Research Article Poly -Hydroxybutyrate Production by ...of PHB was recorded as the loss of the weight a er de nite period of incubation. 3. Results and Discussion.. Isolation of PHB

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 952641 10 pageshttpdxdoiorg1011552013952641

Research ArticlePoly 120573-Hydroxybutyrate Production by Bacillus subtilis NG220Using Sugar Industry Waste Water

Gulab Singh1 Anish Kumari1 Arpana Mittal1 Anita Yadav2 and Neeraj K Aggarwal1

1 Department of Microbiology Kurukshetra University Kurukshetra 136119 India2Department of Biotechnology Kurukshetra University Kurukshetra 136119 India

Correspondence should be addressed to Neeraj K Aggarwal neerajkuk26rediffmailcom

Received 4 April 2013 Revised 16 June 2013 Accepted 17 June 2013

Academic Editor Kannan Pakshirajan

Copyright copy 2013 Gulab Singh et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The production of poly 120573-hydroxybutyrate (PHB) by Bacillus subtilisNG220 was observed utilizing the sugar industry waste watersupplemented with various carbon and nitrogen sources At a growth rate of 014 g hminus1 Lminus1 using sugar industry waste water wassupplemented with maltose (1 wv) and ammonium sulphate (1 wv) the isolate produced 5297 gL of poly 120573-hydroxybutyrateaccumulating 518 (ww) of biomassThe chemical nature of the polymerwas confirmedwith nuclearmagnetic resonance Fouriertransform infrared andGC-MS spectroscopywhereas thermal properties weremonitoredwith differential scanning calorimetry Inbiodegradability study when PHB film of the polymer (made by traditional solvent casting technique) was subjected to degradationin various natural habitats like soil compost and industrial sludge it was completely degraded after 30 days in the compost having25 (ww)moisture So the present study gives insight into dual benefits of conversion of a wastematerial into value added productPHB and waste management

1 Introduction

The diverse group of chemicals used in making of plastic ishighly known to be highly toxic and poses a serious threat tobiosphereThese substances besides hitting hard to ecosystemcan also cause an array of problems like birth defects cancerdamage of nervous and immune systems Polypropylenemdashthe key ingredient for plasticmdashis a petroleum product andbecomes increasingly expensiveWith increasing concern forthe environment biosynthetic and biodegradable biopoly-mers have attracted great interest because of their excellentbiodegradability and being environmentally benign and sus-tainable

The PHAs (hydroxyalkanoic acid) are synthesised bywide range of microorganisms as intracellular carbon andenergy reserve material under nutrient limiting conditions[1] The first identified PHA poly-120573-hydroxybutyrate (PHB)from Bacillus megaterium is drawing much attention due tohaving physical properties similar to petroplastic polypropy-lene [2] with advantage of being completely biodegradable

(into water carbon dioxide and methane under anaerobicconditions) by microorganisms in natural environmentsThehigh production cost of PHB can be curtailed by strain devel-opment improving fermentation and separation processesand using inexpensive carbon source In PHB productionabout 40 of the total production cost is accounted for rawmaterial and thus the use of inexpensive carbon source oreven waste organic materials could be highly significant

Annually themillion tons of organic wastes are producedfrom the wide array of industries and in this context con-tribution from sugar industries is worst in terms of effluentproductionmdashout of all the major sugar manufacturing unitsthat is mill house process house boiler house alcoholproducing unit and distilleries [3] The quantity of wastewater generated ever increasing with sugar productionmdashwhere a typical sugar mills consume around 2000 litres ofwater and generate about 1000 litres of waste water per tonof canemdashcrushed The waste water contains floor washingwaste water and condensate water sugarcane juice syrupand molasses and so forth The sugar mill effluent has a

2 BioMed Research International

high biochemical oxygen demand (BOD) and if dischargeduntreated increased BOD effects aquatic ecosystem and hasultimate impact on human health

The sugar industries need spending lots of money on thetreatment of waste water treatment to adhere to regulatorystandards

Thus the two different problems namely the pollutiondue to synthetic plastic and generation of organic richwaste from sugar industries could uniquely addressed by pro-ducing the bioplastic using the organic waste as nutrientsource

However this carbohydrate waste lacks certain mineralnutrient particularly nitrogen in relation to the amountof oxidizable carbon present and still may require nutrientsupplementation

Here an attemptwasmade to use the sugar industrywastewater after nutritive adjustment for the production of PHBusing an isolate Bacillus subtilis NG220

2 Materials and Methods

21 Bacterial Isolation and Culturing Bacillus subtilisNG220was isolated on carbon rich nutrient agar medium [(wv)glucose 1 beef extract 03 peptone 05 and sodiumchloride 08 agar 15] from the soil sample collectedfrom sugarcane field area The presence of intracellular PHBgranules was confirmed with the help of staining with Sudanblack B and Nile blue A [4] The culture was maintainedon the nutrient agar medium (wv) (beef extract 03peptone 05 sodium chloride 08 and agar 15) andstored at 4∘C

22 Morphological Biochemical and Molecular Character-isation The isolate was morphologically characterized byobserving the standard microbiological methods The bio-chemical characterization of the isolate was done by seriesof biochemical tests including carbohydrate fermentationH2

S production MR-VP test and Catalase test Further formolecular characterization the PCR amplification of 16SrDNA was done at 95∘C for 5min 95∘C for 30 sec 52∘C for30 sec 72∘C for 1min and 72∘C for 10min The universalprimer 16sF-51015840 AGA GTT TGA TCC TGG CTC AG 31015840 and16sR-51015840 ACG GCT ACC TTG TTA CGA CTT 31015840 were usedPCR product was purified by column (Merck bioscienceIndia) and was sequenced with AB instrument 3730XL Theobtained sequencewas subjected to search for closest possiblespecies using distance matrix based on nucleotide sequencehomology (using Kimura-2 parameter) and BLAST toolsavailable at National Centre for Biotechnology Information(NCBI) Phylogenetic analysis was done by using the Phylippackage and ClustalW

23 Preparation of Seed Inoculum One loop full of theculture from slant was inoculated in 5mL of sterile nutrientbroth (beef extract 03 peptone 05 sodium chloride08) After incubation for 24 h at 30∘C 1 (vv) of culturehaving 108 cellsmL was aseptically transferred into 50mLsterile nutrient broth and incubated at 30∘C for 18 h

Table 1 Physiochemical characteristics of sugar industry wastewater

Parameter Concentration (mgL)pH 42ndash69Total solids 1076 plusmn 414Suspended solid 631 plusmn 2274

COD 1395 plusmn 317BOD 1300 plusmn 238CODBOD 16 plusmn 049

BODNP 100 169 069Nitrogen 1148 plusmn 654

Phosphorous 436 plusmn 247

24 Substrate Preprocessing Untreated sugar industry efflu-ent was collected from the Saraswati Sugar Mills Yamu-nanagar Haryana India and was stored at 4∘C until usedThe effluent was filtered through the muslin cloth to removethe undesired solid materials Appropriately diluted filteredwaste water was used as PHB production medium (Table 1)

25 Production Detection Extraction and Purification ofPHB The PHB production was followed in 250mL Erlen-meyer flask containing 50mL sugar industry waste water asproduction medium under stationary condition of growthAfter 72 h of incubation at 30∘C culture broth was cen-trifuged at 8000 rpm for 15min The portion of the palettewas resuspended in 1mL of sterile distilled water sonicated(Hielscher Germany) in 02 aq sol of Nile blue A air-driedthe smear and observed under phase contrast microscope[4] Remaining amount of the pellet obtained was usedfor extraction and recovery of PHB according to Law andSlepecky [5] with minor modifications Briefly the harvestedcells were lyophilized and the dry cell mass weight was notedThe dry cell mass was subsequently incubated with 10mLsodium hypochlorite at 50∘C for 1 h for cells lysis The cellextract obtained was centrifuged at 12000 rpm for 30minthen washed sequentially with distilled water acetone andabsolute ethanol and dissolved in 10mL boiling chloroform(Sigma Aldrich) After evaporation 10mL of conc sulphuricacidwas added and placed inwater bath for 10min at 100∘C toconvert the PHB into crotonic acid On cooling absorbancewas taken at 235 nm [6] using PHB (Sigma Aldrich) asstandard

