isolation and screening of polyhydroxyalkanoates...

Hindawi Publishing CorporationInternational Journal of BiomaterialsVolume 2013, Article ID 752821, 10 pageshttp://dx.doi.org/10.1155/2013/752821

Research ArticleIsolation and Screening of Polyhydroxyalkanoates ProducingBacteria from Pulp, Paper, and Cardboard Industry Wastes

Anish Kumari Bhuwal,1 Gulab Singh,1 Neeraj Kumar Aggarwal,1

Varsha Goyal,1 and Anita Yadav2

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

Correspondence should be addressed to Neeraj Kumar Aggarwal; [email protected]

Received 26 May 2013; Revised 8 August 2013; Accepted 20 August 2013

Academic Editor: Ravin Narain

Copyright © 2013 Anish Kumari Bhuwal et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

Background. Polyhydroxyalkanoates (PHAs) are storagematerials that accumulate by various bacteria as energy and carbon reservematerials. They are biodegradable, environmentally friendly, and also biocompatible bioplastics. Unlike petrochemical-basedplastics that take several decades to fully degrade, PHAs can be completely degradedwithin a year by variety ofmicroorganisms intoCO2and water. In the present study, we aim to utilize pulp, paper, and cardboard industry sludge and waste water for the isolation

and screening of polyhydroxyalkanoates (PHAs) accumulating bacteria and production of cost-effective PHB using cardboardindustry waste water. Results. A total of 42 isolates showed black-blue coloration when stained with Sudan black B, a preliminaryscreening agent for lipophilic compounds, and a total of 15 isolates showed positive result with Nile blue A staining, a morespecific dye for PHA granules.The isolates NAP11 and NAC1 showed maximum PHA production 79.27% and 77.63% with polymerconcentration of 5.236 g/L and 4.042 g/L with cardboard industry waste water. Both of the selected isolates, NAP11 and NAC1, wereclassified up to genus level by studying their morphological and biochemical characteristics and were found to be Enterococcussp., Brevundimonas sp. and, respectively. Conclusion. The isolates Enterococcus sp. NAP11 and Brevundimonas sp. NAC1 can beconsidered as good candidates for industrial production of PHB from cardboard industry waste water.We are reporting for the firsttime the use of cardboard industry waste water as a cultivation medium for the PHB production.

1. Introduction

Plastic materials that have been universally used in ourdaily lives are now causing serious environmental problems.Millions of tons of these nondegradable plastics accumulatein the environment per year. For efficient management ofused-plastic materials, recycling is one solution. Anothersolution to reduce plastic residue is the use of biodegradableplastics [1, 2] and among them polyhydroxyalkanoic acids(PHAs) are drawing much attention. Polyhydroxyalkanoicacids (PHAs) are common intracellular compounds foundin bacteria, archaea, and in few eukaryotes such as yeastsand fungi. PHAs are carbon and energy reserve polymersproduced in some microorganisms when carbon source is inplentiful and other nutrients such as nitrogen, phosphorus,oxygen or sulfur are limited. PHAs are found to accumulate

in varieties of microorganisms as reserve food material forexample, Alcaligenes latus, Ralstonia eutropha, Azotobacterbeijerinckii, Bacillus megaterium, and Pseudomonas oleovo-rans, including some fungi and archaea. Among themembersof PHA family, polyhydroxybutyrate (PHB) is the mostcommon biodegradable polymer and promising alternativeto synthetic nondegradable plastics.These polymers are accu-mulated intracellular membrane enclosed inclusion up to90% of the cell dry weight under conditions of nutrient stressand act as energy reserve material. It has similar mechanicalproperties as those of the oil-derived conventional plasticslike polypropylene or polyethylene which can be molded,made into films, spun into monofilaments, and used to makeheteropolymers with other synthetic polymers and manymore applications in agriculture, packaging, andmedical fieldbeing biodegradable and also immunologically compatible

2 International Journal of Biomaterials

with human tissue [3]. Recently, another application of PHAsis reported as biofuel [4].

In spite of these interesting properties, industrial pro-duction of PHAs is still not well established. In the 1950s,North-American Company W.R. Grace Co. made the firstattempt to produce PHB at commercial level. However, thisprocess was not successful due to low production efficiencyand a lack of suitable purification method. Then, in the1970s, Imperial Chemical Industries (ICI, UK) started pro-ducing PHAs by using a mutant stain Cupriavidus neca-tor, NCIB 11599 from various carbon sources such as 1,4-butanediol, 1,6-hexanediol, and butyrolactone. The com-mercial product was recognized as Biopol. The patentswere later sold to Zeneca, then to Monsanto and are cur-rently the property of Metabolix, Inc. (USA). Commercially,some other biopolyester products with different monomercomposition are also produced with trade names suchas poly(3HB-co-3HV) Nodax, poly(3-hydroxybutyrate-co-3-hydroxyalkanoate) poly(3HB-co-3HA) as Biogreen, andpoly(3HB) as Biomer [5]. But the large scale production washalted at commercial level due to the high production cost ascompared with the oil-derived plastics [1, 6]. With the aim ofcommercializing PHA, great efforts have been employed toreduce the production cost by the development of bacterialstrains and more efficient fermentation/recovery process [7,8]. From the literature, it has been found that the major costin this biopolymer production is the cost of the substrate[9, 10] which accounts for more than 50% of productioncost [5, 11] and makes the difference in price of poly-3-hydroxybutyrate (P3HB) from Biomer about 12 times thecost of polypropylene [12]. To solve this problem, inexpensivesubstrate, renewable substrates, waste material, and wastewater are used as nutrient source for microorganisms forPHA production. Various types of waste products havebeen used for PHB production because it provides dualbenefits of utilizing the waste and cost-effective productionof biodegradable microbial bioplastic.

Cardboard industry is one of the parts of pulp, paper, andpackaging industry. Cardboard industry includes two mainindustries corrugated cardboard industry and noncorrugatedcardboard or paper-board industry. Waste water from chem-ical and mechanical pulping contains 12–25 kg of BOD/tof ADP, but the BOD discharges are 3 to 10 times higherin chemimechanical pulping as compared to mechanicalpulping. Nitrogen and phosphorus are also present in wastewaters and released from the pulping process of raw materialsuch as wood, agricultural waste, and paper waste. Wastewaters released from pulp and paperboard mills are typicallyrich in carbohydrates but poor in fixed nitrogen. If weconsider the scenario of India, pulp and paperboard industryaround 905.8millionm3 of water is consumed and around695.7millionm3 of waste water is discharged annually. Thelargest part of the freshwater is used in sheet formation on thecardboard machine (200m3 h−1) and the smallest quantityis used in stock preparation (90m3 h−1 for thickening). Fur-thermore, the treatment of waste stream to purified effluentneeds much effort and is very difficult, because the wastestream often contains various organic compounds. So instead

of costly treatment, we can exploit the waste water directlyfor cultivation of PHB accumulating microorganisms. Inthis study, polyhydroxyalkanoic acids (PHAs) accumulatingbacterial strains were isolated and screened using cardboardindustry waste water as a sole carbon source with dualbenefit of utilizing the waste and cost-effective production ofbiodegradable microbial bioplastic.

2. Material and Methods

2.1. Isolation of Polyhydroxyalkanoic Acids (PHAs) ProducingBacteria. For the isolation of PHA producing bacteriamactivated sludge and waste water were collected from pulp,kraft, and cardboard manufacturing industry from Khannapulp and paper mills at Amritsar and Topara Kurdh, YamunaNagar, India, respectively. The samples were stored at roomtemperature until analysis. In 99mL sterilized water, 1 gm ofsludge sample was dissolved. Then, the sample was seriallydiluted in sterile distilled water and followed by plating onthe nutrient agar medium with 1% glucose. For isolationfrom waste water sample, 1mL of water sample was addedin 99mL sterilized water. After serial dilution (10−3 to 10−7),1mL of each dilution was spread on carbon rich nutrientagar plate. For the rapid detection and isolation of PHBproducing bacteria, 0.02% alcoholic solution of Sudan blackB was applied to stain bacterial colonies and the plateswere kept undisturbed for 30min. The excess dye was thendecanted and plates were rinsed gently by adding 100%ethanol. Colonies unable to incorporate the Sudan black Bappeared white, while PHB producers appeared bluish black[13].

2.2. Screening for PHA Producing Bacteria. Sudan black Bpositive isolates were checked for PHA production by NileblueA staining, amore specific stain for Polyhydroxyalkanoicacids (PHAs) by a more rapid and sensitive, viable colonymethod [14]. This dye at concentrations of only 0.5𝜇g/mLwas directly included in carbon rich nutrient agar medium(glucose 1%, beef extract 0.3%, peptone 0.5%, sodium chlo-ride 0.8%, and agar 1.5%) and growth of the cells occurredin the presence of the dye. This allowed an estimationof the presence of PHAs in viable colonies at any timeduring the growth experiment and a powerful discriminationbetween PHA-negative and PHA-positive strains. The PHAaccumulating colonies, after Nile blue A staining, showedbright orange fluorescence on irradiation with UV light andtheir fluorescence intensity increased with the increase inPHA content of the bacterial cells.The isolates which showedbright orange fluorescence on irradiation with UV light afterNile blue A staining were selected as PHA accumulators.

2.3. Pretreatment of Cardboard Industry Waste Water. Un-treated cardboard industry effluent was collected from thecardboard industry, Topara Kurdh, Yamuna Nagar, Haryana,India, and stored at 4∘C until used for analysis. The effluentwas first filtered through the muslin cloth and then byrough filter paper to remove the undesired suspended solidmaterials from waste water. After this pretreatment step,

International Journal of Biomaterials 3

cardboard industry waste water was used as quantificationand production medium for PHA production by selectedbacteria.

