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ENHANCED BIOSYNTHESIS OF POLY(3-HYDROXYBUTYRATE) FROM POTATOSTARCH BY Bacillus cereus STRAIN 64-INSIN A LABORATORY-SCALE FERMENTERIftikhar Ali a & Nazia Jamil aa Department of Microbiology and Molecular Genetics , University ofthe Punjab, Quaid-i-Azam Campus , Lahore , PakistanAccepted author version posted online: 26 Nov 2013.Publishedonline: 11 Jul 2014.

To cite this article: Iftikhar Ali & Nazia Jamil (2014) ENHANCED BIOSYNTHESIS OF POLY(3-HYDROXYBUTYRATE) FROM POTATO STARCH BY Bacillus cereus STRAIN 64-INS IN A LABORATORY-SCALE FERMENTER, Preparative Biochemistry and Biotechnology, 44:8, 822-833, DOI:10.1080/10826068.2013.867876

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ENHANCED BIOSYNTHESIS OF POLY(3-HYDROXYBUTYRATE)FROM POTATO STARCH BY Bacillus cereus STRAIN 64-INSIN A LABORATORY-SCALE FERMENTER

Iftikhar Ali and Nazia Jamil

Department of Microbiology and Molecular Genetics, University of the Punjab,Quaid-i-Azam Campus, Lahore, Pakistan

& To decrease the polyhydroxyalkanoate (PHA) production cost by supplying renewable carbonsources has been an important aspect in terms of commercializing this biodegradable polymer.The production of biodegradable poly(3-hydroxyalkanoates) (PHA) from raw potato starch bythe Bacillus cereus 64-INS strain isolated from domestic sludge has been studied in a lab-scalefermenter. The bacterium was screened for the degradation of raw potato starch by a starch hydrolysismethod and for PHA production by Nile blue A and Sudan black B staining. Shake-flask cultures ofthe bacterium with glucose [2% (w=v)] or raw potato starch [2% (w=v)] produced PHA of 64.35%and 34.68% of dry cell weight (DCW), respectively. PHA production was also carried out in a 5-Lfermenter under control conditions that produced 2.78 g=L of PHA and PHA content of 60.53%after 21 hr of fermentation using potato starch as the sole carbon source. Gas chromatography–massspectroscopy (GC-MS) analyses confirmed that the extracted PHA contained poly(3-hydroxybutyrate)(PHB) as its major constituent (>99.99%) irrespective of the carbon source used. The articledescribes, for what we believe to be the first time, PHB production being carried out without anyenzymatic or chemical treatment of potato starch at higher levels by fermentation. More workis required to optimize the PHB yield with respect to starch feeding strategies.

Keywords Bacillus cereus, biodegradable polymer, cheap carbon source, fermentation,GC-MS, potato starch

INTRODUCTION

Polyhydroxyalkanoates (PHAs) are the aliphatic polyesters that areaccumulated intracellular in both Eubacteria and Achaea under theunfavorable conditions. These PHAs not only bear physical and chemicalproperties that are comparable to those of petroleum-based plastics but

Address correspondence to Iftikhar Ali, Department of Microbiology and Molecular Genetics,University of the Punjab, Quaid-i-Azam Campus, Lahore-54590, Pakistan. E-mail: iftikhar_ali_iftikhar@yahoo.com

Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lpbb.

Preparative Biochemistry & Biotechnology, 44:822–833, 2014Copyright # Taylor & Francis Group, LLCISSN: 1082-6068 print/1532-2297 onlineDOI: 10.1080/10826068.2013.867876

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they are also biodegradable, which labels them as ‘‘environment-friendlyplastics’’ and recyclable.[1] These PHAs have already found a number ofapplications in every field of life, such as drug targeting, protein purifi-cation,[2] enzyme-linked assays, and bioimaging;[3] packaging containerssuch as bottles; disposable items such as diapers;[4] as novel biofuels;[5]

and in medicine, food packaging, and industry.[6] A new wave of PHAdevelopment with a focus on new applications is expected soon.[7]

To produce PHA at a price comparable to that of petroleum-basedplastics, one must seek ways that could possibly lower the final price oftheses PHAs at the consumer end. According to Gurieff and Lant,[8] theprice of PHA is more than US$10=kg and it is almost 10 times higher thanthat of petroleum-based plastics (London Metal Exchange, February 2011).A number of factors are responsible for this price difference, such as PHAproductivity, method of recovery, cultivation strategy of microorganism,choice of strain, and carbon substrate cost.[9]

To make polyhydroxyalkanoates production more economical,researchers are focusing on several inexpensive carbon sources suchas plant oils,[10] cheese whey,[11] starch,[12] whey,[13] and municipal sewagesludge[14] to produce PHAs, but still PHA remains underused mostlybecause of its high cost.