26 Polymer Analysis

261 NMR Analysis 1H Nuclear Magnetic Resonance Theidentity of individualmonomer unitwas confirmedby protonnuclear magnetic resonance (1H-NMR) spectroscopy 1H-NMR spectra of PHB sample were recorded in CDCl3 onBruker ACF 300 spectrophotometer at 300MHz by usingldquoTetramethylsilanerdquo as internal standard [7]

262 FT-IR Analyses FT-IR analysis of the polymer samplewas carried out on MB-3000 ABB FTIR spectrophotometerin the range 4000ndash600 cmminus1

BioMed Research International 3

Table 2 Experimental culture conditions for optimization of various parameters under stationary condition of growthlowast

Rigid parameter Name of the parameterIncubation period (h) pH Inoculum age (h) Temperature (∘C)

Incubation period (h) Observed 7 18 30pH 72 Observed 18 30Inoculum age (h) 72 7 Observed 30Temperature (∘C) 72 7 18 ObservedlowastProduction medium containing treated undiluted sugar industry waste water

263 DSC Analysis TA Q10 series instrument was usedto carry out thermal analysis of PHB sample under flow-ing nitrogen atmosphere at a heating rate of 10∘Cminminus1120572-alumina powder was used as reference material usingaluminium sample holder for taking thermogram [8] Inorder to ensure the uniformity of temperature and goodreproducibility small amount (2ndash5mg) of sample was takenTo verify the reproducibility duplicate runs were adoptedunder the same experimental conditions

264 GC-MS Analysis Purified polymer prepared asdescribed before was dissolved in chloroform (5mgPHB mLminus1) and 3 120583L was injected into a GC-MS instrument(Model 6890Hewlett Packard)The column and temperatureprofile used for GC analysis were as described by Schubertet al [9]

27 Optimization of PHB Production The optimization formaximum PHB production by Bacillus subtilis NG220 wascarried out under stationary conditions of growth usingsugar industry waste water as production mediumThe sugarindustry waste water was filtered through muslin cloth andthen rough filter paper before any study to remove any typeof debris or solid waste Several cultural parameters wereevaluated to determine their effect on biomass and PHBproduction using sugar industry waste water The optimizedvalue for each parameter was selected and kept constant forfurther experiments The fermentation was carried out in250mL Erlenmeyer flask containing 50mL sugar industrywaste water as production medium under stationary as wellas shaking condition of growth Several cultural parameterslike time of incubation (24ndash120 h) temperature of incubation(20ndash50∘C) supplementation of various carbon and nitrogensources inoculum age and size (12ndash42 h 1ndash3) effect of pH(4ndash8) and so forth were evaluated to determine their effecton biomass accumulation and PHB production

271 Effect of Carbon andNitrogen Source on PHBProductionProduction of PHB was followed using the optimized valuesof various parameters obtained from different experimentsas per Table 2 PHB production was carried out using pro-ductionmedium supplemented with different carbon sourcesat 1 (wv) namely dextrose xylose sucrose rhamnosemannitol maltose lactose monohydrate mannose galac-tose starch and raffinose pentahydrate PHB productionwas also studied using production medium supplementedwith various inorganic (ammonium chloride ammonium

sulphate di-ammonium hydrogen phosphate and ammo-nium di-hydrogen phosphate ammonium per sulphate) andorganic (peptone urea and tryptone) nitrogen sources at1 (wv) Further effect of the selected carbon and nitrogensource on PHB production was investigated at their differentconcentrations

28 Preparation of Polymer Blend Sheet Conventional sol-vent cast technique [10] was used for preparation of polymerblend sheetThe PHB powder extracted from Bacillus subtilisNG220 was mixed with soluble starch in ratio of 4 1 (ww)and then dissloved in 20mL of chloroformThe solution waspoured into an open flat Petri plate and allowed to evaporateslowly at room temperatureThe sheet was then cut into smallpieces and used for degradation studies

29 Degradation of Polymer Sheet Biodegradability testingwas performed by soil burial method [11] A preweighted30mm pieces were buried either into 200 g of freshly col-lected soil sample compost or sludge in wide mouth glassbeakers The beakers were filled with 200 g of soil sludgeor compost The mouths of the beakers were kept openand incubated at room temperature up to 40 days Differentmoisture content (15 20 25 and 30) in the sampleswas maintained by adding sterile water to study the effectof the moisture on degradation of PHB films Degradationof PHB was recorded as the loss of the weight after definiteperiod of incubation

3 Results and Discussion

31 Isolation of PHB Producing Bacteria More than 300isolates from different ecological niches were obtained onnutrient agar medium The simplest first line screening pro-gram for PHB producing bacteria is the use of Sudan blackB staining The isolates which were found positive for PHBgranules after Staining with Sudan black B (ie showing darkspot inside the pink coloured cells) were further confirmedfor their PHB producing potential with Nile Blue A a morespecific dye for PHB granules in which bright orangecolour fluorescence was observed under the phase contrastmicroscopy

32 Identification of Isolate The isolate NG220 used in thepresent study was morphologically and biochemically char-acterised as per given in Table 3

The isolate on the basis of their morphological and bio-chemical characteristics identified as Bacillus sp (Bergeyrsquos


Table 3 Morphological and biochemical characteristics of isolateNG220

ObservationMorphological characters

Elevation UmbonateEdge Entire with undulateInternal character RoughColour Creamy off-white

Biochemical charactersGram reaction Gram +ve rodGlucose fermentation +veSucrose fermentation +veLactose fermentation minusveH2S production minusve (no gas observed)MR test +veVP test minusveCatalase test +ve

Manual 9th ed) when further characterized with 16S r-RNAgenes for sequence homology (using BLAST and distancematrix based nucleotide sequence homology (Table 4)) iso-late NG220 had the closest matching with the Bacillus subtilis(Figure 1) and was reffered to as Bacillus subtilis NG220 The16S r-RNA sequence of the isolate has been submitted to theNCBI gene bank (Accession no JQ797306)

33 Physiochemical Characterization of Sugar Industry WasteWater The waste water had a high COD (1395 plusmn 317mgL)and BOD (1300 plusmn 238mgL) The nitrogen and phospho-rous concentration in the sugar industry waste water wererecorded as 1148 plusmn 654mgL and 436mgL respectivelyThewastewater has acidic to neutral pH andhave high load ofsuspended solidThis waste water was used for PHB accumu-lation studies by the isolate with or without supplementationof different carbon and nitrogen sources

34 Microbial Growth and PHB Production in Sugar IndustryWaste Water As such there is no report for PHB accumu-lation by microbial cells using sugar industry waste waterIn different concentration of sugar industry waste watergrowth of Bacillus subtilis NG220 was observed (Table 5)and the maximum growth was noted in undiluted (100vv) sugar industry waste water after 72 h of incubation at30∘C Dilution of waste water not only affected the microbialgrowth but also PHB accumulation This may be due to thefact that with increasing dilution of waste water the carbonand other nutrient got diluted and was not able to supportmicrobial growth Under unoptimized conditions Bacillussubtilis NG220 was observed to accumulate about 4928 gLof PHB after 72 h

35 Characterisation of Extracted Polymer

351 1H-NMR Spectroscopy The chemical nature of extract-ed polymers from Bacillus subtilis NG220 was confirmed by1H-NMR spectroscopy The 1H-NMR spectra (Figure 2(a))

showed the presence of three signals characteristic of thepolymer of HB The 1H-NMR spectra of polymer extractedfrom isolate showed a doublets at 085 ppm correspondingto the methyl group (ndashCH3) and two multiplets at 123 and156 ppm corresponding to methylene group (ndashCH2ndash) andmethyne (ndashCHndash) group respectively [12] In this study withreference PHB standard the nature of polymer produced bythe isolate was confirmed