2.4. Extraction and Quantitative Analysis of PHA. The PHBproduction was observed in 250mL Erlenmeyer flask con-taining 50mL of treated cardboard industry waste water, asproduction medium under stationary conditions of growth.After 72 h of incubation at 37∘C, culture brothwas centrifugedat 8000 rpm for 15min. The pellet along with 10mL sodiumhypochlorite was incubated at 50∘C for 1 h for lyses of cells.The cell extract obtained was centrifuged at 12000 rpm for30min and then washed sequentially with distilled water,acetone, and absolute ethanol. After washing, the pellet wasdissolved in 10mL chloroform (AR grade) and incubatedovernight at 50∘C and was evaporated at room temperature[15]. After evaporation, 10mL of sulphuric acid was added toit and placed in water bath for 10min at 100∘C. This convertspolyhydroxyalkanoic acids (PHAs) into crotonic acid, whichgives maximum absorbance at 235 nm [16, 17]. PHB (SigmaAldrich) was used as standard for making standard curve.For quantitative analysis of PHA, cell culture was grown asdescribed earlier and cell pellet was dried to estimate thedry cell weight (DCW) in units of g/L [18]. Residual biomasswas estimated as the difference between dry cell weight anddry weight of extracted PHA [19]. This was calculated todetermine the cellular weight and accumulation other thanPHAs. The percentage of intracellular PHA accumulation isestimated as the percentage composition of PHA present inthe dry cell weight:

Residual biomass (g/L)

= DCW (g/L) − Dryweight of extracted PHA (g/L) ,

PHA accumulation (%)

= Dryweight of extracted PHA (g/L)

× 100%/DCW (g/L) .(1)

2.5. Morphological Characterization and Biochemical Identifi-cation of PHA Producing Bacteria. Microscope Stereo Olym-pus was used to observe themorphology of bacterial coloniesgrown on nutrient agar. The growth characteristics suchas structure, shape, color, margin, surface characteristics,surface upwards, smell, elevation, opacity, end of cells, cell’sarrangement, and Gram-staining of the bacterial colonieswere observed to characterize the bacterial colonies.

Various biochemical tests were performed in selectedPHB producing bacteria, namely, indole production test,methyl red and Voges-Proskauer, citrate utilization test, andH2S production for their biochemical characterization. The

fermentative utilization of various carbohydrates was also fol-lowed for 48 hrs at 37∘C by inoculating the isolates separatelyin the defined medium to which various sugars like xylose,mannose, maltose, sucrose, raffinose, dextrose, trehalose,fructose, glucose, ribose, lactose, rhamnose, esculin, inulin,mannitol, arabinose, sorbitol, and melibiose were added.

2.6. Polymer Analysis

2.6.1. 1H-NMR Spectroscopy and Thermal Gravimetric Anal-ysis (TGA). The identity of individual monomer unit wasconfirmed by proton nuclear magnetic resonance (1H-NMR)spectroscopy. 1H-NMR spectra were acquired by dissolvingthe polymer in deuterochloroform (CDCl

3) at a concen-

tration of 10mg/mL and analyzed on a Bruker AvanceII 500 spectrometer at 22∘C with 7.4ms pulse width (30∘pulse angle), 1 s pulse repetition, 10,330Hz spectral width,and 65,536 data points. Tetramethylsilane was used as aninternal shift standard. Thermal gravimetric analysis (TGA)was performed using a TGA instrument (Mettler-Toledo,TGA/SDTA 851, USA) calibrated with indium. The tem-perature was ramped at a heating rate of 10∘C/min undernitrogen to a temperature (700∘C)well above the degradationtemperature of the polymers.

2.7. FT-IR Analysis. FT-IR analysis of the polymer samplewas carried out on MB-3000, ABB FTIR spectrophotometerin the range 4000–600 cm−1.

2.8. GC-MS Analysis. Purified polymer, prepared as des-cribed before, was dissolved in 2mL of chloroform and then2mL of methanol was added and acidified with 3% (v/v)H2SO4and heated at 100∘C for 3.5 h for depolymerization and

methanolysis of polyesters and 3𝜇L was injected into GCMS-QP 2010 Plusmodel.The samples were injected in the splitlessmode and the injection temperature was 260∘C and columnoven temperature was 100∘C.

3. Result and Discussion

3.1. Isolation and Selection of PHA Producing Bacteria. Awide variety of bacteria are known to accumulate PHA.Today, approximately 150 different hydroxyalkanoic acids areknown to be incorporated into polyhydroxyalkanoates [20],with microbial species from over 90 genera being reportedto accumulate these polyesters [21]. These bacteria havebeen reported from various environments, but only a fewfrom the waste water and sludge ecosystems. For the rapiddetection and isolation of PHB producing bacteria, 0.02%alcoholic solution of Sudan black B and Nile blue A stainingviable colony method [14] was used. The isolation of PHAproducing bacteria was done from cardboard manufacturingindustry waste water and pulp cardboard and kraft industrysludge. A large proportion about 35% of isolated bacteriaproduced PHA as energy reserve material. A total of 42isolates showed black-blue coloration when stained withSudan black B, a preliminary screening agent for lipophiliccompounds, and a total of 15 isolates showed positive resultwith Nile blue A staining (Figure 1), a specific dye for theof PHA granules. Both gram-positive and gram-negativebacteria showed PHA production, but gram-positive bacteriadominated the waste material microflora of pulp, kraft, andcardboard manufacturing industry. Teeka et al. [22] used thismethod to screen the potential PHA producing bacteria fromsoil, and Ramachandran and Abdullah [23] also observed


(a)

(b)

Figure 1: (a) Pink/orange florescence under UV light by PHBproducer isolate NAP11. (b) No florescence under UV light by Non-PHB producer with Nile blue A staining by viable colony method.

the colonies formed on nutrient rich medium under ultra-violet light (UV) to screen for the pink fluorescence whichindicated the presence of PHA producers. Kitamura andDoi [24] first demonstrated this viable colony method onagar plates; they induced the isolates to accumulate PHA byculturing in E

2medium containing 2% (w/v) glucose before

Nile blue A staining. The PHA accumulating colonies, afterNile blue A staining, showed bright orange fluorescence onirradiation with UV-light and their fluorescence intensityincreased with increase in PHA content of the bacterial cells.

3.2. Production, Extraction, and Estimation of PHA withCardboard Industry Waste Water as a Sole Carbon Source.The PHA-positive isolates selected after Nile blue A stainingwere grown in 50mL cardboard industry waste water inErlenmeyer flasks and were employed to extract PHA after

2 days of incubation under stationary conditions of growth.The PHA from the isolates was extracted by the hypochloriteand chloroformmethod [15] as described earlier. The isolatesNAP11 andNAC1 showedmaximumPHAproduction 79.27%and 77.63% (Table 1) with cardboard industry waste waterand were selected for further biochemical identification andchemical characterization.

Organic matter from wastes and waste waters has highBOD and COD values, and hence microorganisms can grow,utilizing the nutrient present in waste water, and can convertthem into valuable compounds and polymers. Based on thisidea, many researchers reported the PHA production fromvarious industrial waste materials. PHA production by A.vinelandii from swine waste liquor was studied by [25]. Theraw liquor supported the production of only 0.43 g/L PHA,at a polymer content of 37% w/w, whereas twofold dilutionand supplementation with 30 g glucose/L allowed a PHAconcentration of 5.5 g/L at a 58.3% w/w polymer content.Few researchers have proposed coupling PHA productionto biological waste water treatment [26–28]. Ceyhan andOzdemir [29] reported polyhydroxybutyrate (PHB) produc-tion from domestic waste water using Enterobacter aerogenes12Bi strain with good yield ranging from 16.66 to 96.25%(w/w). The use of pure C. necator cultures to produce PHAsfrom waste waters has been explored by Ganzeveld et al.[30]. They used a supernatant, obtained by centrifugingfermented organic waste, as the sole carbon source for theproduction of P(3HBco-3HV), and obtained a maximumpolymer concentration of 1.13 g/L at a polymer content of40.8% in 45 h. Cardboard industry waste water is typicallyrich in carbohydrates but poor in fixed nitrogen, due to thehigh C/N ratio. This high carbon-nitrogen ratio favors thegrowth of PHA producing bacteria. It is the first time thatcardboard industry waste water is used for the isolation,screening, and production of polyhydroxyalkanoates. Thiswaste has high BOD and COD values 680–1250mg/L and3400–5780mg/L andCOD/BOD ratio between 3.9 and 5 [31],which is suitable for microbial growth.

Extracted PHA of selected isolates was quantified andits efficiencies were compared with the standard. Standardpure culture of Ralstonia eutrophus MTCC no. 1473 was usedfor PHA production with cardboard waste water producinga polymer concentration of 2.974 g/L and PHB content upto 41.30% with cardboard industry waste water. The selectedisolates NAP11 from pulp sludge have produced 79.27%w/w PHA with polymer concentration of 5.236 g/L usingcardboard waste water which are 37% higher as comparedto standard stain of Ralstonia eutrophus (Figure 2). Theother NAC1 isolates showed PHA production up to 77.63%with polymer concentration of 4.042 g/L under stationaryconditions of growth.

3.3. Morphological and Biochemical Characterization ofSelected Isolates. By using Bergey’s Manual of DeterminativeBacteriology [32] and by ABIS Online-Advanced BacterialIdentification Software, bothisolates were classified up togenus level using the morphological and biochemical char-acteristics (Table 2). NAP11 and NAC1 were found to be


Table 1: List of PHA accumulating bacteria with source of isolation.

Name of isolate Source of isolate Gramreaction

PHA concentration(g/L) PHA content (%)

NAP11 Pulp industry sludge +ve 5.236 79.27 ± 0.3

NAP4 Pulp industry sludge +ve 3.682 65.75 ± 0.2

NAW2 Waste water from cardboard manufacturing industry −ve 4.012 76.26 ± 0.26

NAW24 Waste water from cardboard manufacturing industry +ve 4.018 62.90625 ± 0.11



NAW27 Waste water from cardboard manufacturing industry −ve 2.966 51.13793 ± 0.16


NAC1 Waste sludge from cardboard manufacturing industry −ve 4.042 77.63 ± 0.3

NAC24 Waste sludge from cardboard manufacturing industry +ve 4.006 65.75 ± 0.12

NAC10 Waste sludge from cardboard manufacturing industry −ve 3.97 64.80 ± 0.16

NAC9 Waste sludge from cardboard manufacturing industry +ve 3.802 70.92 ± 0.13NAC12 Waste sludge from cardboard manufacturing industry +ve 3.682 68.28 ± 0.04

NAK8 Kraft industry sludge +ve 3.83 71.53571 ± 0.05



Ralstonia eutrophaMTCC no. 1473 MTCC −ve 2.974 41.86 ± 0.1

PHB production90

80

70

60

50

40

30

20

10

0

PHB

(%)

79.27 77.63

41.86

NAP11 NAC1 Ralstonia eutrophaIsolates

Series1

Figure 2: Comparison of PHB production from selected isolateswith Ralstonia eutropha.