Bacillus sp. have been well known for their ability to produce PHA, andsome of them have been well reported in this context, such as Bacillusthrungiensis,[15] Bacillus spp.,[16,17] B. subtilis,[18] and B. megaterium strainOU303A.[19] The production of PHA from potato starch has beenreported[20–23] to emphasize the need to utilize the second most abundantstarch source for the useful biopolymers production, but processing ofstarch has been an issue. Here we used potato starch residue as sole carbonsource to grow locally isolated Bacillus cereus 64-INS. The aim of thisresearch was to isolate a microorganism capable of utilizing raw potatostarch as sole carbon source for its growth and for the subsequentproduction of PHA in a chemically defined nitrogen-limited medium.

MATERIALS AND METHODS

Isolation, Purification, and Screening of PHA-ProducingBacteria

Various bacterial colonies were isolated from a local domestic sludge sam-ple on LB agar medium plates by a standard serial dilution method. The pur-ified bacterial isolates were then screened for their ability to produce PHA bySudan black B staining.[9] For starch hydrolysis activity, bacteria were grownon starch agar (0.2%); plates were incubated at 37�C for 24hr, followed byflooding with iodine solution to check the starch hydrolyzing ability.[16]

The PHA-producing bacterial isolate 64-INS used in this study was chosen

Enhanced Biosythesis of Poly(3-hydrobutyrate) From Potato Starch 823

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for its better starch-hydrolyzing ability and was maintained monthly on solidLB agar medium containing (%, w=v): tryptone, 1.0; yeast extract, 0.5; NaCl,1.0; and agar 1.5, pH 7.0. The bacterium was also characterized physiologi-cally and biochemically for the colony morphology, growth requirements likepH and temperature, and ability to utilize various sugars such as lactose, malt-ose, sucrose, fructose, and so on[24]

Bacterial Growth on Various Carbon Sources

Bacillus cereus strain 64-INS was grown in 250-mL Erlenmeyer flaskscontaining 50mL of nitrogen-limited mineral medium [(NH4)2SO4, 2 g=L;KH2PO4, 13.3 g=L; MgSO4 � 7H2O, 1.2 g=L; citric acid, 1.7 g=L; and traceelements solution 10ml=L] with different carbon sources (fructose 1%;lactose 1%; sodium gluconate 10mM; molasses 0.1% and sodium octanoate10mM) at 30�C and 200 rpm. The cultures were centrifuged, washedwith 0.85% saline, and freeze-dried for dry cell weight (DCW) estimation,subsequent PHA extraction, and analysis.

Pretreatment of Potato Waste

The solid waste of potatoes was obtained from a local market andcrushed to fine powder following its dissolution to distilled water. Themixture was heated at 100�C for 10min. After centrifugation the supernatantwas analyzed for its starch content against a standard curve. Supernatant(0.5ml) was added with 5mL of 0.0007 N iodine and the optical density ofthe mixture was measured at 660nm to quantify the starch.[25]

Fermentative Production of PHA From Potato Starch

Bacillus cereus 64-INS was grown for 24 hr at 30�C and 150 rpm in 250-mLErlenmeyer flask containing 50mL of rich medium containing tryptone1.0 g, yeast extract 0.5 g, NaCl 1.0 g, and peptone 1.0 g=L. Bacterial cells wereharvested by centrifugation at 4000� g for 10min, briefly dried, and 1%(v=v) inoculum of cells was given to minimal medium containingpotato starch at final concentrations of 2% (w=v) in a 5-L fermenter(Bioengineering, Wald, Switzerland) with a working capacity of 3 L. Thefermentation temperature was maintained at 30�C and pH was adjusted to7.0 by the automatic addition of 2 N KOH solution. The dissolved oxygenwas set to 20% while airflow rate was 18L=h with continuous agitation rateof 600 rpm. A parallel set of experiments was conducted in 500-mL flaskscontaining 100mL of the already-describedminimal medium supplementedwith either glucose or potato starch at 2% (w=v). The flasks were alsoincubated in a reciprocal orbital shaker set at 200 rpm and 30�C.