352 Fourier Transforms Infrared Spectroscopy (FTIR) Poly-mer extracted from Bacillus subtilis NG220 was used forrecording IR spectra in the range 4000ndash600 cmminus1 IR spectra(Figure 2(b)) showed two intense absorption band at 1705 and1034 cmminus1 specific for C=O and CndashO stretching vibrationsrespectively The absorption bands at 2916 and 2955 cmminus1are due to CndashH stretching vibrations of methyl methylenegroups These prominent absorption bands confirm thestructure of poly-120573-hydroxybutyrate

353 DSC Analysis of PHB Polymer The melting tempera-ture of the extracted PHB sample obtained was determinedusing DSC The thermal properties of the polymer such asthe melting temperature (119879

119898

) are crucial for polymer pro-cessingThemelting temperature of extractedPHB (13254∘C)(Figure 2(c)) was lower as compared to that was reported inthe literature (173ndash180∘C) The melting point near to 130∘Cshowed that the extracted PHB contained the nearly 15molHV in the PHB polymer [13]

354 GC-MS Analysis of Extracted PHB In this study thePHB was methanolysed in the presence of sulphuric acidandmethanol and the methanolysed 3HB was then analyzedby GC-MS Figure 2(d) showed that a common molecularfragment of the 3HB methyl ester ion chromatogram of thePHB produced A predominant peak corresponding to the3HB methyl ester was noted at 21min while four othersmall peaks were observed at 27 33 35 and 38min Ofthese the peak at 33min was speculated to be an impuritybased on its ion fragment pattern The retention timesand ion fragment patterns of the peaks at 27 and 38minwere identical to those of themethyl esters of 3HV and 3HHxrespectively Nevertheless the 3HHx content was negligiblein this copolymer (less than 01mol) The peak at 35minalso had an ion fragment pattern similar to that of the 3HHxmethyl ester however its retention time was not identical tothat of 3HHx

36 Optimization of PHB Production

361 PHB Production versus Incubation Time The PHB pro-duction in sugar industry waste water was followed for 96 hunder stationary growth conditions The production of PHBincreased up to 72 h (5191 gL) and thereafter got reduced(5125 gL after 96 h) (Figure 3(a)) The decrease of PHBproduction after 72 h might indicate that the bacteria usedPHB as nutrient source Matavulj andMolitoris [14] reportedthat the highest PHB level in Agrobacterium radiobacterwas achieved during stationary growth phase after 96 h Theobservation was supported by Yuksekdag et al [15]


Table 4 Distance matrix analysis for homology (Kitmura-parameter-2) for isolate NG220

B lichnif 0B sp BTB C 0 0B subtilis 001 001 0Query NG220 001 001 0 0B amyloliq 001 001 0 0 0B atropae 002 002 001 001 001 0B pumilus A 003 003 003 003 003 002 0B cereus AT 006 006 006 006 006 006 006 0B thuringi 006 006 006 006 006 006 006 0 0B Anthraci 006 006 006 006 006 006 006 0 0 0B coahuile 006 006 006 006 006 006 006 004 004 004

Bacillussubtilis

BacillusamyloliquefaciensDSM

Bacillusatrophaeuschromosome

BacilluslicheniformisATCCchromosome

Bacilluscoahuilensism

BacilluscereusATCC

BacillusthuringiensisstrAlHakamchromosome

BacilluspumilusATCCBATContig

BacillusanthracisstrABAOContig

Query NG220

BacillusspBTB CTcont

Figure 1 Phylogenetic tree of isolate NG220

Table 5 Growth and PHB production with sugar industry wastewater

sugar industry waste water (vv) Biomasslowast (gL) PHBlowast (gL)20 048 mdash40 066 mdash60 082 mdash80 442 0012100 10211 4928lowastBiomass and PHB production after 72 h of incubation at 30∘C with prepro-cessed sugar industry waste water

362 Effect of Incubation Temperature and pH on PHB Pro-duction The maximum PHB production of 5201 gL wasrecorded at 40∘C after 72 h The increase of temperaturebeyond 40∘C has negative impact on PHB production(Figure 3(b)) The decrease in PHB production at hightemperature could be due to low PHB polymerase enzymeactivity [15]

The effect of pH variations on PHB production is shownin Figure 3(c) From analysis it is clear that pH 7 wasfavourable for PHB production by Bacillus subtilis NG220 insugar industry waste water The current observation was in


728

1

214

91

624

160

31

581

156

01

540

145

91

437

140

81

352

130

31

275

123

51

213

102

21

000

088

00

859

001

9

083

140

100

159

083

140

100

159

13 12 11 10 9 8 7 6 5 4 3 2 1 0 minus1(ppm)

(a)

100

95

90

85

80

4000 3750 3500 3250 3000 2750 2500 2250 2000 1750 1500 1250

3742

3618

2955

2916

2361

1744

1705

1651

1520

1458

1373

1311

1219

1165

1034

895

841

771

663

609T

()

(cmminus1)

(b)

02

00

minus02

minus04

minus06

13214∘C

0 100 200 300 400 500

1032 Jg2843 Jg

8740∘C

13254∘C

Hea

t flow

(Wg

)

Temperature (∘C)Exo up

(c)

Figure 2 Continued


200 600 1000 1400 1800 2200 2600 3000 3400Time

Abun

danc

e

1060

109913241800

3804

7020

1019311505 15836

15903

17260

18550

18717

19000

20357

21264

21330

227382301824382

25038

25737

26469

26530

266362749928036

28243

28344

29301

2990730472

31917

33327

34431

2

6

10

14

18

22

26

30

34

38

42

times104

4

8

12

16

20

24

28

32

36

40

44

(d)

Figure 2 (a) NMR spectra (b) FTIR spectra (c) thermogram by DSC analysis and (d) GC-MS spectra of extracted PHB polymer

agreement with Aslim et al [16] where the authers observedthat the maximum PHB was produced (001 to 05 gL) atpH 7 by Rhizobium strain grown on yeast extract mannitolbroth while studying the effect of different pH on exo-polysaccharide and PHB production in two strains of Rhi-zobium meliloti Tavernier et al [17] reported that these twostrains showed higher PHB content at pH 70 Shivakumar[18] reported optimum pH between 68 and 80 for PHBproduction by Alcaligenes eutrophus

363 Age of Inoculum The maximum PHB productionhas been achieved after 72 h when the sugar industry wastewater was inoculated with 18 h old inoculum (1 vv) at40∘C (Figure 3(d)) Kumbhakar et al [19] also reported thatroot nodule bacteria have produced maximum PHB with12 h and 16 h old inoculum Ramadas et al [20] achievedthe maximum PHB production by Bacillus sphaericus whenproduction medium was inoculated with 16 h old inoculum

364 Effect of Different Carbon and Nitrogen Source Sup-plementation on PHB Production To find the best availablecarbon source and its optimum concentration for maxi-mum PHB production commercial carbohydrates dextrosexylose sucrose rhamnose mannitol maltose lactose mono-hydrate mannose galactose starch and raffinose were triedas a sole carbon source supplement to sugar industry wastewater under stationary culture conditions From Figure 3(e)analysis it is clear that maltose supplementation to pro-duction media gave PHB yield of 5254 gL Hori et al[21] reported that PHB content in B megaterium reachedmaximum level in a medium containing glucose as carbon

source The study conducted by Wu et al [22] reportedthat Bacillus sp JMa5 accumulated 25ndash35 (ww) PHBduring sucrose fermentation [23] Working with differentcarbon sources in MSM broth Khanna and Srivastava[24] observed higher PHB yield on fructose by A Eutro-phus [24] Out of various organic and inorganic nitrogensources ammonium sulphate (1 wv) supported the maxi-mum (5297 gL) PHB production followed by peptone andtryptone (Figure 3(f)) In the literature ammonium sulphateis reported to be the best nitrogen source for PHB productionin differentmicroorganism such asAlcaligenes eutrophus [25]Methylobacterium sp [26] and Sinorhizobium fredii [27]Thehighest PHB was obtained in ammonium sulphate by halo-tolerant photosynthetic bacteria Rhodobacter Sphaeroideswhen cultivated under aerobic and dark conditions [28] Thehighest level of PHB accumulationwas observed in themediawith protease peptone as nitrogen sources by Aslim et al [16]in B subtilis 25 and in B megaterium 12