Enterococcus sp., and Brevundimonas sp., respectively. Otherresearchers also reported these genuses from the wasteeffluents. Silva et al. [33] studied the ecology of Enterococciand related bacteria in raw and treated waste water from atreatment plant receiving domestic and pretreated industrialeffluents. The predominant species found in the raw wastewater were Enterococcus hirae, Enterococcus faecium, andEnterococcus faecalis. Jiang et al. [34] isolated 3,851 in totalEnterococci isolates from eight individual source categoriesincluding feces fromanimals and birds, soil, and sewagewatersamples to establish antibiotic resistance analysis (ARA).Reddy andMohan [35] also reported the Enterococcus italicussp. inmixed consortia in waste water treatment and produced

PHA up to 71.4%. During their study of influence of substrateload and nutrient concentration (nitrogen and phosphorous)on PHA production using waste water as substrate andmixedculture as biocatalyst, they found that PHA accumulationwas high at higher substrate load (40.3% of dry cell weight(DCW)), low nitrogen (45.1% DCW), and low phosphorous(54.2% DCW) conditions by mixed consortia containg inEnterococcus sp. this paper confirms that Rani et al. [36]reported Brevundimonas with other bacteria as the domi-nant cultured bacteria in microbial diversity in functionalpesticide effluent treatment plants (ETPs). Brevundimonasaveniformis sp.nov. A stalked species, was isolated fromactivated sludge by Ryu et al. [37]. Brevundimonas sp. MIFCand Brevundimonas diminuta was isolated from refineryactive sludge and olive mill waste water, respectively, [38]and a Brevundimonas sp. were isolated from tannery wastetreatment plant [39]. PHA production also reported up to64% from the acid hydrolyzed saw dust (hydrolyzed wood)by Brevundimonas vesicularis. They also optimized the C :Nratio for PHA production in Brevundimonas vesicularis sp.and found that C :N proportion of 100 : 3.5 yieldedmaximumPHA up to 64% of cell dry weight. Thus, they concludedthat acid hydrolyzed saw dust can be used as substrate byBrevundimonas vesicularis sp. for PHA production [40].

3.4. Polymer Analysis by 1H-NMR Spectroscopy. Based on thecharacterization of the PHA produced by NAP11 and NAC1through NMR comparison with the standard PHB (Sigma),it was observed that the PHA obtained from NAP11 andNAC1 is having properties similar to that of the standard PHB(Sigma) (Figure 3(a)), so the PHA produced by both bacteriais polyhydroxybutyrate (PHB). The structures of polyesters


4.180

4.171

4.104

4.040

3.980

3.957

3.919

2.599

2.342

2.228

2.166

2.127

2.061

1.938

1.868

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1.274

1.053

0.868

0.724

7.280

10 9 8 7 6 5 4 3 2 1 0

1.85

1.00

(ppm)

YGL-2

(a)

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1.00

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(ppm)

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7.363

7.279

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2.496

2.463

2.314

1.573

1.438

1.340

1.292

1.267

1.046

0.848

0.079

0.008

CWCP

(b)

10 9 8 7 6 5 4 3 2 1 0

1.00

(ppm)

7.279

1.581

1.442

1.349

1.301

1.276

1.054

0.857

0.087

0.018

CW1

1.72

(c)

Figure 3: 1H NMR spectra of extracted PHB from isolates: (a) PHB standard (PHB Sigma Aldrich), (b) NAP11, and (c) NAC1.

were investigated by1H NMR. The

1H NMR spectra of

the PHAs extracted from Enterococcus sp. NAP11 show thefollowing resonance signals: HC=CH bond at 5.25 ppm,CH2O–COOH bond at 2.580 ppm, a high signal at 1.26 ppm

that belongs to the hydrogen of methylene in the saturatedlateral chain, and a terminal –CH3 group at 0.8 ppm; the1H NMR spectra (Figure 3(b)) of the PHAs extracted fromBrevundimonas sp. NAC1 (Figure 3(c)) show the followingresonance signals: HC=CHbond at 5.30 ppm,CH

2O–COOH

bond at 2.574 ppm, a high signal at 1.30 ppm that belongsto the hydrogen of methylene in the saturated lateral chain,and a terminal –CH3 group at 0.857 ppm [15]. The 1HNMR spectra of the samples and the standard are almostidentical, conferring that extracted intracellular compoundsare polyhydroxybutyrates (PHBs).

3.5. Fourier Transform Infrared Spectroscopy (FTIR). Polymerextracted from NAP11 and NAC1 was used for recording IR

spectra in the range 4000–600 cm−1. IR spectra (Figure 4)showed two intense absorption bands at 1720 and 1281 cm−1of NAP11 and at 1720 and 1273 of NAC1 specific for C=O andC–Ostretching vibrations, respectively.The absorption bandsat 2932 and 2954 cm−1 are due to C–H stretching vibrationsof methyl, methylene groups. These prominent absorptionbands confirm the structure of poly-𝛽-hydroxybutyrate.

3.6. Thermogravimetric Analysis (TGA). TGA results ofNAM5 showed that the 𝑇

𝑚is 171.33∘C and the enthalpy of

PHA fusion is 85.56 J/g.The result showed similarity with thedata obtained from standard PHB (176.29∘C and 86.49 J/g)[41] and from other studies from the literature also [42, 43].

3.7. GC-MS Analysis of Extracted PHA. In this study, thePHB was methanolysed in the presence of sulphuric acidandmethanol, and the methanolysed 3HB was then analyzed


Table 2: Morphological and biochemical characters of selectedisolates.

Morphological characters NAP11 NAC1Colony color White CreamColony texture Smooth Smooth-elevatedGram reaction +ve −veCell shape Cocci-shaped Rod-shapedCell arrangement Chain ChainSpore formation −ve −ve

Biochemical testsCitrate utilization test −ve −veIndole test +ve +veMethyl red test −ve −veV-P test −ve −veH2S production test +ve +veAmylase production test −ve −veSucrose +ve −veDextrose +ve +veMannitol +ve +veTrehalose +ve +veD-Arabinose −ve −veMaltose +ve +veGlucose +ve +veRaffinose +ve +ve with gas productionXylose −ve −veSorbitol −ve −veLactose −ve −veMannose −ve −veFructose +ve −veRibose −ve −veInulin −ve −veEsculin −ve −veMellibiose −ve −veRamanose −ve −ve

by GC-MS. Figures 5(a) and 5(b) showed that a commonmolecular fragment of the 3HB methyl ester ion chro-matogram of the PHB was produced. A predominant peakcorresponding to the dimer 3HB methyl ester was notedat 13.63 to 13.667min, respectively, in GC purified productfrom NAP11 and NAC1, while 3 other major peaks wereobserved at 11.5, 12.2, and 12.3min. The retention times andion fragment patterns of the peaks at 11.6 and 12.2min wereidentical to those of the dimer methyl esters of 3HV and3HBV, respectively, but in very low percentage up to 5% inisolate NAP11 and 20% and 11% in NAC1 isolate, respectively.From the data obtained by GC-MS, the molecular weightofPHB obtained from isolate NAP11 is 256 kDa and from isolateNAC1 is 242 kDa.

4000

3750

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3433

3394

3286

2932

1720

1651

1520

1450

1381

1273

1227

1180

11341095

1049

980

895

825

671

T(%

)

(cm−1)

(a)

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1381

1273

1227

1180

11341095

1049

980

895

825

671

T(%

)

(cm−1)

(b)

Figure 4: FTIR graph of extracted polymer of (a) NAP11 and (b)NAC1 isolates.

4. Conclusion

In this study, inexpensive cardboard industry waste waterwas tried as a carbon source to produce PHA. Differentbacterial strains were isolated from cardboard industry wastewater and pulp, kraft, and cardboardmanufacturing industrysludge and screened for polyhydroxyalkanoate productionusing cardboard manufacturing industry waste water as acarbon source.The bacterial isolates NAP11 and NAC1 can beregarded as potential strains for the conversion of cardboardindustry waste water into PHB. Both of the selected isolatesutilized cardboard industry waste water as sole carbon sourcefor growth and PHB biosynthesis, accumulating PHB upto 79.27% and 77.63% of the cell dry mass, respectively. Asa conclusion, isolates NAP11 and NAC1 can be consideredas good candidates for industrial production of PHB fromcardboard industry waste water. Based on the morphologicaland biochemical characterization, NAP11 and NAC1 wereidentified up to genus level as Enterococcus sp. and Brevundi-monas sp., respectively. Currently, these bacterial strains arefurther investigated to increase the productivity of PHB bythe optimization of the process parameters and making thewhole process more cost-effective.

Conflict of Interests

The authors declare that they have no competing interests.


11.0 12.0 13.0 14.0 14.5

11.610

12.251

12.335

13.668

13.882

(min)

13.000.000

TIC∗1.00

(a)

11.0 12.0 13.0 14.0

(min)15.0

11.588

12.248

12.335

12.446

13.639

13.888

5.000.000

TIC∗1.00

(b)

100

41

43

60 73

85

98 115

129

143

157171 185

199 227

256

OHO

213

20

40

60

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100

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180

200

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220

(c)

Figure 5: GC/MS analysis of poly(3HB) purified from NAP11 and NAC1 isolates. (a) GC of NAP11 showing main peak at 13.667min of 3HBdimer. (b) GC of NAC1 showing main peak at 13.667min of 3HB dimer and 3 other peaks indicating the presence of 3HV in the purifiedpolymer. (c) MS graph showing that the major peak atm/z 73 is of hexadecanoic acid.

Authors’ Contribution

All authors havemade extensive contribution into the design,experiments and data analysis, paper preparation, and thereview and finalization of this paper. All authors read andapproved the final paper.

Acknowledgments

The authors express their sincere gratitude to the Rana card-board industry, Yamuna Nagar, for providing the untreatedwaste water.The authors are very thankful to the Departmentof Chemistry, Kurukshetra University, Kurukshetra, India,for providing the necessary facilities for NMR analysis of thepolymer.

References

[1] D. Byrom, “Polymer synthesis by microorganisms: technologyand economics,” Trends in Biotechnology, vol. 5, no. 9, pp. 246–250, 1987.

