extent. The interaction of citrate andincubation period at zero level of acetate and K2HPO4 wassignificant, as visualized from Fig. 4c where PHB yield wasfound to increase with a decreasing level of incubationperiod, but the citrate set near to the zero level. Figure 4dexplains the interaction of acetate with K2HPO4 where withdecreasing acetate concentrations and K2HPO4 level, thePHB production achieved a quadratic gain. Other interac-tion model terms were not shown graphically since theywere found insignificant.

After knowing the possible direction for maximizing thepolymer production, optimization was done using the

0

10

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40

0 7 14 21 28 35

PH

B c

on

ten

t (%

dcw

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Incubation period (days)

(a)

0

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20

30

40

0 7 14 21 28 35

PH

B c

on

ten

t (%

dcw

)P

HB

co

nte

nt

(% d

cw)

Incubation period (days)

0 7 14 21 28 35Incubation period (days)

(b)

0

20

40

60

80(c)

�Fig. 3 Interaction of various concentrations of acetate with glucose,fructose, and citrate on the PHB accumulation potential of A. fertilissima.a Acetate with glucose: control (white circle), 0.1% (black diamond),0.2% (black square), 0.3% (black triangle), and 0.5% (x mark). bAcetate with fructose: control (white circle), 0.1% (black diamond),0.3% (black triangle), 0.5% (x mark), and 1.0% (white square). cAcetate with citrate: control (white circle), 0.1% (black diamond), 0.2%(black square), 0.3% (black triangle), and 0.5% (x mark)

808 J Appl Phycol (2012) 24:803–814

“point optimization” technique with the help of the samesoftware. A maximum polymer content of 86.7% (dcw) waspredicted at 0.26% citrate, 0.28% acetate, and 5.58 mg L−1

K2HPO4 for 5 days of incubation period (Table 6). Theresiduals from the least square fit play an important role injudging the model adequacy (Myers and Montgomery2002). By constructing a normal probability plot of theresiduals, a check was made for the normality assumption

(Fig. 5). The normality assumption was satisfied as theresidual plot approximated along a straight line. A plot ofresiduals versus the predicted response suggested that thevariance of the original observation is constant for allvalues of Y (data not shown). Both the plots were foundhighly satisfactory, thus depicting good reliability of themodel. Furthermore, experiments were performed in triplicateunder the above optimized condition to verify the model. Itcould be seen from Table 6 that the predicted 86.7% (dcw)and experimental 85.1% (dcw) PHB content after optimiza-tion were well in agreement. Cells cultivated in 1% fructose-supplemented medium to obtain maximum biomass concen-tration were further kept under the optimized condition forpolymer accumulation. Thus, after optimization, accumula-tions of 1,986.5 and 1,597.1 mg L−1, respectively, forbiomass and polymer concentrations were obtained (Table 6).

Properties of the polymer

The polymer extracted from A. fertilissima exhibited Tm andTg values of 174 and 0.6°C, respectively (Table 7). The Xcvalue was 60.7%. The tensile strength was 37.6 MPa, whilethe elongation-to-break value and Young’s modulus wererespectively 4.9% and 3.4 GPa.

Discussion

The time course study indicated that PHB is a growth-associated product in A. fertilissima, and its accumulationincreased significantly when the culture reached theexponential phase until the stationary phase. The maximumvalue, 6.4% (dcw), was achieved on the 14th day ofcultivation. After 20 days, the decrease in the PHB contentmight be due to the mobilization of PHB for cellular

Carbon concentration (%, w/v) P deficiency N deficiency

PHB content PHB content

(mg L−1) (% dcw) (mg L−1) (% dcw)

BG-11 0.0 32.6±0.42a 10.5±0.21a 25.8±0.22a 9.8±0.32a

Acetate 0.1 125.7±0.72ef 43.8±0.92d 194.4±1.42h 62.2±1.67d

0.3 134.1±0.91f 51.2±0.76e 187.0±1.31g 65.3±1.57d

0.5 160.1±1.02g 77.2±1.89f 143.5±1.09ef 58.6±1.33d

Citrate 0.1 120.1±0.99e 61.2±0.81e 141.1±1.03e 70.1±1.87e

0.3 105.4±0.95d 56.3±0.66e 125.2±0.99d 68.4 ±1.99de

0.5 73.1±0.82c 44.6±0.19d 101.9±0.92b 61.1±1.78d

Fructose 0.1 76.0±0.86c 33.7±0.71c 149.8±1.02f 48.1±1.13c

0.3 60.4±0.55b 20.1±0.52b 120.3±0.95c 31.7±1.09b

0.5 59.5±0.51b 14.3±0.44a 118.9±0.89c 28.2±1.13b

Table 4 Interaction of P and Ndeficiencies with mixotrophy onPHB accumulation potential ofA. fertilissima on day 4of incubation