37 Evaluation of Kinetics Parameters The evaluation ofkinetic parameter for batch fermentation was studied withrespect to PHB and biomass production using sugar industrywaste water as productionmediaThe Bacillus subtilisNG220grew at the rate 0141 g Lminus1 hminus1 of production media andaccumulated 518 of PHB at the rate of 00072 ggminus1(biomass) hminus1 Coats et al have reported that in some mixedcultures or activated sludge PHAproduction could be as highas over 50 of the cell dry weight (CDW) [29]

38 Degradation of PHB Sheet Polymers in natural environ-ment are degraded through hydrolysis mechanical thermal


5148 5151

5191

5125

50851

512514516518

52

24 48 72 96

PHB

(gL

)

Time of incubation (h)

(a)

484 488

5201

49285

46474849

515253

20 30 40 50

PHB

(gL

)

Incubation temperature (∘C)

(b)

508 5075103 5103

5213519

5108

5

495

505

51

515

52

525

5 55 6 65 7 75 8

PHB

(gL

)

pH

(c)

51225219

5148 5128 50975054

495

515253

12 18 24 30 36 42PH

B (g

L)

Inoculum age (h)

(d)

474849

5515253

PHB

(gL

)

Dex

trose

Xylo

se

Sucr

ose

Rham

nose

Man

nito

l

Mal

tose

Lact

ose

Man

nose

Gal

acto

se

Star

ch

491749654982

5254

49855017 50175022502849284931

Carbon source supplement (1)

(e)

49315005 5005

5297

49914942 4931

498

474849

551525354

Ure

a

Tryp

tone

Pept

one

(NH

4) 2

SO4

(NH

4)H

2PO

4

(NH

4) 2

HPO

4

NH

4S 2

O8

NH

4Cl

PHB

(gL

)

Nitrogen source supplement (1)

(f)

Figure 3 Optimization of cultural conditions for Bacillus subtilis NG220

oxidative and photochemical destruction and biodegrada-tion One of the valuable properties of PHB is its biodegrad-ability and it was studied in simulated natural environment(in soil compost and industrial sludge) in laboratory Alltest pieces of PHB starch blend sheet lost weight duringincubation but the degree of weight loss varied widely withenvironment in which the polymer sheets were incubatedThe compost was found to be the better for degradation ofPHB films compared to soil and industrial sludge It is furthersupported by increase microbial load (50 times 106 cfumLminus1 tomore than 30 times 109 cfumLminus1g) (Table 6) in compostafter 30 days where PHB could serve as nutrient sourceby microflora The microbial population was increased incompost after 30 daysThe bacteria detected on the degradedPHBfilmswere dominated by the generaPseudomonasBacil-lus Azospirillum Mycobacterium Streptomyces Aspergillus

and Penicillium [30]The rate of degradation of the PHBfilmswas 253 and 502 within 30 days in compost containing10 and 15 moisture respectively while in the compostcontaining the 20 and 25 moisture the PHB films werefound to be degraded into fine pieces in 30 days resultinginto 802 and 100 loss in wt of PHB films (Figure 4)indicating the importantance of moisture content of thecompost Similar observation was noted by Wang et al [11]Woolnough et al [31] and Kulkarni et al [10]

4 Conclusion

The isolate Bacillus subtilis NG220 from sugarcane field areawas able to efficiently utilize the sugar industry waste water asnutrient source for PHB production The first line of impres-sion of this study concludes that the sugar industry waste


Table 6 Degradation of polymer sheet in natural samples

Natural sample Incubation (days) weight loss of polymer sheet Microbial population

Soil

0 0 30 times 106

10 302 plusmn 05 40 times 107

20 435 plusmn 12 49 times 107

30 706 plusmn 26 52 times 108

40 983 plusmn 21 54 times 108

Compost

0 0 5 times 106

10 502 plusmn 05 2 times 107

20 802 plusmn 05 12 times 108

30 100 30 times 109

Industrial sludge

0 0 25 times 105

10 263 plusmn 12 32 times 106

20 576 plusmn 15 49 times 106

30 736 plusmn 15 56 times 106

40 972 plusmn 22 60 times 106

lowastDegradation study was followed in lab conditions at room temperature

0

20

40

60

80

100

120

10 15 20 25

Wei

ght l

oss (

)

Moisture content (ww)

Figure 4 Effect of moisture content on PHB degradation in com-post

water could directly serve as an inexpensive nutrient sourcefor production of biodegradable plastic Thus this studymay solve the problem of costly treatment of sugar industrywaste water as well as high production cost of biodegradablebioplastic and help in conservation of petroleum productswhich were used in the commercial production of plasticproduction

Acknowledgments

Theauthors express their sincere gratitude to theDepartmentof Science and Technology Government of India for provid-ing the financial aid for this research work The authors alsoacknowledge the Saraswati Sugar Mills (SSM) Ltd YamunaNagar for providing the untreated waste water The authorsare very thankful to the Department of Chemistry Kuruk-shetra University Kurukshetra for providing the necessaryfacilities for NMR FTIR and DSC analysis of the polymer

References

[1] A J Anderson andEADawes ldquoOccurrencemetabolismmet-abolic role and industrial uses of bacterial polyhydroxyalka-noatesrdquo Microbiological Reviews vol 54 no 4 pp 450ndash4721990

[2] E RHowells ldquoOpportunities in biotechnology for the chemicalindustryrdquo Chemistry amp Industry vol 8 pp 508ndash511 1982

[3] C Wood Environment Impact Asseesment A ComparativeReview Longman Harlow UK 1995

[4] S Banziger P D N Tobler and H Brandl H The Formationof Reserve Polymers in Bacillus megaterium Microbial EcologyCourse University of Zurich Zurich Switzerland 2001

[5] J H Law and R A Slepecky ldquoAssay of poly-beta-hydroxybutyr-ic acidrdquo Journal of Bacteriology vol 82 pp 33ndash36 1961

[6] I Y Lee H N Chang and Y H Park ldquoA simple method forrecovery of microbial poly 120573-hydroxybutrate by alkaline solu-tion treatmentrdquo Journal of Microbiology and Biotechnology vol5 pp 238ndash240 1995

[7] H Yoshie S Ozasa K Adachi H Nagai M Shiga and YNakamura ldquoNuclear magnetic resonance of 59Co in Gd

2

Co7

rdquoJournal of Magnetism and Magnetic Materials vol 104ndash107 no2 pp 1449ndash1450 1992

[8] L M W K Gunaratne R A Shanks and G AmarasingheldquoThermal history effects on crystallisation and melting ofpoly(3-hydroxybutyrate)rdquo Thermochimica Acta vol 423 no 1-2 pp 127ndash135 2004

[9] P Schubert N Kruger and A Steinbuchel ldquoMolecular analysisof the Alcaligenes eutrophus poly(3-hydroxybutyrate) biosyn-thetic operon identication of the N terminus of poly(3-hydroxybutyrate) synthase and identification of the promoterrdquoJournal of Bacteriology vol 173 no 1 pp 168ndash175 1991

[10] S O Kulkarni P P Kanekar J P Jog et al ldquoCharacterisation ofcopolymer poly (hydroxybutyrate-co-hydroxyvalerate) (PHB-co-PHV) produced by Halomonas campisalis (MCM B-1027)its biodegradability and potential applicationrdquoBioresource Tech-nology vol 102 no 11 pp 6625ndash6628 2011