[2] S. Y. Lee and H. N. Chang, “Production of poly(3-hydroxybut-yric acid) by recombinant Escherichia coli strains: genetic andfermentation studies,”Canadian Journal ofMicrobiology, vol. 41,no. 1, pp. 207–215, 1995.

[3] B. Hazer and A. Steinbuchel, “Increased diversification of poly-hydroxyalkanoates by modification reactions for industrial andmedical applications,” Applied Microbiology and Biotechnology,vol. 74, no. 1, pp. 1–12, 2007.

[4] X. Gao, J. Chen, Q. Wu, and G. Chen, “Polyhydroxyalkanoatesas a source of chemicals, polymers, and biofuels,” Current Opin-ion in Biotechnology, vol. 22, no. 6, pp. 768–774, 2011.

[5] E. Rudnik, “Definitions, structures and methods of prepara-tion,” in Compostable Polymer Materials, pp. 10–36, Elsevier,Amsterdam, The Netherlands, 1st edition, 2008.

[6] J. Choi and S. Y. Lee, “Process analysis and economic evalua-tion for poly(3-hydroxybutyrate) production by fermentation,”Bioprocess Engineering, vol. 17, no. 6, pp. 335–342, 1997.

[7] I. M. Tamer, M. Moo-Young, and Y. Chisti, “Optimization ofpoly(𝛽-hydroxybutyric acid) recovery from Alcaligenes latus:combined mechanical and chemical treatments,” BioprocessEngineering, vol. 19, no. 6, pp. 459–468, 1998.

[8] E. Grothe, M. Moo-Young, and Y. Chisti, “Fermentation opti-mization for the production of poly(𝛽-hydroxybutyric acid)microbial thermoplastic,” Enzyme and Microbial Technology,vol. 25, no. 1-2, pp. 132–141, 1999.

[9] T. Yamane, “Cultivation engineering of microbial bioplasticsproduction,” FEMS Microbiology Reviews, vol. 103, no. 2–4, pp.257–264, 1992.

[10] T. Yamane, “Yield of poly-D(−)-3-hydroxybutyrate from var-ious carbon sources: a theoretical study,” Biotechnology andBioengineering, vol. 41, no. 1, pp. 165–170, 1993.

[11] P.M.Halami, “Production of polyhydroxyalkanoate from starchby the native isolate Bacillus cereus CFR06,” World Journal ofMicrobiology and Biotechnology, vol. 24, no. 6, pp. 805–812,2008.


[12] L. R. Castilho, D. A. Mitchell, and D. M. G. Freire, “Productionof polyhydroxyalkanoates (PHAs) fromwastematerials and by-products by submerged and solid-state fermentation,” Biore-source Technology, vol. 100, no. 23, pp. 5996–6009, 2009.

[13] M. L. Juan, L. W. Gonzalez, and G. C. Walker, “A novel screen-ing method for isolating exopolysaccharide-deficient mutants,”Applied and Environmental Microbiology, vol. 64, no. 11, pp.4600–4602, 1998.

[14] P. Spiekermann, B. H. A. Rehm, R. Kalscheuer, D. Baumeister,and A. Steinbuchel, “A sensitive, viable-colony staining methodusing Nile red for direct screening of bacteria that accumulatepolyhydroxyalkanoic acids and other lipid storage compounds,”Archives of Microbiology, vol. 171, no. 2, pp. 73–80, 1999.

[15] G. Singh, A. Mittal, A. Kumari, V. Goel, N. K. Aggarwal, and A.Yadav, “Optimization of poly-B-hydroxybutyrate productionfrom Bacillus species,” European Journal of Biological Sciences,vol. 3, no. 4, pp. 112–116, 2011.

[16] J. Law and R. A. Slepecky, “Assay of poly-beta-hydroxybutyricacid,” Journal of Bacteriology, vol. 82, pp. 52–55, 1961.

[17] I. Y. Lee, H. N. Chang, and Y. H. Park, “A simple methodfor recovery of microbial poly-3-hydroxybutyrate by alkalinesolution treatment,” Journal of Microbiology and Biotechnology,vol. 5, no. 4, pp. 238–240, 1995.

[18] G. Du, J. Chen, J. Yu, and S. Lun, “Continuous production ofpoly-3-hydroxybutyrate by Ralstonia eutropha in a two-stageculture system,” Journal of Biotechnology, vol. 88, no. 1, pp. 59–65, 2001.

[19] M. R. Zakaria, H. J. Ariffin, N. A. M. Aziz, S. A. Nishida, H.Y. Shirai, and M. A. Hassan, “Biosynthesis and characterizationof poly(3-hydroxybutyrate-co-3- hydroxyvalerate) copolymerfrom wild-type Comamonas sp. EB172,” Polymer Degradationand Stability, vol. 95, no. 8, pp. 1382–1386, 2010.

[20] A. Steinbuchel, “Perspectives for biotechnological productionand utilization of biopolymers: metabolic engineering of poly-hydroxyalkanoats biosynethesis pathways as a successful exam-ple,”Macromolecular Bioscience, vol. 1, pp. 1–24, 2001.

[21] M. Zinn, B. Witholt, and T. Egli, “Occurrence, synthesis andmedical application of bacterial polyhydroxyalkanoate,”Advanced Drug Delivery Reviews, vol. 53, no. 1, pp. 5–21, 2001.

[22] J. Teeka, T. Imai, X. Cheng et al., “Screening of PHA producingbacteria using biodiesel-derived waste glycerol as a sole carbonsource,” Journal of Water and Environment Technology, vol. 8,pp. 371–381, 2010.

[23] H. Ramachandran and A. A. Abdullah, “Isolation of PHA-producing bacteria from Malaysian environment,” in Proceed-ings of the 7th IMT-GT UNINET and the 3rd International PSU-UNS Conferences on Bioscience, pp. 178–179, 2010.

[24] S. Kitamura and Y. Doi, “Staining method of poly(3-hydro-xyalkanoic acids) producing bacteria by nile blue,” Biotechnol-ogy Techniques, vol. 8, no. 5, pp. 345–350, 1994.

[25] K. Cho, H. W. Ryu, C. Park, and P. R. Goodrich, “Poly(hydro-xybutyrate-co-hydroxyvalerate) from swine waste liquor byAzotobacter vinelandiiUWD,” Biotechnology Letters, vol. 19, no.1, pp. 7–10, 1997.

[26] J. Yu, “Production of PHA from starchy wastewater via organicacids,” Journal of Biotechnology, vol. 86, no. 2, pp. 105–112, 2001.

[27] H. Salehizadeh and M. C. M. van Loosdrecht, “Productionof polyhydroxyalkanoates by mixed culture: recent trends andbiotechnological importance,” Biotechnology Advances, vol. 22,no. 3, pp. 261–279, 2004.

[28] J. M. L. Dias, P. C. Lemos, L. S. Serafim et al., “Recent advancesin polyhydroxyalkanoate production bymixed aerobic cultures:from the substrate to the final product,” Macromolecular Bio-science, vol. 6, no. 11, pp. 885–906, 2006.

[29] N. Ceyhan and G. Ozdemir, “Poly-𝛽-hydroxybutyrate (PHB)production from domestic wastewater using Enterobacter aero-genes 12Bi strain,” African Journal of Microbiology Research, vol.5, no. 6, pp. 690–702, 2011.

[30] K. J. Ganzeveld, A. Van Hagen, M. H. Van Agteren, W. DeKoning, and A. J. M. S. Uiterkamp, “Upgrading of organicwaste: production of the copolymer poly-3-hydroxybutyrate-co-valerate by Ralstonia eutrophus withorganic waste as solecarbon source,” Journal of Cleaner Production, vol. 7, no. 6, pp.413–420, 1999.

[31] M.N. Rao andA.K.Datta,WasteWater Treatment, pp. 203–208,Oxford and IBH Publishing, New Delhi, India, 2007.

[32] G. H. John, N. R. Krieg, S. Peter, H. A. Staley, T. W. Satnley,and T. James, Bergey’s Manual of Determinative Bacteriology,Williams andWilkins, Philadelphia, Pa, USA, 9th edition, 2009.

[33] M. F. da Silva, I. Tiago, A. Verıssimo, R. A. Boaventura, O. C.Nunes, and C. M. Manaia, “Antibiotic resistance of Enterococciand related bacteria in an urban wastewater treatment plant,”FEMS Microbiology Ecology, vol. 55, no. 2, pp. 322–329, 2005.

[34] S. C. Jiang,W.Chu, B.H.Olson et al., “Microbial source trackingin a small southern California urban watershed indicates wildanimals and growth as the source of fecal bacteria,” AppliedMicrobiology andBiotechnology, vol. 76, no. 4, pp. 927–934, 2007.

[35] M. V. Reddy and S. V. Mohan, “Effect of substrate load andnutrients concentration on the polyhydroxyalkanoates (PHA)production using mixed consortia through wastewater treat-ment,” Bioresource Technology, vol. 114, pp. 573–582, 2011.

[36] A. Rani, S. Porwal, R. Sharma, A. Kapley, H. J. Purohit, andV. C. Kalia, “Assessment of microbial diversity in effluenttreatment plants by culture dependent and culture independentapproaches,” Bioresource Technology, vol. 99, no. 15, pp. 7098–7107, 2008.

[37] S. H. Ryu, M. Park, J. R. Lee, P. Yun, and C. O. Jeon, “Brevu-ndimonas aveniformis sp. nov., a stalked species isolated fromactivated sludge,” International Journal of Systematic and Evolu-tionary Microbiology, vol. 57, no. 7, pp. 1561–1565, 2007.

[38] C. Ruggeri, A. Franzetti, G. Bestetti et al., “Isolation and char-acterisation of surface active compound-producing bacteriafrom hydrocarbon-contaminated environments,” InternationalBiodeterioration and Biodegradation, vol. 63, no. 7, pp. 936–942,2009.

[39] G. Tsiamis, G. Tzagkaraki, A. Chamalaki et al., “Olive-millwastewater bacterial communities display a cultivar specificprofile,” Current Microbiology, vol. 64, no. 2, pp. 197–203, 2012.

[40] J. A. Silva, L. M. Tobella, J. Becerra, F. Godoy, and M. A. Mart-ınez, “Biosynthesis of poly-𝛽-hydroxyalkanoate by Brevundi-monas vesicularis LMG P-23615 and Sphingopyxis macro-goltabida LMG 17324 using acid-hydrolyzed sawdust as carbonsource,” Journal of Bioscience and Bioengineering, vol. 103, no. 6,pp. 542–546, 2007.