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Extraction and Purification of Polyhydroxyalkanoates

Samples were drawn at regular intervals from the PHA-producing culturesand cells were harvested by centrifugation at 4000� g for 15min at 8�C. Thecells were resuspended in deionized distilled water twice and were freeze-driedin a lyophilizer for 24hr. These cells were mixed by sodium hypochlorite(13%) and chloroform solution (1:1) and agitated at 150 rpm for 100minat ambient temperatures to release the PHA granules from the cells. The mix-ture was centrifuged and the lower dense organic phase containing the PHAwas precipitated by cold acetone (1:3) to precipitate the biopolymer. The PHApolymer was obtained by centrifugation and dried at room temperature.[26]

Analytical Procedure for PHA Composition

A small amount of purified PHA (8mg) was methanolyzed by heating at100�C for 140min in a mixture of 1.0mL chloroform, 0.85mL methanol,and 0.15mL concentrated sulfuric acid.[27] A 1-mL sample of methanolyzedPHA sample was injected in a gas chromatograph—mass spectrometer(GCMS) QP2010 (Shimadzu) via split injection mode (70:30), andnitrogen was used as the carrier gas at a flow rate of 3mL=min. The oventemperature was programmed as follows: 60�C for 2min at the start andthen ramped at a rate of 5�C per min to 260�C, and held for 15min.The temperature of the injector was set at 260�C and column flow ratewas 0.57 cm=s. A DB-5 MS column (30m, 0.25 mm, and 0.25mm) was used.The gas chromatography system was coupled with a mass spectrometrysystem (GCMS QP2010 with D1), in which the temperature of the ion sourcewas set at 200�C, to analyze the monomers produced in this PHA sample.

16S rRNA and Phylogenetic Analysis of Bacillus cereus 64-INS

Genomic DNA of bacterium was isolated and 16S rRNA gene wasamplified using the 27F and 1492R primers following the gene sequencingwith 518F and 800R primers by Macrogen, Inc., Korea. The 16S rRNAsequence obtained in this study was deposited in NCBI GenBank underaccession number JQ013099.1. A phylogenetic tree was constructed bythe MEGA software (version 5.0) using the neighbor-joining method anda 1000-replicates bootstrap analysis.

RESULTS

Screening for PHA-Producing Bacteria and BiochemicalCharacterization

The majority of bacterial colonies that were isolated from the sludgesample were unable to produce PHA content in general. Only 11% of

Enhanced Biosythesis of Poly(3-hydrobutyrate) From Potato Starch 825

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bacteria were found to be positive for PHA production as depicted by thescreening methodology. Sludge environment has already been reportedfor the presence of PHA-producing bacteria.[14] Bacterial isolates capableof PHA production (positive for both Sudan black B and Nile blue A tests)were characterized biochemically, and isolate 64-INS was selected due to itsbest starch hydrolysis activity in terms of showing the biggest clearing zoneon starch-supplemented minimal medium agar (Supplemental Table S1).

Shake-Flask Studies With Glucose and Other Carbon Sources

PHA was produced by the Bacillus cereus strain 64-INS at its maximumlevels (64.35% of DCW) when glucose was used as sole carbon source at30�C after 24 hr of incubation. The bacterium also produced PHA frommolasses (16.59%) and sodium gluconate (9.84%) but in relatively loweramounts as compared to glucose (Figure 1). PHA contents of 3.86% and1.12% were obtained from fructose and sodium octanoate, respectively,but no PHA was produced from lactose. The PHA quantity varied in caseof different concentrations of potato starch and it was at its maximum level(34.68% of DCW) for 2% (w=v) solution of extracted starch (Table 1).

Fermentative Production of PHA by Bacillus cereusStrain 64-INS

Growth of bacterium in a bioreactor showed a typical exponentialphase when grown on potato starch followed by biopolymer accumulation.The quantity of PHA produced increased in the fermenter as the levels ofstarch decreased, most likely due to bacterial amylase production. It can beseen from the Figure 2 that cell dry weight increased steadily over thepassage of time. The production of PHA was related to the decreasedamount of starch present in the fermenter. The highest production of

FIGURE 1 Production of PHA by Bacillus cereus strain 64-INS on different carbon sources after 24 hr ofincubation at 30�C and 200 rpm in shake-flask experiments. Experiments were performed in duplicatesand standard deviation is represented here as error bars.

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PHA was at 21 hr when dry biomass was 4.59 g=L and PHA content was60.53% of its DCW (Figure 2). Once the cells had reached considerable celldensity, the dissolved oxygen levels reached their minimum value andbacterial activity for the PHA accumulation was its peak. The yield ofPHA also reached its maximum value of 2.78 g=L when the starch in thevessel had been consumed in considerable amounts (Figure 3).

TABLE 1 PHA Production by Bacillus cereus 64-INS Grown on Starch and Glucose

Carbon Source (w=v) Growth DCW (g=L) PHA %a PHA (g=L) PHA Monomer

2% Glucose Shake flasks 2.52� 0.31 64.35� 0.78 1.62� 0.23 3HB2% Potato starch Shake flasks 1.72� 0.23 34.68� 0.27 0.59� 0.12 3HB

aPHA content in terms of bacterial dry cell weight (DCW).