[11] S Wang C Song W Mizuno et al ldquoEstimation on biode-gradability of poly(3-hydroxybutyrate-co-3-hydroxyvalerate)(PHBV) and numbers of aerobic PHBV degrading microor-ganisms in different natural environmentsrdquo Journal of Polymersand the Environment vol 13 no 1 pp 39ndash45 2005

[12] M Thirumala S V Reddy and S K Mahmood ldquoProductionand characterization of PHB from two novel strains of Bacillusspp isolated from soil and activated sludgerdquo Journal of Indus-trial Microbiology and Biotechnology vol 37 no 3 pp 271ndash2782010

[13] H Mitomo P J Barham and A Keller ldquoCrystallization andmorphology of poly(120573-hydroxybutyrate) and its copolymerrdquoPolymer Journal vol 19 no 11 pp 1241ndash1253 1987

[14] M Matavulj and H P Molitoris ldquoFungal degradation of poly-hydroxyalkanoates and a semiquantitative assay for screeningtheir degradation by terrestrial fungirdquo FEMS MicrobiologyReviews vol 103 no 2-4 pp 323ndash331 1992

[15] Z N Yuksekdag B Aslim Y Beyatli and N Mercan ldquoEffectof carbon and nitrogen sources and incubation times on poly-beta-hydroxybutyrate (PHB) synthesis by Bacillus subtilis 25and Bacillus megaterium 12rdquo African Journal of Biotechnologyvol 3 no 1 pp 63ndash66 2004

[16] B Aslim Z N Yuksekdag and Y Beyatli ldquoDetermination ofgrowth quantities of certain Bacillus species isolated from soilrdquoTurkish Electronic Journal of Biotechnology pp 24ndash30 2002

[17] P Tavernier J-C Portais J E Nava Saucedo J Courtois BCourtois and J-N Barbotin ldquoExopolysaccharide and poly-120573-hydroxybutyrate coproduction in two Rhizobium melilotistrainsrdquo Applied and Environmental Microbiology vol 63 no 1pp 21ndash26 1997

[18] S Shivakumar ldquoPolyhydroxybutyrate (PHB) production usingagro-industrial residue as substrate by Bacillus thuringiensisIAM 12077rdquo International Journal of ChemTech Research vol 4no 3 pp 1158ndash1162 2012

[19] S Kumbhakar P K Singh and A S Vidyarthi ldquoScreening ofroot nodule bacteria for the production of polyhydroxyalka-noate (PHA) and the study of parameters influencing the PHAaccumulationrdquo African Journal of Biotechnology vol 11 no 31pp 7934ndash7946 2012

[20] N V Ramadas S K Singh C R Soccol and A Pandey ldquoPoly-hydroxybutyrate production using agro-industrial residue assubstrate by Bacillus sphaericus NCIM 5149rdquo Brazilian Archivesof Biology and Technology vol 52 no 1 pp 17ndash23 2009

[21] K Hori M Kaneko Y Tanji X-H Xing and H Unno ldquoCon-struction of self-disruptive Bacillus megaterium in responseto substrate exhaustion for polyhydroxybutyrate productionrdquoAppliedMicrobiology and Biotechnology vol 59 no 2-3 pp 211ndash216 2002

[22] Q Wu H Huang G Hu J Chen K P Ho and G-Q ChenldquoProduction of poly-3-hydroxybutrate by Bacillus sp JMa5cultivated in molasses mediardquo Antonie van Leeuwenhoek Inter-national Journal of General andMolecular Microbiology vol 80no 2 pp 111ndash118 2001

[23] M Singh ldquoCane sugar industry in Indiardquo in Proceedings of theXXIII ISSCT Congress New Delhi India 1999

[24] S Khanna and A K Srivastava ldquoStatistical media optimizationstudies for growth and PHB production by Ralstonia eutrophardquoProcess Biochemistry vol 40 no 6 pp 2173ndash2182 2005

[25] A A Koutinas Y Xu R Wang and C Webb ldquoPolyhydroxybu-tyrate production from a novel feedstock derived from a wheat-based biorefineryrdquo Enzyme and Microbial Technology vol 40no 5 pp 1035ndash1044 2007

[26] M-S Kim J-S Baek and J K Lee ldquoComparison of H2 accu-mulation by Rhodobacter sphaeroides KD131 and its uptakehydrogenase and PHB synthase deficient mutantrdquo InternationalJournal of Hydrogen Energy vol 31 no 1 pp 121ndash127 2006

[27] Z Liangqi X Jingfan F Tao and W Haibin ldquoSynthesis of poly(3-hydroxybutyrate-co-3-hydroxyoctanoate) by a Sinorhizo-bium fredii strainrdquo Letters inAppliedMicrobiology vol 42 no 4pp 344ndash349 2006

[28] K Sangkharak and P Prasertsan ldquoNutrient optimization forproduction of polyhydroxybutyrate from halotolerant photo-synthetic bacteria cultivated under aerobic-dark conditionrdquoElectronic Journal of Biotechnology vol 11 no 3 2008

[29] E R Coats F J Loge M P Wolcott K Englund and A GMcDonald ldquoSynthesis of polyhydroxyalkanoates in municipalwastewater treatmentrdquoWater Environment Research vol 79 no12 pp 2396ndash2403 2007

[30] A P Bonartsev V L Myshkina D A Nikolaeva et al ldquoBio-synthesis biodegradation and application of poly(3-hydrox-ybutyrate) and its copolymers natural polyesters producedby diazotrophic bacteriardquo in Communicating Current Researchand Educational Topics and Trends in Applied Microbiology AMendez-Vilas Ed pp 295ndash307 Formatex Badajoz Spain 2007

[31] C A Woolnough L H Yee T Charlton and L J R FosterldquoEnvironmental degradation and biofouling of lsquogreenrsquo plasticsincluding short and medium chain length polyhydroxyalka-noatesrdquo Polymer International vol 59 no 5 pp 658ndash667 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


high biochemical oxygen demand (BOD) and if dischargeduntreated increased BOD effects aquatic ecosystem and hasultimate impact on human health

The sugar industries need spending lots of money on thetreatment of waste water treatment to adhere to regulatorystandards

Thus the two different problems namely the pollutiondue to synthetic plastic and generation of organic richwaste from sugar industries could uniquely addressed by pro-ducing the bioplastic using the organic waste as nutrientsource

However this carbohydrate waste lacks certain mineralnutrient particularly nitrogen in relation to the amountof oxidizable carbon present and still may require nutrientsupplementation

Here an attemptwasmade to use the sugar industrywastewater after nutritive adjustment for the production of PHBusing an isolate Bacillus subtilis NG220

2 Materials and Methods

21 Bacterial Isolation and Culturing Bacillus subtilisNG220was isolated on carbon rich nutrient agar medium [(wv)glucose 1 beef extract 03 peptone 05 and sodiumchloride 08 agar 15] from the soil sample collectedfrom sugarcane field area The presence of intracellular PHBgranules was confirmed with the help of staining with Sudanblack B and Nile blue A [4] The culture was maintainedon the nutrient agar medium (wv) (beef extract 03peptone 05 sodium chloride 08 and agar 15) andstored at 4∘C

22 Morphological Biochemical and Molecular Character-isation The isolate was morphologically characterized byobserving the standard microbiological methods The bio-chemical characterization of the isolate was done by seriesof biochemical tests including carbohydrate fermentationH2