[41] V. Tanamool, T. Imai, P. Danvirutai, and P. Kaewkannetra,“Biosynthesis of polyhydroxyalkanoate (PHA) by Hydro-genophaga sp. Isolated from soil environment during batchfermentation,” Journal of Life Sciences, vol. 5, no. 12, pp. 1003–1012, 2011.

[42] A. Yezza, A. Halasz,W. Levadoux, and J. Hawari, “Production ofpoly-𝛽-hydroxybutyrate (PHB) by Alcaligenes latus frommaple


sap,” Applied Microbiology and Biotechnology, vol. 77, no. 2, pp.269–274, 2007.

[43] W. M. Pachekoski, J. A. M. Agnelli, and L. P. Belem, “Thermal,mechanical and morphological properties of poly (hydroxybu-tyrate) and polypropylene blends after processing,” MaterialsResearch, vol. 12, no. 2, pp. 159–164, 2009.

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Bhuwal et al. Bioresources and Bioprocessing 2014, 1:9http://www.bioresourcesbioprocessing.com/content/1/1/9

RESEARCH Open Access

Poly-β-hydroxybutyrate production andmanagement of cardboard industry effluent bynew Bacillus sp. NA10Anish Kumari Bhuwal1, Gulab Singh1, Neeraj Kumar Aggarwal1*, Varsha Goyal1 and Anita Yadav2

Abstract

Background: In the present study, we aim to utilize the ecological diversity of soil for the isolation and screeningfor poly β-hydroxybutyrate (PHB)-accumulating bacteria and production of cost-effective bioplastic using cardboardindustry effluent.

Results: A total of 120 isolates were isolated from different soil samples and a total of 62 isolates showed positiveresults with Nile blue A staining, a specific dye for PHB granules and 27 isolates produced PHB using cardboardindustry effluent. The selected isolate NA10 was identified as Bacillus sp. NA10 by studying its morphological,biochemical, and molecular characteristics. The growth pattern for the microorganism was studied by logisticmodel and exactly fitted in the model. A maximum cell dry weight (CDW) of 7.8 g l−1 with a PHB concentration of5.202 g l−1 was obtained when batch cultivation was conducted at 37°C for 72 h, and the PHB content was up to66.6% and productivity was 0.072 g l−1 h−1 in 2.0 L fermentor. Chemical characterization of the extracted PHB wasdone by H1NMR, Fourier transform infrared spectroscopy (FTIR), thermal gravimetric analysis (TGA), Gaschromatography–mass spectrometry (GC-MS) analysis to determine the structure, melting point, and molecularmass of the purified PHB. The polymer sheet of extracted polymer was prepared by blending the polymer withstarch for packaging applications.

Conclusions: The isolate NA10 can be a good candidate for industrial production of PHB from cardboard industrywaste water cost-effectively and ecofriendly.

Keywords: Polyhydroxybutyrate (PHB); Bioplastic; Cardboard industry waste water; Bacillus sp.

BackgroundToday, plastics have become a necessary part of contem-porary life due to their durability and resistance to degrad-ation. Worldwide production of petroleum-based syntheticpolymer was approximately 270.0 million tons in 2007 [1],and these synthetic polymers are found to be recalcitrantto microbial degradation [2]. Problems related to solid wastemanagement of these petrochemical-derived plastics posea serious threat to global environment. Therefore, currentconcern about the environmental fate of polymeric materialshave created much interest in the development of biodegrad-able plastic (bioplastic), such as starch derivatives, polylacticacid, cellulosic polymers and polyhydroxyalkanoates, which

* Correspondence: [email protected] of Microbiology, Kurukshetra University, Kurukshetra, Haryana136119, IndiaFull list of author information is available at the end of the article

© 2014 Bhuwal et al.; licensee Springer. This isAttribution License (http://creativecommons.orin any medium, provided the original work is p

plays an important role. In addition to being biodegradable,they have further advantage of being produced from re-newable resources [3]. Among the various biodegradablepolymer materials, polyhydroxyalkanoates (PHAs) providea good fully degradable alternative to petrochemical plas-tics [4,5]. Polyhydroxybutyrate (PHB) was the first PHA tobe discovered and is also the most widely studied and bestcharacterized PHA. It is accumulated as a membraneenclosed inclusion in many bacteria at up to 80% of thedry cell weight and nearly 90% in recombinant E. coli [6].In addition to the easy biodegradability and biocompatibil-ity, it has mechanical properties that are very similar toconventional plastics like polypropylene or polyethyleneand have many domestic and commercial applications suchas food packing films, biodegradable carriers for medicinesand insecticides, disposable cosmetic products, absorbablesurgical devices and being immunologically compatible

an Open Access article distributed under the terms of the Creative Commonsg/licenses/by/4.0), which permits unrestricted use, distribution, and reproductionroperly cited.

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Bhuwal et al. Bioresources and Bioprocessing 2014, 1:9 Page 2 of 11http://www.bioresourcesbioprocessing.com/content/1/1/9

with human tissue can form microspheres and microcap-sules [7]. Recently, PHA has been found useful as a newtype of biofuel [8]. Besides all these properties and appli-cations, wider use of PHAs is prevented mainly due totheir high production cost compared with the oil-derivedplastics [9]. High production cost of PHB production ismainly devoted to the expensive carbon substrates and te-dious production procedures [10]. Due to the large impactof the high price of carbon sources on production costs,one of the most important approaches to reduce costs isto use wastes and by-products as raw material for thefermentation process. Novel technologies have been devel-oped to produce PHAs from organic matters in wastewa-ter [12-14], industrial wastes [15,16], municipal waste [17],food wastes [18], and activated sludge of paper and pulpmills [19]. Hence, replacement of non-biodegradable withbiodegradable plastic from organic waste can provide mul-tiple benefits to the environment and contribute to sus-tainable development [20]. Therefore, organic waste fromcardboard industry waste water could be a good approachfor cost effective production PHA.

MethodsIsolation of PHA-producing bacteriaFor the isolation of PHA-producing bacteria, various soilsamples were collected from different ecological niches.The samples were stored at room temperature until ana-lysis. In 99 mL sterilized water, 1 g of soil sample wasmixed. Then, the sample was serially diluted in sterile dis-tilled water and followed by plating on the carbon-richnutrient agar medium (beef extract 0.3%, peptone 0.5%,sodium chloride 0.5%, glucose 1%, and agar 2%). For therapid detection and isolation of PHB-producing bacteria,0.02% alcoholic solution of Sudan black B was applied tostain bacterial colonies and the plates were kept undis-turbed for 30 min. The excess dye was then decanted, andplates were rinsed gently by adding 100% ethanol. Col-onies unable to incorporate the Sudan black B appearedwhite, while PHB producers appeared bluish black [21].

Rapid screening for PHA-producing bacteriaThe Sudan black B-positive isolates were further screenedby Nile blue A, a more specific stain for PHA by a morerapid and sensitive, viable colony method [22]. This dyeat concentrations of only 0.5 μg/mL was directly includedin carbon-rich nutrient agar medium, and growth of thecells occurred in the presence of the dye. The PHA-accumulating colonies, after Nile blue A staining, showedbright orange fluorescence on irradiation with UV lightand their fluorescence intensity increased with the increasein PHA content of the bacterial cells. The isolates whichshowed bright orange fluorescence on irradiation with UVlight after Nile blue A staining were selected as PHA accu-mulators. The selected PHA accumulators after Nile blue

A staining were checked for growth and PHA productionin both nutrient broth (beef extract 0.3%, peptone 0.5%,sodium chloride 0.5%) and cardboard industry effluent.

Pretreatment of cardboard industry waste waterUntreated cardboard industry effluent was collected fromthe cardboard industry, Yamunanagar (Haryana), India,and stored at 4°C until used for analysis. The effluent wasfirst filtered through the muslin cloth and then throughrough filter paper to remove the undesired suspendedsolid materials from waste water. After this pretreatmentstep, cardboard industry waste water was used as quan-tification and production medium for PHA productionby selected bacterial isolate.

Morphological and biochemical characterization-selectedbacteriaMicroscope Stereo Olympus (America) was used to ob-serve the morphology of bacterial colonies grown on nutri-ent agar. The growth characteristics such as structure,shape, color, margin, surface characteristics, elevation, cell'sarrangement, and Gram staining of the bacterial colonieswere observed to characterize the bacterial colonies. Vari-ous biochemical tests were performed in selected PHB-producing bacteria, namely, indole production test, methylred and Voges-Proskauer, citrate utilization test, and H2Sproduction for their biochemical characterization. Thefermentative utilization of various carbohydrates (xylose,mannose, maltose, sucrose, raffinose, dextrose, trehalose,fructose, glucose, ribose, lactose, rhamnose, esculin, inulin,mannitol, arabinose, sorbitol, and melibiose) were alsofollowed for 48 h at 37°C by inoculating the selected iso-late separately in the defined medium to which varioussugars were added.

Molecular identificationColony PCR (16S rRNA gene amplification)Colony polymerase chain reaction (Colony PCR) of theisolate was performed according to Gen Elute™ Bacterialgenomic DNA kit (Sigma, St. Louis, MO, USA), and thecolonies (approximately 1 mm in diameter) were pickedup with a sterilized toothpick and directly transferred tothe PCR tubes as DNA templates. The PCR amplifica-tion of 16S rRNA was done at 94°C for 4 min, 94°C for20 s, 52°C for 30 s, 72°C for 2 min, and 72°C for 7 minwith hold at 4°C. The universal primer 16sF-5′ AGAGTT TGA TCC TGG CTC AGA 3′ and 16sR-5′ ACGGCT ACC TTG TTA CGA CTT 3′ were used.

Detection of PCR productsPCR-amplified DNA fragments were observed by agarosegel electrophoresis in 1% ± agarose gels (FMC). Ten mi-croliters of each amplified mixture and the molecularmass marker were subjected to agarose gel electrophoresis


and ethidium bromide staining and tracked by 0.25% ofbromophenol blue. The ampli®ed DNA fragments werevisualized by gel documentation box (Genie, Redmond,WA, USA).