Values are the mean±SE, n=3

Values in the column followed bydifferent letters are significantly(P<0.05) different from eachother (Duncan’s new multiplerange test). Separate analysiswas done for each column

Table 3 Chemoheterotrophic accumulation of PHB in A. fertilissima

Treatment PHB content

(mg L−1) (% dcw)

Control (light/dark cycles) 32.3±0.56a 6.2±0.27a

Dark 77.8±0.63d 13.5±0.28bc

Dark+A (0.3%) 141.4±1.10gh 24.4±0.21h

Dark+A (0.5%) 96.8±0.92e 16.6±0.19d

Dark+G (0.3%) 96.9±0.98e 12.8±0.13b

Dark+G (0.5%) 129.6±0.99f 15.4±0.42cd

Dark+S (0.5%) 139.3±1.06g 21.3±0.52f

Dark+S (1.0%) 128.0±0.90f 14.6±0.52c

Dark+C (0.3%) 48.9±0.46b 15.6±0.50cd

Dark+C (0.5%) 61.4±0.57cd 19.7±0.97e

Dark+C (1.0%) 56.9±0.45c 19.9±0.25e

Dark+F (0.3%) 54.4±0.43c 11.2±0.21b

Dark+F (0.5%) 211.3±1.15i 23.1±0.09g

Dark+F (1.0%) 144.9±0.98h 14.7±1.63c

Cells were grown for 14 days under light/dark cycles followed by adark incubation period of 5 days

Values are the mean±SE, n=3

Values in the column followed by different letters are significantly(P<0.05) different from each other (Duncan’s new multiple rangetest). Separate analysis was done for each column

A acetate, G glucose, S sucrose, C citrate, F fructose

J Appl Phycol (2012) 24:803–814 809

metabolism to regain the full capacity of photoautotrophicgrowth as a source of carbon storage material (Stal 1992).A pH of 8.5 and a temperature range of 28–32°C werefound optimum for PHB accumulation.

A. fertilissima is quite flexible in utilizing carbonsources. Intracellular accumulation of PHB, however,appeared to be carbon source-specific (Table 2), andmaximum rise, i.e. an ∼6-fold increase in PHB content(up to 34% dcw), was observed in cultures supplementedwith 3 g L−1 citrate. As observed by Chen et al. (2010), theaddition of citrate decreased the activity of phosphofructo-kinase by chelating Mg2+, which blocks the glycolyticpathway, especially the reaction from fructose-6-phosphateto fructose-1,6-bisphosphate, thus resulting into the elevated

pool of fructose-6-phosphate. An ultimate rise in glucose-6-phosphate pool was observed by the conversion of fructose-6-phosphate to glucose-6-phosphate by isomerase, which led tothe increased activity of 6-phosphoglucose dehydrogenase,the first enzyme of the pentose phosphate pathway (PPP), thusfacilitating the production of a more reduced cofactor,NADPH via PPP. NADPH is reported to be a prerequisitefor the activity of the enzyme acetoacetyl-CoA reductase forconversion of acetoacetyl-CoA to β-hydroxybutyryl-CoA.Therefore, the increased PHB accumulation following citratesupplementation could be due to the enhanced availability ofNADPH.

Citrate supplementation was found to inhibit biomassconcentration significantly (Table 2). Contrary to this,fructose supplementation was stimulatory for biomassproduction. By increasing the concentration up to10 g L−1, growth and PHB production increased up to2.39 and 0.38 g L−1, respectively, against 0.50 and0.03 g L−1 in the control culture. A further increase infructose concentration inhibited the growth of A. fertil-issima. This might be due to the increased osmotic pressureat high fructose concentration and the imbalance betweenglycolysis and metabolic oxidation in cyanobacterial cells(Tabandeh and Vasheghani-Farahani 2003). The stimulatoryeffect of acetate on PHB accumulation, i.e., 27.1% (dcw)against 6.4% control, could be due to a direct utilization ofacetate to increase the intracellular acetyl-CoA pool at theexpense of free CoA by means of the usual pathwayoperating in cyanobacteria (Gibson 1981; Panda andMallick 2007). Glucose utilization in cyanobacteria, how-ever, occurs via PPP. Thus, the positive effect of glucose onPHB production could be attributable to the increasedsupply of the reduced cofactor, NADPH (Lee et al. 1995).Similar explanation could also be valid for the increasedPHB contents in fructose-, sucrose-, and maltose-supplemented cultures. Interestingly, co-feeding A. fertil-issima cultures with 3 g L−1 citrate and 3 g L−1 acetatestimulated PHB accumulation up to 65.9% (dcw). Thiscould be explained by the combined effects of the precursoravailable in plenty, i.e., acetate, and the cofactor NADPH(Fig. 3c).