S production MR-VP test and Catalase test Further formolecular characterization the PCR amplification of 16SrDNA was done at 95∘C for 5min 95∘C for 30 sec 52∘C for30 sec 72∘C for 1min and 72∘C for 10min The universalprimer 16sF-51015840 AGA GTT TGA TCC TGG CTC AG 31015840 and16sR-51015840 ACG GCT ACC TTG TTA CGA CTT 31015840 were usedPCR product was purified by column (Merck bioscienceIndia) and was sequenced with AB instrument 3730XL Theobtained sequencewas subjected to search for closest possiblespecies using distance matrix based on nucleotide sequencehomology (using Kimura-2 parameter) and BLAST toolsavailable at National Centre for Biotechnology Information(NCBI) Phylogenetic analysis was done by using the Phylippackage and ClustalW

23 Preparation of Seed Inoculum One loop full of theculture from slant was inoculated in 5mL of sterile nutrientbroth (beef extract 03 peptone 05 sodium chloride08) After incubation for 24 h at 30∘C 1 (vv) of culturehaving 108 cellsmL was aseptically transferred into 50mLsterile nutrient broth and incubated at 30∘C for 18 h

Table 1 Physiochemical characteristics of sugar industry wastewater

Parameter Concentration (mgL)pH 42ndash69Total solids 1076 plusmn 414Suspended solid 631 plusmn 2274

COD 1395 plusmn 317BOD 1300 plusmn 238CODBOD 16 plusmn 049

BODNP 100 169 069Nitrogen 1148 plusmn 654

Phosphorous 436 plusmn 247

24 Substrate Preprocessing Untreated sugar industry efflu-ent was collected from the Saraswati Sugar Mills Yamu-nanagar Haryana India and was stored at 4∘C until usedThe effluent was filtered through the muslin cloth to removethe undesired solid materials Appropriately diluted filteredwaste water was used as PHB production medium (Table 1)

25 Production Detection Extraction and Purification ofPHB The PHB production was followed in 250mL Erlen-meyer flask containing 50mL sugar industry waste water asproduction medium under stationary condition of growthAfter 72 h of incubation at 30∘C culture broth was cen-trifuged at 8000 rpm for 15min The portion of the palettewas resuspended in 1mL of sterile distilled water sonicated(Hielscher Germany) in 02 aq sol of Nile blue A air-driedthe smear and observed under phase contrast microscope[4] Remaining amount of the pellet obtained was usedfor extraction and recovery of PHB according to Law andSlepecky [5] with minor modifications Briefly the harvestedcells were lyophilized and the dry cell mass weight was notedThe dry cell mass was subsequently incubated with 10mLsodium hypochlorite at 50∘C for 1 h for cells lysis The cellextract obtained was centrifuged at 12000 rpm for 30minthen washed sequentially with distilled water acetone andabsolute ethanol and dissolved in 10mL boiling chloroform(Sigma Aldrich) After evaporation 10mL of conc sulphuricacidwas added and placed inwater bath for 10min at 100∘C toconvert the PHB into crotonic acid On cooling absorbancewas taken at 235 nm [6] using PHB (Sigma Aldrich) asstandard

26 Polymer Analysis

261 NMR Analysis 1H Nuclear Magnetic Resonance Theidentity of individualmonomer unitwas confirmedby protonnuclear magnetic resonance (1H-NMR) spectroscopy 1H-NMR spectra of PHB sample were recorded in CDCl3 onBruker ACF 300 spectrophotometer at 300MHz by usingldquoTetramethylsilanerdquo as internal standard [7]

262 FT-IR Analyses FT-IR analysis of the polymer samplewas carried out on MB-3000 ABB FTIR spectrophotometerin the range 4000ndash600 cmminus1





























119898








Bacillussubtilis





BacilluscereusATCC




Query NG220








728

1

214

91

624

160

31

581

156

01

540

145

91

437

140

81

352

130

31

275

123

51

213

102

21

000

088

00

859

001

9

083

140

100

159

083

140

100

159

13 12 11 10 9 8 7 6 5 4 3 2 1 0 minus1(ppm)

(a)

100

95

90

85

80

4000 3750 3500 3250 3000 2750 2500 2250 2000 1750 1500 1250

3742

3618

2955

2916

2361

1744

1705

1651

1520

1458

1373

1311

1219

1165

1034

895

841

771

663

609T

()

(cmminus1)

(b)

02

00

minus02

minus04

minus06

13214∘C

0 100 200 300 400 500

1032 Jg2843 Jg

8740∘C

13254∘C

Hea

t flow

(Wg

)


(c)

Figure 2 Continued


200 600 1000 1400 1800 2200 2600 3000 3400Time

Abun

danc

e

1060

109913241800

3804

7020

1019311505 15836

15903

17260

18550

18717

19000

20357

21264

21330

227382301824382

25038

25737

26469

26530

266362749928036

28243

28344

29301

2990730472

31917

33327

34431

2

6

10

14

18

22

26

30

34

38

42

times104

4

8

12

16

20

24

28

32

36

40

44

(d)









5148 5151

5191

5125

50851

512514516518

52

24 48 72 96

PHB

(gL

)


(a)

484 488

5201

49285

46474849

515253

20 30 40 50

PHB

(gL

)


(b)

508 5075103 5103

5213519

5108

5

495

505

51

515

52

525

5 55 6 65 7 75 8

PHB

(gL

)

pH

(c)

51225219

5148 5128 50975054

495

515253

12 18 24 30 36 42PH

B (g

L)

Inoculum age (h)

(d)

474849

5515253

PHB

(gL

)

Dex

trose

Xylo

se

Sucr

ose

Rham

nose

Man

nito

l

Mal

tose

Lact

ose

Man

nose

Gal

acto

se

Star

ch

491749654982

5254

49855017 50175022502849284931


(e)

49315005 5005

5297

49914942 4931

498

474849

551525354

Ure

a

Tryp

tone

Pept

one

(NH

4) 2

SO4

(NH

4)H

2PO

4

(NH

4) 2

HPO

4

NH

4S 2

O8

NH

4Cl

PHB

(gL

)


(f)




4 Conclusion





Soil

0 0 30 times 106

10 302 plusmn 05 40 times 107

20 435 plusmn 12 49 times 107

30 706 plusmn 26 52 times 108

40 983 plusmn 21 54 times 108

Compost

0 0 5 times 106

10 502 plusmn 05 2 times 107

20 802 plusmn 05 12 times 108

30 100 30 times 109

Industrial sludge

0 0 25 times 105

10 263 plusmn 12 32 times 106

20 576 plusmn 15 49 times 106

30 736 plusmn 15 56 times 106

40 972 plusmn 22 60 times 106


0

20

40

60

80

100

120

10 15 20 25

Wei

ght l

oss (

)




Acknowledgments


References








2

Co7


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





























119898








Bacillussubtilis





BacilluscereusATCC




Query NG220








728

1

214

91

624

160

31

581

156

01

540

145

91

437

140

81

352

130

31

275

123

51

213

102

21

000

088

00

859

001

9

083

140

100

159

083

140

100

159

13 12 11 10 9 8 7 6 5 4 3 2 1 0 minus1(ppm)

(a)

100

95

90

85

80

4000 3750 3500 3250 3000 2750 2500 2250 2000 1750 1500 1250

3742

3618

2955

2916

2361

1744

1705

1651

1520

1458

1373

1311

1219

1165

1034

895

841

771

663

609T

()

(cmminus1)

(b)

02

00

minus02

minus04

minus06

13214∘C

0 100 200 300 400 500

1032 Jg2843 Jg

8740∘C

13254∘C

Hea

t flow

(Wg

)


(c)

Figure 2 Continued


200 600 1000 1400 1800 2200 2600 3000 3400Time

Abun

danc

e

1060

109913241800

3804

7020

1019311505 15836

15903

17260

18550

18717

19000

20357

21264

21330

227382301824382

25038

25737

26469

26530

266362749928036

28243

28344

29301

2990730472

31917

33327

34431

2

6

10

14

18

22

26

30

34

38

42

times104

4

8

12

16

20

24

28

32

36

40

44

(d)









5148 5151

5191

5125

50851

512514516518

52

24 48 72 96

PHB

(gL

)


(a)