FIGURE 2 Growth and production of PHB by Bacillus cereus strain 64-INS in the presence of potatostarch as sole carbon source in a bioreactor. Experiments were performed in duplicates, and standarddeviation is represented here as error bars.

FIGURE 3 PHBproduction and utilization of potato starch in the bioreactor by Bacillus cereus strain 64-INS.Experiments were performed in duplicates, and standard deviation is represented here as error bars.

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Characterization of Purified PHA

PHA isolated and purified from the shake-flask and fermentationstudies was analyzed by GC-MS. The chromatograms showed that themethyl esters of poly(3-hydroxybutyric) acid were the dominant monomersfor the case of both glucose and starch as carbon sources.

Identification of the Bacterium

DNA sequence for the 16S rRNA gene of strain 64-INS was comparedwith the already deposited sequences in GenBank by NCBI BLAST, and itwas observed that this organism had 98% homology with the Bacillus cereusstrain CFR06 (Figure 4).

FIGURE 4 A phylogenetic tree of Bacillus cereus strain 64-INS constructed by neighbor-joining methodwith 1000 bootstrap using MEGA5.0 software with Halolactibacillus alkaliphilus strain NBRC103919 asan outgroup.

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DISCUSSION

With the diminution of crude oil source to produce conventionalplastics, there is a possibility of producing biodegradable plastics fromrenewable sources like cellulose, starch, and sugars.[28] Since 50% of thecost of PHA production is due to its carbon source,[29] various inexpensivecarbon sources have been employed by researchers to combat the highproduction costs of PHA. The present work reports the productionand characterization of bioplastic by using potato starch as a cheap carbonsource from a bacterium isolated from a sludge sample. Due to itsabundance, renewability, and low cost, starch has been used in differentforms, such as granular starch, plasticized starch, modified starch, andstarch blends.[30] So far, Bacillus sp. has been in use for the utilization ofstarch due to its a-amylase production.[31–33] Moreover, Bacillus cereus hasbeen exploited to produce PHA from different unusual carbon sources likepea-shells slurry, in addition to H2 production.[34,35]

The bacterium isolated in this study was identified as a Bacillus cereusstrain according to 16 s rRNA gene similarity to already reported speciesat the NCBI GenBank. Bacillus cereus 64-INS was able to utilize the molassesand sodium gluconate as well and produced DCW of 0.53 and 0.75 g=L,respectively, but the amount of PHA produced was very low. It producedPHA content of 16.59 and 9.84%, which was very low as compared to64.35% when grown on glucose, while other carbon sources (especiallylactose) could sustain neither healthy biomass for bacterium nor thePHA (Figure 1). The bacterium produced very little amount of PHA fromfructose and sodium octanoate, but it seems to prefer more simplifiedmonomers (as present in molasses) for its growth and PHA production.

There have been a number of ways reported to utilize starch in either itsraw form or its processed form to produce PHA. The processing of starchinvolves liquification and saccharification processes that are costly due totheir enzyme consumption. Hence, the idea has been to use a bacteriumthat has both starch hydrolysis and PHA biosynthetic capabilities so thatwe may save the extra cost of starch hydrolysis. In shake-flask studies,strain 64-INS showed good bacterial growth on potato starch as well, thatis, 1.72 g=L as compared to 2.52 g=L when grown on glucose, while PHAcontent was almost double in the case of glucose (Table 1).

Strain 64-INS continuously used the starch up to 21hr of fermentation,resulting in >60% poly(3-hydroxybutyrate) (PHB) of its DCW, althoughsome starch content was still there that was never used. In fact, the bacterialmetabolism was shifted at this point and resulted in consumption of PHB aswell. This was the point where we obtained maximum yield of PHB (2.78g=L),as indicated in Figure 3. Bacillus cereus 64-INS in this study produceda considerable amount of 60% PHA in a relatively short period of time(21 hr), as compared to other studies where nonprocessed starch has been

Enhanced Biosythesis of Poly(3-hydrobutyrate) From Potato Starch 829

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TABLE2

ProductionofPHAUsingVariousForm

sofStarch

byDifferentBacteriaRep

orted

inLiterature

Organ

ism

CarbonSo

urce

Cultivation

PHA(%

)aPHA

(g=L)

PHAtype

Referen

ce

Bacillusmegaterium

Hydrolyzedcassavastarch

Shakeflask

29.70

1.48

PHB

[33]