Sequencing and analysis of 16S rRNA geneAfter that, the PCR product was purified by GenElute™gel extraction kit (Sigma) method and sequencing ofPCR product was done by Sanger sequencing method at96°C for 1 min, at 96°C for 15 s, at 52°C for 30 s, and at60°C for 4 min for 25 cycles with a hold at 4°C. The ABIPrism Big Dye Terminator Cycle Sequencing ReadyReaction Kit (Applied Biosystems, Foster City, CA, USA)was used for the sequencing of the PCR product. A com-bination of universal primers was chosen to sequence thegene sequence. Samples were run on an ABI Prism3130×1 Genetic Analyzer (Applied Biosystems). The chro-matogram was made by Chromas 2.4. software. The ob-tained sequence was subjected to search for closest possiblespecies by BLAST tools and distance matrix tree toolavailable at National Centre for Biotechnology Information(NCBI). Phylogenetic tree was constructed by MEGA5.2phylogenetic tree analysis software.

Optimization of various growth parameters for PHBproduction in shake flask cultureThe optimization for maximum PHA production by se-lected isolate was carried in 250-ml Erlenmeyer flask usingpreprocessed cardboard industry waste water as produc-tion medium at 100 rpm. Several cultural parameters wereevaluated to determine their effect on biomass and PHBproduction using cardboard industry waste water. The op-timized value for each parameter was selected and keptconstant for further experiments. Several cultural parame-ters like production media concentration (25%–100%)time of incubation (24–96 h), temperature of incubation(25°C–55°C), and effect of pH (5.5–8) were evaluated todetermine their effect on biomass accumulation and PHAproduction.

Scale up of poly-β-hydroxybutyrate production andgrowth kineticsThe production of PHAs was carried out in a 2.0-L fer-menter (Minifors CH 4103, Switzerland) with a workingvolume of 1.25 L using the optimized parameters and pro-duction medium. Initially, the production medium (pre-processed undiluted cardboard industry waste water) wasadded into the fermenter, the pH was adjusted to 7.0, andthe medium was sterilized in situ. Dissolved oxygen wasmaintained at 80%–100% air saturation; at the start ofthe process, 1% (v/v) of overnight culture (at log phase)was inoculated aseptically and the impeller speed wasmaintained at 100 rpm and temperature at 37°C. ThepH was maintained at 7.0 using 1 N NaOH and 1 N

HCL. Antifoam (silicone oil) at a concentration of 1:10(v/v) in water was added after 36th and 54th hour.The growth curves (DCW vs. time) were prepared to

determine the start and end point of exponential phasefor NA10 in batch fermentation. Various other growthparameters such as maximum specific growth rate (μ)and doubling time (Td) were determined according to themethod provided by Painter and Marr [24] and Levasseuret al. [25].

μ K0 ¼ In wt2=wt1ð Þ= t2 > t1; t2t1

where wt2 and wt1 are the dry cell weight at the differenttime points (t1 and t2), respectively.

Td ¼ In2=μm

where Td is the doubling time and μm is the maximumspecific growth rate.Cell dry weight (CDW) and PHB yield coefficient relative

to cell dry weight (Yp/x, g/g, defined as gram PHBproduced per gram dry cell mass produced) [26], PHBconcentration (g/L, defined as g PHB measured in 1 Lculture), PHB content [g/g, defined as the ratio of PHBconcentration (g/L) to dry cell concentration (g/L)], andPHB productivity (g/L/h) [10,11] were measured and cal-culated per definition during the fermentation process.All experiments were performed in triplicate to check

the reproducibility. The results were analyzed statisticallyby determining standard deviations values and performinganalysis of variance (ANOVA) test.

Extraction and quantitative analysis of PHBFor extraction and quantitative analysis of PHB, culturebroth was centrifuged at 8,000 rpm for 15 min after 72 hof incubation at 37°C. The pellet dissolved in 10 mL so-dium hypochlorite was incubated at 50°C for 1 h for ly-ses of cells. The cell extract obtained was centrifuged at12,000 rpm for 30 min and then washed sequentiallywith distilled water, acetone, and absolute ethanol. Afterwashing, the pellet was dissolved in 10 mL chloroform(AR grade) and incubated overnight at 50°C [23]. Afterevaporation at 50°C, 10 mL of sulfuric acid was added toit and placed in water bath for 10 min at 100°C. Thisconverts PHB into crotonic acid, which gives absorbancemaximum at 235 nm [24,25]. PHB (Sigma Aldrich) wasused as standard for making standard curve.

Polymer analysis1H-NMR spectroscopy and FTIR analysisThe identity of individual monomer unit was confirmedby proton nuclear magnetic resonance (1H-NMR) spec-troscopy. 1H-NMR spectra were acquired by dissolvingthe polymer in deuterated chloroform (CDCl3) at a con-centration of 10 mg/ml and analyzed on a Bruker Avance


II 500 spectrometer (Madison, WI, USA) at 22°C with7.4 ms pulse width (30° pulse angle), 1 s pulse repetition,10,330 Hz spectral width, and 65,536 data points. Tetra-methylsilane was used as an internal shift standard. FTIRanalysis of the polymer sample was carried out on MB-3000, ABB Fourier transform infrared (FTIR) spectropho-tometer in the range 4,000 to 600 cm−1.

TGAThermal gravimetric analysis (TGA) was performed usinga TGA instrument (Perkin Elmer, Diamond TG/DTAanalyzer, USA) calibrated with indium. The temperaturewas ramped at a heating rate of 10°C/min in nitrogen-ous environment to a temperature (700°C) well abovethe degradation temperature of the polymers.

GC-MS analysisFor molecular analysis of purified polymer, a coupledGas chromatography–mass spectrometry (GC-MS) wasperformed using a GC-MS-QP 2010 Plus model, withcapillary Column-Rtx-5 MS (30 m × 0.25 mm i.d. × 0.25μm film thickness). The samples were injected (3 μL) inthe splitless mode, and the injection temperature was260°C and column oven temperature was 100°C. The massspectra obtained were compared with the Nist-08 andWilley-08 mass spectral library.

Results and discussionIsolation and selection of PHA-producing bacteriaA wide variety of bacteria are known to accumulate PHAgranules intracellularly as an energy reserve material.Microbial species from over 90 genera have been reportedto accumulate approximately 150 different hydroxyalka-noic acids as polyhydroxyalkanoate polyesters granules[27,28]. These bacteria have been reported from variousenvironments. For the rapid detection and isolation ofPHB-producing bacteria, 0.02% alcoholic solution ofSudan black B and Nile blue A, viable colony method [20]was used. The isolation of PHA-producing bacteria wasdone from various ecological niches. A total of 120 isolatesshowed black-blue coloration when stained with Sudanblack B, a preliminary screening agent for lipophilic com-pounds, and a total of 62 isolates showed positive resultwith Nile blue A staining, a specific dye for the presenceof PHA granules. Teeka et al. [29] used this method toscreen the potential PHA-producing bacteria from soil,and Ramachandra and Abdullah [30] also observed thecolonies formed on nutrient-rich medium under ultravio-let light (UV) to screen for the pink fluorescence which in-dicated the presence of PHA producers. Kitamura andDoi [31] first demonstrated this viable colony method onagar plates; they induced the isolates to accumulate PHAby culturing in E2 medium, containing 2% (w/v) glucose be-fore Nile blue A staining. The PHA-accumulating colonies,

after Nile blue A staining, showed bright orange fluores-cence on irradiation with UV light and their fluorescenceintensity increased with increase in PHA content of thebacterial cells.

Production of PHB in nutrient broth and cardboardindustry waste waterThe PHA-positive isolates selected after Nile blue A stain-ing were screened in nutrient broth and best 27 producerswere grown in processed cardboard industry waste waterin 250-mL Erlenmeyer flasks and were employed to ex-tract PHB after 72 h of incubation under stationary condi-tions of growth. The PHB from the isolates was extractedby the hypochlorite and chloroform method [21] as de-scribed earlier. The isolate NA10 showed maximum PHBproduction in both nutrient broth and cardboard industrywaste water (3.951 g/L) (Table 1) and were selected forfurther optimization of PHB production.Organic matter from waste and wastewater have high

biological oxygen demand (BOD) and carbon oxygen de-mand (COD) values and, hence, microorganisms cangrow, utilizing the nutrient present in waste water and canconvert them into valuable compounds and polymers.Based on this idea, many researchers reported the PHAproduction from various industrial waste materials. Manyresearchers have proposed coupling PHA production tobiological wastewater treatment [32-34]. Rebah et al. [14]utilized two types of industrial wastewater (starch andslaughterhouse wastewater from a plant located aroundQuebec region) and a secondary sludge (Quebec munici-pal waste water treatment plant) for PHB production bycultivating fast-growing Rhizobia but the PHB productionwas very low up to 10%. In an another study, Mockoset al. [35] reported that selectively enriched pulp millwaste activated sludge can serve as an inoculum for PHAproduction from methanol-rich pulp mill effluents, andthe enriched cultures accumulated nearly 14% PHA on adry weight basis under nitrogen-limited conditions.Cardboard industry waste water is typically rich in car-

bohydrates but poor in fixed nitrogen due to the high C/Nratio. This high carbon-nitrogen ratio favors the growth ofPHA-producing bacteria. This waste have high BOD andCOD values 680–1,250 mg/L and 3,400–5,780 mg/L andCOD/BOD ratio between 3.9–5 [36], which is suitable formicrobial growth.