A marginal rise (13.5% dcw) in PHB accumulationunder chemoheterotrophy was also observed (Table 3). Thiscould possibly be due to the degradation of glycogen tosupply acetyl-CoA, the substrate for PHB biosynthesis, orthe sudden decrease in ATP pool by transferring the culturesto dark aerobic condition (Smith 1982). Addition of 3 g L−1

acetate at the initiation of dark incubation was found toraise PHB accumulation up to 24.4% (dcw). This agreeswell with the finding of Sharma and Mallick (2005) wherethe PHB content was raised up to 43% (dcw) inN. muscorum under chemoheterotrophy supplemented with4 g L−1 acetate. However, the yield was maximum under

Table 5 Central composite design matrix with predicted and actualresponses of PHB accumulation (Y)

Run Process variable Response (Y)

A B C D Predicted Actual

1 0.25 0.35 5 5 73.52 70.30

2 0.30 0.30 10 8 73.72 71.04

3 0.25 0.25 15 5 35.11 37.42

4 0.30 0.30 10 8 73.72 75.23

5 0.40 0.30 10 8 36.91 33.73

6 0.25 0.35 15 5 46.91 49.38

7 0.35 0.25 5 5 44.58 48.89

8 0.25 0.25 5 11 75.57 76.85

9 0.30 0.30 10 8 73.72 75.02

10 0.30 0.30 10 8 73.72 75.70

11 0.30 0.30 10 14 57.64 59.50

12 0.25 0.35 5 11 61.21 61.65

13 0.35 0.35 15 11 59.31 58.38

14 0.35 0.25 5 11 20.04 17.00

15 0.30 0.20 10 8 31.02 28.52

16 0.30 0.30 10 2 87.99 84.50

17 0.35 0.25 15 11 26.66 32.07

18 0.35 0.35 5 11 29.25 29.13

19 0.30 0.30 20 8 30.51 29.31

20 0.35 0.35 15 5 80.07 80.98

21 0.20 0.30 10 8 59.28 60.84

22 0.30 0.30 10 8 73.72 71.09

23 0.30 0.40 10 8 52.03 52.90

24 0.25 0.35 15 11 41.10 38.98

25 0.25 0.25 5 5 85.16 85.53

26 0.35 0.25 15 5 44.70 43.70

27 0.35 0.35 5 5 56.51 58.97

28 0.30 0.30 10 8 73.72 74.23

29 0.30 0.30 0 8 50.50 50.07

30 0.25 0.25 15 11 32.02 29.00

810 J Appl Phycol (2012) 24:803–814

5 g L−1 fructose supplementation (211.3 mg L−1), whichwas about 6-fold higher against the control (Table 3). Anexplanation for this could be the PHB biosynthesis which isexpected to increase under dark incubation with carbon

supplementation through increased NADPH production, asreported for Aphanocapsa 6714 (Pelroy et al. 1976). In thisstudy, the PHB content of A. fertilissima reached the levelof 77.2% of dry cell weight when subjected to P deficiency

Table 6 PHB accumulation before and after optimization of critical variables

Variable Before After PHB content Fructose (1%)+optimized condition

Before After optimization

(% dcw) Predicted(% dcw)

Experimental(% dcw)

Biomass concentration(mg L−1 dcw)

PHB content

(mg L−1) (% dcw)

Citrate (%, w/v) 0.0 0.26

Acetate (%, w/v) 0.0 0.28

KH2PO4 (mg L−1) 40.0 5.58 6.4±0.05 86.7 85.1±0.94 1,986.5±6.23 1,597.1±5.92 80.4±0.79

Incubation period (days) 14 5

Values are the mean±SE, n=3

Fig. 4 Three-dimensional response surface: Interactive effects of variedacetate and citrate concentrations at zero level of K2HPO4 and incubationperiod (a), varied K2HPO4 and citrate concentrations at zero level ofacetate and incubation period (b), varied citrate concentrations and

incubation periods at zero level of acetate and K2HPO4 (c), and variedK2HPO4 and acetate concentrations at zero level of citrate andincubation periods (d)

J Appl Phycol (2012) 24:803–814 811

in the presence of 5 g L−1 acetate for 4 days. The rise inPHB content under mixotrophy and nutrient deficiencycorroborates the finding of Panda et al. (2006) where thecombined effects of mixotrophy and P deficiency werefound stimulatory for PHB accumulation in Synechocystissp. PCC 6803.