484 488

5201

49285

46474849

515253

20 30 40 50

PHB

(gL

)


(b)

508 5075103 5103

5213519

5108

5

495

505

51

515

52

525

5 55 6 65 7 75 8

PHB

(gL

)

pH

(c)

51225219

5148 5128 50975054

495

515253

12 18 24 30 36 42PH

B (g

L)

Inoculum age (h)

(d)

474849

5515253

PHB

(gL

)

Dex

trose

Xylo

se

Sucr

ose

Rham

nose

Man

nito

l

Mal

tose

Lact

ose

Man

nose

Gal

acto

se

Star

ch

491749654982

5254

49855017 50175022502849284931


(e)

49315005 5005

5297

49914942 4931

498

474849

551525354

Ure

a

Tryp

tone

Pept

one

(NH

4) 2

SO4

(NH

4)H

2PO

4

(NH

4) 2

HPO

4

NH

4S 2

O8

NH

4Cl

PHB

(gL

)


(f)




4 Conclusion





Soil

0 0 30 times 106

10 302 plusmn 05 40 times 107

20 435 plusmn 12 49 times 107

30 706 plusmn 26 52 times 108

40 983 plusmn 21 54 times 108

Compost

0 0 5 times 106

10 502 plusmn 05 2 times 107

20 802 plusmn 05 12 times 108

30 100 30 times 109

Industrial sludge

0 0 25 times 105

10 263 plusmn 12 32 times 106

20 576 plusmn 15 49 times 106

30 736 plusmn 15 56 times 106

40 972 plusmn 22 60 times 106


0

20

40

60

80

100

120

10 15 20 25

Wei

ght l

oss (

)




Acknowledgments


References








2

Co7


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology














119898








Bacillussubtilis





BacilluscereusATCC




Query NG220








728

1

214

91

624

160

31

581

156

01

540

145

91

437

140

81

352

130

31

275

123

51

213

102

21

000

088

00

859

001

9

083

140

100

159

083

140

100

159

13 12 11 10 9 8 7 6 5 4 3 2 1 0 minus1(ppm)

(a)

100

95

90

85

80

4000 3750 3500 3250 3000 2750 2500 2250 2000 1750 1500 1250

3742

3618

2955

2916

2361

1744

1705

1651

1520

1458

1373

1311

1219

1165

1034

895

841

771

663

609T

()

(cmminus1)

(b)

02

00

minus02

minus04

minus06

13214∘C

0 100 200 300 400 500

1032 Jg2843 Jg

8740∘C

13254∘C

Hea

t flow

(Wg

)


(c)

Figure 2 Continued


200 600 1000 1400 1800 2200 2600 3000 3400Time

Abun

danc

e

1060

109913241800

3804

7020

1019311505 15836

15903

17260

18550

18717

19000

20357

21264

21330

227382301824382

25038

25737

26469

26530

266362749928036

28243

28344

29301

2990730472

31917

33327

34431

2

6

10

14

18

22

26

30

34

38

42

times104

4

8

12

16

20

24

28

32

36

40

44

(d)









5148 5151

5191

5125

50851

512514516518

52

24 48 72 96

PHB

(gL

)


(a)

484 488

5201

49285

46474849

515253

20 30 40 50

PHB

(gL

)


(b)

508 5075103 5103

5213519

5108

5

495

505

51

515

52

525

5 55 6 65 7 75 8

PHB

(gL

)

pH

(c)

51225219

5148 5128 50975054

495

515253

12 18 24 30 36 42PH

B (g

L)

Inoculum age (h)

(d)

474849

5515253

PHB

(gL

)

Dex

trose

Xylo

se

Sucr

ose

Rham

nose

Man

nito

l

Mal

tose

Lact

ose

Man

nose

Gal

acto

se

Star

ch

491749654982

5254

49855017 50175022502849284931


(e)

49315005 5005

5297

49914942 4931

498

474849

551525354

Ure

a

Tryp

tone

Pept

one

(NH

4) 2

SO4

(NH

4)H

2PO

4

(NH

4) 2

HPO

4

NH

4S 2

O8

NH

4Cl

PHB

(gL

)


(f)




4 Conclusion





Soil

0 0 30 times 106

10 302 plusmn 05 40 times 107

20 435 plusmn 12 49 times 107

30 706 plusmn 26 52 times 108

40 983 plusmn 21 54 times 108

Compost

0 0 5 times 106

10 502 plusmn 05 2 times 107

20 802 plusmn 05 12 times 108

30 100 30 times 109

Industrial sludge

0 0 25 times 105

10 263 plusmn 12 32 times 106

20 576 plusmn 15 49 times 106

30 736 plusmn 15 56 times 106

40 972 plusmn 22 60 times 106


0

20

40

60

80

100

120

10 15 20 25

Wei

ght l

oss (

)




Acknowledgments


References








2

Co7


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Bacillussubtilis





BacilluscereusATCC




Query NG220








728

1

214

91

624

160

31

581

156

01

540

145

91

437

140

81

352

130

31

275

123

51

213

102

21

000

088

00

859

001

9

083

140

100

159

083

140

100

159

13 12 11 10 9 8 7 6 5 4 3 2 1 0 minus1(ppm)

(a)

100

95

90

85

80

4000 3750 3500 3250 3000 2750 2500 2250 2000 1750 1500 1250

3742

3618

2955

2916

2361

1744

1705

1651

1520

1458

1373

1311

1219

1165

1034

895

841

771

663

609T

()

(cmminus1)

(b)

02

00

minus02

minus04

minus06

13214∘C

0 100 200 300 400 500

1032 Jg2843 Jg

8740∘C

13254∘C

Hea

t flow

(Wg

)


(c)

Figure 2 Continued


200 600 1000 1400 1800 2200 2600 3000 3400Time

Abun

danc

e

1060

109913241800

3804

7020

1019311505 15836

15903

17260

18550

18717

19000

20357

21264

21330

227382301824382

25038

25737

26469

26530

266362749928036

28243

28344

29301

2990730472

31917

33327

34431

2

6

10

14

18

22

26

30

34

38

42

times104

4

8

12

16

20

24

28

32

36

40

44

(d)









5148 5151

5191

5125

50851

512514516518

52

24 48 72 96

PHB

(gL

)


(a)

484 488

5201

49285

46474849

515253

20 30 40 50

PHB

(gL

)


(b)

508 5075103 5103

5213519

5108

5

495

505

51

515

52

525

5 55 6 65 7 75 8

PHB

(gL

)

pH

(c)

51225219

5148 5128 50975054

495

515253

12 18 24 30 36 42PH

B (g

L)

Inoculum age (h)

(d)

474849

5515253

PHB

(gL

)

Dex

trose

Xylo

se

Sucr

ose

Rham

nose

Man

nito

l

Mal

tose

Lact

ose

Man

nose

Gal

acto

se

Star

ch

491749654982

5254

49855017 50175022502849284931


(e)

49315005 5005

5297

49914942 4931

498

474849

551525354

Ure

a

Tryp

tone

Pept

one

(NH

4) 2

SO4

(NH

4)H

2PO

4

(NH

4) 2

HPO

4

NH

4S 2

O8

NH

4Cl

PHB

(gL

)


(f)




4 Conclusion





Soil

0 0 30 times 106

10 302 plusmn 05 40 times 107

20 435 plusmn 12 49 times 107

30 706 plusmn 26 52 times 108

40 983 plusmn 21 54 times 108

Compost

0 0 5 times 106

10 502 plusmn 05 2 times 107

20 802 plusmn 05 12 times 108

30 100 30 times 109

Industrial sludge

0 0 25 times 105

10 263 plusmn 12 32 times 106

20 576 plusmn 15 49 times 106

30 736 plusmn 15 56 times 106

40 972 plusmn 22 60 times 106


0

20

40

60

80

100

120

10 15 20 25

Wei

ght l

oss (

)