Ralstonia

eutropha

NCIM

B11

599

Saccharifiedwaste

potato

starch

Fed

-batch

52.51b

94.00

PHB

[39]

Halom

onas

boliviensisLC1

Starch

hydrolysate

Shakeflask

56.00

NA

PHB

[40]

Batch

ferm

enter

35.00

NA

PHB

Haloferax

mediterranei

Extruded

cornstarch

Rep

eatedfed-batch

38.70

24.20

NA

[41]

Haloferax

mediterranei

Enzymatic

extruded

starch

Fed

-batch

50.80

NA

PHBV

[12]

Alcaligenes

eutrophu

sDSM

545

Enzymetreatedpotato

waste

Batch

ferm

enter

77.00

5.00

PHB

[21]

Pseudomonas

aeruginosaNCIB

950

Cassava

starch

hydrolysate

Batch

ferm

enter

57.70

NA

PHB

[38]

Saccharophagusdegradan

s

ATCC

4396

1Raw

starch

Fed

-batch

17.46

2.71

PHB

[36]

Batch

ferm

enter

7.12

0.53

PHB

Pseudomonas

fluorescens

Cassava

starch

hydrolysate

Batch

ferm

enter

71.66

1.25

PHB

[42]

BacilluscereusCFR06

Soluble

starch

Shakeflask

48.00

0.48

PHB

[31]

Bacillussp.CFR67

Corn

starch

Shakeflask

2.93

b0.09

NA

[37]

Potato

starch

Shakeflask

17.14b

0.24

NA

Topioca

starch

Shakeflask

30.00b

0.39

NA

Soluble

starch

Shakeflask

18.5

b0.37

NA

Alcaligenes

eutrophu

sAcidoge

nic

starch

ywastewater

Batch

ferm

enter

34.10

1.20

PHB

[43]

Azotobacter

chroococcum

23So

luble

starch

Shakeflask

74.00

NA

PHB

[44]

Soluble

starch

Batch

ferm

enter

44.00

NA

PHB

Soluble

starch

Fed

-batch

46.00

NA

PHB

Bacilluscereus64

-INS

Potato

starch

Batch

ferm

enter

60.53

2.78

PHB

Thisstudy

aPercentage

amountofPHAin

itsDCW.

b Calcu

latedonbasisofgivendatain

respective

articles.

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used as carbon source[31,36,37]; hence, it is suggested as a potentialorganism to produce PHB from raw potato starch as compared to otherbacteria (Table 2). Alcaligenes eutrophus DSM 545 has produced PHBcontent up to 77% in batch fermentation as well, but it was fed with enzy-matically treated potato starch.[21] Pseudomonas aeruginosa NCIB 950 couldalso produce PHB in batch fermentation but in the presence of cassavastarch hydrolysate.[38] The raw starch has been used in fed-batch fermen-tation by Gonzalez-Garcia et al.[36] using Saccharophagus degradans ATCC43961, but it could only produce a PHA content of about 18% of itsDCW. Other processed starch feeding has also been reported, but the moresimplified and cost-effective form of starch may be the most economical wayof converting this cheap carbon source into biotechnologically importantpolymers like PHB. As we know that the presence of 3-hydroxybutyrate(3HB) monomers contributes to the brittle nature of PHA polymer, thisrequires a sufficient amount of other monomers (e.g., 3-hydroxyvalerate[3HV] and 3-hydroxyhexanoate [3HHx]) to form copolymers that maybe used as a thermoplastic polymer. Hence, it is necessary to find the waysand develop strategic fermentation technologies for the conversion ofabundant quantities of starch present in the environment into morethermoplastic PHA polymer.

CONCLUSION

The newly isolated bacterium in this study, Bacillus cereus 64-INS, hasshown the ability to produce biodegradable polymer PHB (poly-3-hydroxy-butyrate) in considerable amounts by using potato starch, especially inbatch fermentation. The novelty in this work is found in both the strainof biocatalyst used and the fact that this is the first account of a PHA-producing bacterium that can utilize untreated starch as a carbon source,suggesting the presence of some highly active amylase enzymes. The useof potato starch for this purpose would certainly reduce the costs of carbonsources at industrial level to produce PHA. This is for the first time, to thebest of our knowledge, that PHB production has been carried out withoutany enzymatic or chemical treatment of potato starch, and it gave high PHAcontent as well; however, further studies are still necessary to optimize thePHB yield by varying the starch feeding strategies.

FUNDING

The authors are thankful to the Higher Education Commission (HEC),Pakistan, and the University of the Punjab, Lahore, Pakistan, for providingsupport and funds to complete this research work.

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SUPPLEMENTAL DATA

Supplemental data for this article can be accessed on the publisher’swebsite.

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