Biochemical and molecular characterization of selectedisolateBy using Bergey's manual of determinative bacteriology[37] and by ABIS online, advanced bacterial identificationsoftware, the selected isolate was classified up to genuslevel using the morphological and biochemical character-istics (Tables 2 and 3) and was identified as Bacillus sp.For further characterization, almost complete 16S rRNA

Table 1 List of best PHA accumulating bacteria screened in nutrient broth and cardboard industry effluent

Isolate Nutrient broth Cardboard industry effluent

PHB (g/L) Cell dry weight (g/L) PHB (g/L) Cell dry weight (g/L)

NA2 2.382 ± 0.012 6.2 ± 0.23 1.73 ± 0.021 4.4 ± 0.12

NA10 3.512 ± 0.011 7.8 ± 0.17 3.951 ± 0.024 8.6 ± 0.13

NA11 3.504 ± 0.019 6.8 ± 0.13 2.604 ± 0.033 8.8 ± 0.16

NA12 4.15 ± 0.014 8.7 ± 0.28 3.387 ± 0.027 9.2 ± 0.23

NA38 2.634 ± 0.017 5.8 ± 0.21 3.128 ± 0.041 7.3 ± 0.17

NA46 2.574 ± 0.021 8.4 ± 0.13 2.822 ± 0.02 8.2 ± 0.33

NA49 1.034 ± 0.033 4.2 ± 0.19 1.87 ± 0.032 4.8 ± 0.13

NA52 3.342 ± 0.014 6.8 ± 0.21 2.373 ± 0.036 4.2 ± 0.18

NA61 1.42 ± 0.031 5 ± 0.23 2.132 ± 0.022 5 ± 0.21

NA67 1.334 ± 0.033 4.4 ± 0.11 2.121 ± 0.017 5 ± 0.31

NA71 2.628 ± 0.016 4.8 ± 0.27 1.732 ± 0.015 3.6 ± 0.42

NA97 3.154 ± 0.009 7.6 ± 0.33 3.132 ± 0.028 10.2 ± 0.28

NA99 1.214 ± 0.018 4.4 ± 0.21 1.73 ± 0.024 4.8 ± 0.26

AN23 2.152 ± 0.032 5.4 ± 0.17 2.143 ± 0.033 8.6 ± 0.22

AN8 4.13 ± 0.033 6.6 ± 0.12 3.658 ± 0.035 7.5 ± 0.33

Growth and production of PHA in nutrient broth and preprocessed cardboard industry waste water after 72 h of incubation at 37°C.

Table 3 Biochemical characteristics and carbohydratetests of selected isolate NA10

Biochemical/carbohydrate test Results Carbohydrate test Results

Citrate utilization test − Glucose +

Indole test + Raffinose +

Methyl red test − Xylose −


gene sequences were determined. The obtained sequenceswere aligned and compared with the bacterial sequencesavailable in the GenBank database. The phylogenetic ana-lysis (Figure 1) was done using MEGA 5.2 software byneighbor-joining tree and distance matrix-based nucleo-tide sequence homology which revealed that isolate NA10is Bacillus sp. Yilmaz et al. [38] isolated 29 strains of thegenus Bacillus from different soil samples which weretaken from grasslands of Ankara, Turkey, and the highestPHB production and productivity percentage was foundin Bacillus brevis M6 (41.67%w/v).

Growth pattern and PHB production in cardboardindustry waste waterAs such, there is no report available from literature forPHB accumulation by microbial cells using cardboardindustry waste water. The growth of Bacillus NA10 wasrecorded in different concentration of cardboard indus-try waste water (Figure 2), and the maximum growthwas noted in undiluted (100% v/v) cardboard industry

Table 2 Morphological characteristics of selected isolateNA10

Morphological characters Results

Colony color White

Colony texture Smooth

Gram reaction +

Cell shape Rod shaped

Cell arrangement Isolated colonies

Spore formation +

waste water after 72 h of incubation at 37°C. Dilution ofwaste water not only affected the microbial growth butalso the PHB accumulation. This may be due to the factthat with increasing dilution of waste water, the carbonand other nutrient got diluted and was not able to sup-port microbial growth [38]. By submerged fermentation,Bacillus sp.NA10 accumulated highest PHB (3.952 g/L)from undiluted processed cardboard industry waste waterin unoptimized cultural conditions. Quagliano et al. [39]studied the effect of molasses concentration on PHB byincreasing the molasses concentration from 1% to 5% (w/v),and a maximum of 54% PHA was obtained with 5% mo-lasses concentration with highest Mw 640 kDa. Pozo et al.

V-P Test − Sorbitol −

H2S production test + Lactose −

Amylase production test − Mannose −

Sucrose + Fructose +

Dextrose + Ribose −

Mannitol + Inulin −

Trehalose + Esculin -

D-Arabinose − Mellibiose −

Maltose + Rammanose −

Figure 1 Neighbor-joining phylogenic tree made by MEGA 5.2 software of isolate NA10.


[40] found that NH4 medium amended with 60% alpechinshowed the maximum PHA production and concludedthat alpechin was tolerated by A. chroococcum strain H23at high concentrations and that it acts as a substrate tosupport the growth of the microorganisms.

Optimization of growth parametersPHB production versus incubation timeThe PHB production in cardboard industry waste waterwas followed for 120 h by submerged fermentation in250 mL Erlenmeyer flask at an interval of 24 h. The pro-duction of PHB increased up to 72 h (4.456 g/L) and,thereafter, got reduced (3.26 g/L after 72 h) (Figure 3a).This reduction in PHB production after 72 h may bedue to lack of micronutrients as well as increase in me-tabolites that might have negative effect on the PHB pro-duction. The observation was supported by Yüksekdağet al. [41] Alcaligenes eutrophus [42] and A. vinelandii [43].

4.8

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Figure 2 Growth pattern and PHB production in cardboardindustry waste water.

Klüttermann et al. [44] also reported that the highest PHBlevel in Agrobacterium radiobacter was achieved after 96 h.After this time, the PHB contents in fermentation brothdecreased which might indicate that the bacteria used PHBas a nutrient source due to inadequate nitrogen and carbonsources in the medium.

Effect of incubation temperature on PHB productionThe maximum PHB production of 4.274 g/L was recordedat 35°C after 72 h. The increase of temperature beyond35°C has negative impact on PHB production (Figure 3b).This decrease in PHB production at high temperatureis also supported by studies of Grothe et al. [10] andYüksekdağ et al. [41]. The decrease in PHB productionat high temperature could be due to low PHB polymeraseenzyme activity [45]. Tamdoğan and Sidal [46] also re-ported that higher and lower temperatures than 30 C leadto decrease in cell biomass and PHB synthesis by Bacillussubtilis ATCC6633.This result coincides with that repre-sented by Aslim et al. [47], Hamieh et al. [48], who re-ported that optimum incubation temperature for PHBproduction by Bacillus subtilis , Bacillus pumilis andBacillus thuringiensis was 37 C.

Effect of pH of the production medium on PHB productionTypically, metabolic process was highly susceptible toeven slight changes in pH. Therefore, proper control ofpH was critical. The experimental results showed thathighest PHB content (4.492 g L−1) was obtained at pH 7.5by isolate NA10 (Figure 3c). The current observation wasin agreement with Sindhu et al. [49] who observed thatthe maximum PHB was produced (0.01 to 0.5 g/L) at

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Figure 3 Optimization of various cultural and growth parameters for Bacillus sp. NA10. (a) time (b) temperature (c) pH for Bacillus sp. NA10.


pH 7–7.5 by Bacillus sphaericus NII 0838 from crude gly-cerol. Flora et al. [50] revealed that the maximum PHBproduction (25%) by B. sphaericus was at pH range from6.5–7.5, and the reduction of polymer accumulation athigher pH values is due to the effect on the degradativeenzymes of polymer breakdown, so that PHB is utilized ata rate almost equal to the rate of its synthesis. Shivakumar[51] also reported that pH 6.8 to 8.0 was optimum forPHB production by A. eutrophus.

Scale up study and growth kineticsGrowth kinetics of the culture was studied in 2.0 L labscale bioreactor. The profiles of PHB accumulation wereanalyzed in batch cultivation by growing on the cardboardindustry waste water under optimized growth conditionsobtained at flask level (Figure 4). The growth pattern forthe microorganism was studied by logistic model andexactly fitted as presented in Figure 5. The bacteria NA10was found to accumulate cell dry weight and specific

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Figure 4 Cell dry weight, specific product yield (Yp/x), and PHB production by Bacillus sp. NA10 in 2.0 L fermenter.


product yield after the initial logarithmic growth (24 h)and reached a maximum 7.8 g/l at a rate 0.028 h−1 in sta-tionary growth phase at 72 h. Similarly specific productyield (Yp/x) increased by about 50% from initial exponen-tial growth to stationary phase, suggesting that cells accu-mulated PHB while growing and aging; hence, anystrategy that prolongs the stationary phase is likely to fur-ther enhance the PHB yield per unit biomass. A maximumCDW of 7.8 g l−1 with a PHB concentration of 5.202 g l−1

was obtained when batch cultivation was conducted at 37°C after 72 h, and the PHB content was up to 66% andproductivity was 0.072 g l−1 h−1. Tanamool et al. [52] re-ported that maximum 0.920 g/l of cell dry mass and0.034 g/l PHB using sweet sorghum juice by Ralstoniaeutropha with yield and productivity of 0.037 g PHB/g drycell and 0.0019 g/(l h), respectively. Similarly, El-Sayedet al. [53] found specific growth rate of 0.055 h−1on su-crose media while a specific growth rate of 0.053 h−1 wasobtained with R. eutropha on glucose medium.

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Figure 5 Logarithmic growth curve of Bacillus sp. NA10 incardboard industry effluent.

Polymer analysis1H-NMR spectroscopyThe structures of polyesters were investigated by 1HNMR (Figure 6a). The 1H NMR spectra of the PHAsextracted from Bacillus sp. NA10 shows a doublet at1.26 ppm which is attributed to the methyl group coupledto one proton, a doublet of quadruplet at 2.580 ppmwhich is attributed to a methylene group adjacent to anasymmetric carbon atom bearing a single proton, and amultiplet at 5.25 ppm characteristic of the methyne group.Two other signals are also observed, the broad one at1.04 ppm which is due to water, and another one at7.27 ppm which is attributed to chloroform. From theseresults, it can be concluded that Bacillus sp. NA10 cellsgrown with cardboard industry effluent as the carbonsource produce PHA exclusively in the form of PHB.

FTIRPolymer extracted from NA10 was used for recordingIR spectra in the range 4,000 to 600 cm−1. IR spectra(Figure 6b) showed two intense absorption band at1,720 and 1,273 cm−1 which are specific for C = O andC–O stretching vibrations, respectively. The absorptionbands at 2,932 and 2,962 cm−1 are due to C-H stretchingvibrations of methyl and methylene groups. These prom-inent absorption bands confirm the structure of poly-β-hydroxybutyrate.

TGATGA curves were obtained in the temperature range of30°C to 700°C for PHB. TGA results of Bacillus sp. NA10showed that the Tm is 182.34°C, and the enthalpy of PHAfusion is 83.62 J/g. The result showed similarity with thedata obtained from standard PHB (174.29°C and 86.49 J/g)and from other works [54,55]. The PHA gave a rapidthermal degradation between 250°C and 290°C while

Figure 6 (a) NMR spectra (b) FTIR spectra (c, d) GC-MS of extractedpolymer.


the standard PHB represented at between 250°C and295°C. It is indicated that biopolymeric material obtainedcan possibly be further used in a large-scale processing ofbioplastic [56,57].