Acknowledgments


References








2

Co7


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


728

1

214

91

624

160

31

581

156

01

540

145

91

437

140

81

352

130

31

275

123

51

213

102

21

000

088

00

859

001

9

083

140

100

159

083

140

100

159

13 12 11 10 9 8 7 6 5 4 3 2 1 0 minus1(ppm)

(a)

100

95

90

85

80

4000 3750 3500 3250 3000 2750 2500 2250 2000 1750 1500 1250

3742

3618

2955

2916

2361

1744

1705

1651

1520

1458

1373

1311

1219

1165

1034

895

841

771

663

609T

()

(cmminus1)

(b)

02

00

minus02

minus04

minus06

13214∘C

0 100 200 300 400 500

1032 Jg2843 Jg

8740∘C

13254∘C

Hea

t flow

(Wg

)


(c)

Figure 2 Continued


200 600 1000 1400 1800 2200 2600 3000 3400Time

Abun

danc

e

1060

109913241800

3804

7020

1019311505 15836

15903

17260

18550

18717

19000

20357

21264

21330

227382301824382

25038

25737

26469

26530

266362749928036

28243

28344

29301

2990730472

31917

33327

34431

2

6

10

14

18

22

26

30

34

38

42

times104

4

8

12

16

20

24

28

32

36

40

44

(d)









5148 5151

5191

5125

50851

512514516518

52

24 48 72 96

PHB

(gL

)


(a)

484 488

5201

49285

46474849

515253

20 30 40 50

PHB

(gL

)


(b)

508 5075103 5103

5213519

5108

5

495

505

51

515

52

525

5 55 6 65 7 75 8

PHB

(gL

)

pH

(c)

51225219

5148 5128 50975054

495

515253

12 18 24 30 36 42PH

B (g

L)

Inoculum age (h)

(d)

474849

5515253

PHB

(gL

)

Dex

trose

Xylo

se

Sucr

ose

Rham

nose

Man

nito

l

Mal

tose

Lact

ose

Man

nose

Gal

acto

se

Star

ch

491749654982

5254

49855017 50175022502849284931


(e)

49315005 5005

5297

49914942 4931

498

474849

551525354

Ure

a

Tryp

tone

Pept

one

(NH

4) 2

SO4

(NH

4)H

2PO

4

(NH

4) 2

HPO

4

NH

4S 2

O8

NH

4Cl

PHB

(gL

)


(f)




4 Conclusion





Soil

0 0 30 times 106

10 302 plusmn 05 40 times 107

20 435 plusmn 12 49 times 107

30 706 plusmn 26 52 times 108

40 983 plusmn 21 54 times 108

Compost

0 0 5 times 106

10 502 plusmn 05 2 times 107

20 802 plusmn 05 12 times 108

30 100 30 times 109

Industrial sludge

0 0 25 times 105

10 263 plusmn 12 32 times 106

20 576 plusmn 15 49 times 106

30 736 plusmn 15 56 times 106

40 972 plusmn 22 60 times 106


0

20

40

60

80

100

120

10 15 20 25

Wei

ght l

oss (

)




Acknowledgments


References








2

Co7


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


200 600 1000 1400 1800 2200 2600 3000 3400Time

Abun

danc

e

1060

109913241800

3804

7020

1019311505 15836

15903

17260

18550

18717

19000

20357

21264

21330

227382301824382

25038

25737

26469

26530

266362749928036

28243

28344

29301

2990730472

31917

33327

34431

2

6

10

14

18

22

26

30

34

38

42

times104

4

8

12

16

20

24

28

32

36

40

44

(d)









5148 5151

5191

5125

50851

512514516518

52

24 48 72 96

PHB

(gL

)


(a)

484 488

5201

49285

46474849

515253

20 30 40 50

PHB

(gL

)


(b)

508 5075103 5103

5213519

5108

5

495

505

51

515

52

525

5 55 6 65 7 75 8

PHB

(gL

)

pH

(c)

51225219

5148 5128 50975054

495

515253

12 18 24 30 36 42PH

B (g

L)

Inoculum age (h)

(d)

474849

5515253

PHB

(gL

)

Dex

trose

Xylo

se

Sucr

ose

Rham

nose

Man

nito

l

Mal

tose

Lact

ose

Man

nose

Gal

acto

se

Star

ch

491749654982

5254

49855017 50175022502849284931


(e)

49315005 5005

5297

49914942 4931

498

474849

551525354

Ure

a

Tryp

tone

Pept

one

(NH

4) 2

SO4

(NH

4)H

2PO

4

(NH

4) 2

HPO

4

NH

4S 2

O8

NH

4Cl

PHB

(gL

)


(f)




4 Conclusion





Soil

0 0 30 times 106

10 302 plusmn 05 40 times 107

20 435 plusmn 12 49 times 107

30 706 plusmn 26 52 times 108

40 983 plusmn 21 54 times 108

Compost

0 0 5 times 106

10 502 plusmn 05 2 times 107

20 802 plusmn 05 12 times 108

30 100 30 times 109

Industrial sludge

0 0 25 times 105

10 263 plusmn 12 32 times 106

20 576 plusmn 15 49 times 106

30 736 plusmn 15 56 times 106

40 972 plusmn 22 60 times 106


0

20

40

60

80

100

120

10 15 20 25

Wei

ght l

oss (

)




Acknowledgments


References








2

Co7


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


5148 5151

5191

5125

50851

512514516518

52

24 48 72 96

PHB

(gL

)


(a)

484 488

5201

49285

46474849

515253

20 30 40 50

PHB

(gL

)


(b)

508 5075103 5103

5213519

5108

5

495

505

51

515

52

525

5 55 6 65 7 75 8

PHB

(gL

)

pH

(c)

51225219

5148 5128 50975054

495

515253

12 18 24 30 36 42PH

B (g

L)

Inoculum age (h)

(d)

474849

5515253

PHB

(gL

)

Dex

trose

Xylo

se

Sucr

ose

Rham

nose

Man

nito

l

Mal

tose

Lact

ose

Man

nose

Gal

acto

se

Star

ch

491749654982

5254

49855017 50175022502849284931


(e)

49315005 5005

5297

49914942 4931

498

474849

551525354

Ure

a

Tryp

tone

Pept

one

(NH

4) 2

SO4

(NH

4)H

2PO

4

(NH

4) 2

HPO

4

NH

4S 2

O8

NH

4Cl

PHB

(gL

)


(f)




4 Conclusion





Soil

0 0 30 times 106

10 302 plusmn 05 40 times 107

20 435 plusmn 12 49 times 107

30 706 plusmn 26 52 times 108

40 983 plusmn 21 54 times 108

Compost

0 0 5 times 106

10 502 plusmn 05 2 times 107

20 802 plusmn 05 12 times 108

30 100 30 times 109

Industrial sludge

0 0 25 times 105

10 263 plusmn 12 32 times 106

20 576 plusmn 15 49 times 106

30 736 plusmn 15 56 times 106

40 972 plusmn 22 60 times 106


0

20

40

60

80

100

120

10 15 20 25

Wei

ght l

oss (

)




Acknowledgments


References








2

Co7


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Soil

0 0 30 times 106

10 302 plusmn 05 40 times 107

20 435 plusmn 12 49 times 107

30 706 plusmn 26 52 times 108

40 983 plusmn 21 54 times 108

Compost

0 0 5 times 106

10 502 plusmn 05 2 times 107

20 802 plusmn 05 12 times 108

30 100 30 times 109

Industrial sludge

0 0 25 times 105

10 263 plusmn 12 32 times 106

20 576 plusmn 15 49 times 106

30 736 plusmn 15 56 times 106

40 972 plusmn 22 60 times 106


0

20

40

60

80

100

120

10 15 20 25

Wei

ght l

oss (

)




Acknowledgments


References








2

Co7


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Poly -Hydroxybutyrate Production by ...of PHB was recorded as the loss of the weight a er de nite period of incubation. 3. Results and Discussion.. Isolation of PHB