It is important to optimize the significant factors affectingPHB accumulation in A. fertilissima for low-cost industrialapplications. The model obtained for PHB production wasadequate enough as depicted from the high F value (73.89),insignificant lack of fit (probability>F=0.109), and R2 closeto unity (0.986), thus indicating that 98.6% of variability in

the response could be explained by this model. According tothe second-order polynomial equation, the linear coefficientsA (citrate), C (K2HPO4), and D (incubation period) showednegative effects on PHB yield, whereas B (acetate) exhibiteda positive impact. PHB accumulation up to 85.1% (dcw) wasachieved at a reduced level of citrate, acetate, and phosphate.This result agrees well with earlier reports where phosphatelimitation was more preferred rather than complete Pdeficiency for PHB accumulation (Nishioka et al. 2001).

A comparison of the material properties of PHB polymerproduced by A. fertilissima with the commercial PHB alongwith others reported in the literature is presented in Table 7.The Tg and Tm values were 0.6°C and 174°C, respectively,which are within the range (Anderson and Dawes 1990;Vincenzini and De Philippis 1999). A. fertilissima PHBpolymer exhibited a crystallinity of 60.7%, which is lowerthan the value reported for N. muscorum (Bhati et al. 2010).Lower crystalline polymers are usually less brittle and havea wide range of applications. Young’s modulus, tensilestrength, and elongation-to-break are the three importantmechanical properties of polymer. The lower Young’smodulus demonstrates better flexibility of the polymerproduced by the test cyanobacteruim than N. muscorum.Moreover, the elongation-to-break value was found to becomparable with the commercial polymer (Table 7). Thisadvocates its potential application in various fields.

PHB accumulation in Synechococcus sp. MA19 reachedup to 55% (dcw) under P-limited condition (Nishioka et al.2001), which was the highest value reported so far forcyanobacterial cultures. In heterotrophic bacteria, PHBaccumulation up to 80% (dcw) has been reported (Junget al. 2000; Reddy et al. 2003). A. fertilissima is the firstcyanobacterium where PHB accumulation reached up to85% (dcw) by manipulating the nutrient status of the

Table 7 Comparative accounts on the properties of PHB polymer from A. fertilissima with the commercially available polymers and the polymersobtained from other cyanobacterial sources

Property PHB fromAulosirafertilissimaa

Commercial PHB (Holmes 1988; Brandlet al. 1990; Doi 1990; Vincenzini and DePhilippis 1999)

PHB from Nostocmuscorum (Bhati et al.2010)

PHB from Spirulinasubsala (Shrivastav et al.2010)

Tm (°C) 174 171–182 176 261

Tg (°C) 0.6 0–5 0.8 4.5

Xc (%) 60.7 60–80 62.4 nr

Tensile strength(MPa)

37.6 40 32.4 nr

Young’s modulus(GPa)

3.4 3.5 3.9 nr

Elongation-to-break (%)

4.9 5 4.7 nr

nr not reporteda Polymer from this study. Values are means of three independent observations

Fig. 5 Plot of normal probability of internally studentized residuals

812 J Appl Phycol (2012) 24:803–814

culture medium. However, biomass productivity of A.fertilissima was affected under acetate–citrate supplemen-tation with P limitation. This necessitates a two-stagecultivation practice where at the first stage A. fertilissimashould be grown to reach high cell density, for example infructose-supplemented medium, followed by the secondstage under optimized condition to boost PHB accumulation.In this study, PHByield reached up to 1.59 g L−1, a value ∼50-fold higher than the control. The exogenous carbonrequirement has been found to be profoundly lower in A.fertilissima as compared with heterotrophic bacteria (10-foldless); thus, low-cost production can be envisaged. This maylead to the introduction of PHB films from cyanobacteriainto various fields.

Acknowledgments Financial support from the Department ofBiotechnology, Ministry of Science and Technology, New Delhi,India, is thankfully acknowledged.

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