GC-MS analysis of extracted PHBGC-MS analysis helps in elucidating the structure ofcomponents. The key compounds of concern were identi-fied based on their retention peak. PHA from Bacillus sp.NA10 cultured in processed cardboard industry waste sig-nificantly contained hexadecanoic acid, methyl ester (56%),and tetradecanoic acid (32%). These compounds signifythat the monomer chains were of biodegradable polyesterfamily [8]. Figure 6c,d shows that a common molecularfragment of the 3HB ion chromatogram of the PHB wasproduced. A predominant peak corresponding to the tetra-mer of 3HB (hexadecanoic acid) was noted at 13.639 to13.88 min, respectively, in GC-purified product fromBacillus sp. NA10. The retention times and ion fragmentpatterns of the peaks at 11.6, 12.24, and 15.73 min wereidentical to those of the dimer methyl esters of 3HV and3HBV, respectively, but in low percentage up to 32% and11%, respectively. The similar results of GC-MS were ob-served by He et al. [58] with 3-hydroxydecanoate (HD orC10) 63% and 3-hydroxyoctanoate (HO or C8) 21% withother medium chain length (mcl) monomers. From thedata obtained by GC-MS, the molecular weight of PHBobtained from isolate Bacillus sp. NA10 is 242 kDa while,the commercial PHB have a molecular weight of 275 kDa.Galego et al. [59] extracted the PHB with a molecularweight of about 177 kDa. This lowering in molecularweight is due to the presence of other polyhydroxyalk-anoic acids e.g. 3HV and 3HBV up to a low percentage(32%) with major polymer 3HB.

Figure 7 PHB-starch blended sheet.


Preparation of polymer blended (PHB-starch) sheetThe PHB produced was found to be brittle and breakseasily. Blending PHB with other polymers is an economicway to improve its mechanical properties. The PHB blendsheet (Figure 7) was prepared by mixing extracted poly-mer with soluble starch following the conventional solventcast technique. Starch is used for blending because it hasfew advantage of being biodegradable, biocompatible,cheap, and also readily available while all other biodegrad-able polymer such as polyethylene glycol and polylacticacid are costly. Starch-blended sheet shows better mech-anical and thermal properties making it more reliablefor packaging industry. As PHB and starch was notcompletely miscible, the blend showed insoluble particleaggregation on the surface. Identical observations werereported by Choi et al. [60] in which they prepared PHBsheet with EPB blends. The blends showed a higherthermal stability compared to PHB sheet. Parra et al.[61] reported PHB blend preparation with polyethylene-glycol (PEG) with improved properties.

ConclusionsIn this study, inexpensive cardboard industry waste waterwas tried as a carbon source to produce PHB. Differentbacterial strains were isolated from soil and screened forpolyhydroxybutyrate production using cardboard manu-facturing industry waste water as a carbon source. Thebacterial isolate Bacillus sp. NA10 can be regarded as po-tential strain for conversion of cardboard industry wastewater into PHB. The selected isolate efficiently utilizedcardboard industry waste water as sole carbon source forgrowth and PHB biosynthesis, accumulating PHB up to66.6% of the cell dry mass with 0.072 g l−1 h−1 productivity.Currently, this bacterial strain is further studied to increasethe productivity of PHB by studying the effect of variouscarbon and nitrogen supplementation in cardboard indus-try waste water.

Competing interestsThe authors declare that they have no competing interests.

Authors' contributionsAKB carried out all the experimental work such as isolation, screening,fermentation studies, chemical, and statistical analysis and drafted themanuscript. GS (Gulab Singh) collected the substrate and helped infermentor running and chemical and statistical analysis. NKA guided all theresearch work and participated in the design of the study. VG and AY helpedin isolation and screening and provided the moral support in the wholestudy. All authors read and approved the final manuscript.

AcknowledgementsThe authors express their sincere gratitude to the Rana Cardboard industry,Yamuna Nagar, for providing the untreated waste water. The authors arevery thankful to the Department of Chemistry, Kurukshetra University,Kurukshetra, for providing the necessary facilities for NMR, FTIR, and TGAanalysis of the polymer.

Author details1Department of Microbiology, Kurukshetra University, Kurukshetra, Haryana136119, India. 2Department of Biotechnology, Kurukshetra University,Kurukshetra 136119, India.

Received: 11 April 2014 Accepted: 30 June 2014Published: 31 July 2014

References1. Lazarevic D, Aoustin E, Buclet N, Brandt N (2010) Plastic waste management

in the context of a European recycling society: comparing results anduncertainties in a life cycle perspective. Resour Conserv Recyclingvol 55:246–259

2. Flechter A (1993) Plastics from bacteria and for bacteria: PHA as natural,biodegradable polyesters. Springer, New York, pp 77–93

3. Nath A, Dixit M, Bandiya A (2008) Enhanced PHB production and scale upstudies using cheese whey in fed batch culture of Methylobacterium sp. ZP24. Bioresource Technol 99:5749–5755

4. Anderson AJ, Dawes EA (1990) Occurrence, metabolism, metabolic role, andindustrial uses of bacterial polyhydroxyalkanoates. Microbiol Rev 54:450–472

5. Yu J, Stahl H (2008) Microbial utilization and biopolyester synthesis ofbagasse hydrolysates. Bioresource Technol 99:8042–8048

6. Lee IY, Chang HN, Park YH (1995) A simple method for recovery ofmicrobial poly-3-hydroxybutyrate by alkaline solution treatment. J MicrobiolBiotechnol 5(4):238–240

7. Gao X, Chen JC, Wu Q, Chen GQ (2011) Polyhydroxyalkanoates as a sourceof chemicals, polymers and biofuels. Curr Opin Biotechnol 22:768–774

8. Choi JI, Lee SY (1997) Process analysis and economic evaluation for poly(3-hydroxybutyrate) production by fermentation. Bioprocess Eng 17:335–342

9. Yamane T, Chen XF, Ueda S (1996) Growth associated production ofpoly(3-hydroxyvalerate) from n-pentanol by a methylotrophic bacterium,Paracoccus denitificans. Appl Environ Microbiol 62:380–384

10. Grothe E, Moo-Young M, Chisti Y (1999) Fermentation optimization for theproduction of poly (ß-hydroxybutyric acid) microbial thermoplastic. EnzymeMicrob Technol 25:132–141

11. Wen Q, Chen Z, Tian T, Chen W (2010) Effects of phosphorus and nitrogenlimitation on PHA production in activated sludge. J Environ Scien 22(10):1602–1607

12. Ceyhan N, Ozdemir G (2011) Polyhydroxybutyrate (PHB) production fromdomestic wastewater using Enterobacter aerogenes 12Bi strain. Afr JMicrobiol Res 5(6):690–702

13. Wong HH, Lee SY (1998) Poly-(3-hydroxybutyrate) production from whey byhigh- density cultivation of recombinant Escherichia coli. Appl MicrobiolBiotechnol 50:30–33

14. Rebah FB, Prévost D, Tyagi RD, Belbahri L (2009) Poly-β-hydroxybutyrateproduction by fast-growing Rhizobia cultivated in sludge and in industrialwastewater. Appl Biochem Biotech 158(1):155–163

15. Reddy VST, Thirumala M, Mahmood KS (2009) Production of P HB and P(3HB-co-3HV) biopolymers of Bacillus megaterium strain OU303A isolatedfrom municipal sewage sludge. W J Microbiol Biotechnol 25:391–397

16. Koller MB, C hiellini E, Fernandes EG, Horvat P, K utschera C, Hesse P,Braunegg G (2008) Polyhydroxyalkanoate production from whey byPseudomonas hydrogenovora. Bioresour Technol 99(11):4854–4863

17. Bengtsson S, Werker A, Welander T (2008) Production ofpolyhydroxyalkanoates by glycogen accumulating organisms treating apaper mill wastewater. Water Scien Technol 58:323–330

18. Du G, Yu J (2002) Green technology for conversion of food scraps tobiodegradable thermoplastic polyhydroxyalkanoates. Environ Sci Technol36:5511–5516

19. Juan ML, Gonzalez LW, Walker GC (1998) A novel screening method forisolating exopolysaccharide-deficient mutants. Appl Environ Microbiol64:4600–4602

20. Spiekermann P, Rehm BH, Kalscheuer R, Baumeister D, Steinbüchel A (1999)A sensitive, viable-colony staining method using Nile red for directscreening of bacteria that accumulate polyhydroxyalkanoic acids and otherlipid storage compounds. Arch Microbiol 171(2):73–80

21. Singh G, Mittal A, Kumari A, Goel V, Aggarwal NK, Yadav A (2011)Optimization of poly-β-hydroxybutyrate production from Bacillus species. EurJ Biol Scien 3(4):112–116

22. Law J, Slepecky RA (1961) Assay of poly-hydroxybutyric acid. J Bacteriol82:52–55


23. Lee YU, Yoo YJ (1991). Kinetics for the growth of Alcaligenes eutrophus andthe biosynthesis of poly-β-hydoxybutyrate. Korean J Appl. Microbiol.Biotechnol 19:186–92

24. Painter PR, Marr AG (1963) Mathematics of microbial populations. Ann RevMicrobiol 22:219–221

25. Levasseur M, Thompson PA, Harrison Paul J (1993) Physiological acclimationof marine phytoplankton to different nitrogen sources. J Phycol29(5):587–595

26. Wang F, Lee SY (1997) Poly(3-hydroxybutyrate) production with highproductivity and high polymer content by a fed-batch culture of Alcaligeneslatus under nitrogen limitation. Appl Environ Microbiol 63(9):3703–3706

27. Du G, Yu J, Chen J, Lun S (2001) Continuous production of poly-3-hydroxybutyrate by Ralstonia eutropha in a two stage culture system. JBiotechnol 88:59–65

28. Steinbüchel A (2001) Perspectives for biotechnological production andutilization of biopolymers: metabolic engineering of polyhydroxyalkanoatsbiosynthesis pathways as a successful example. Macromol Bioscien 1:1–24

29. Teeka J, Cheng I, Xuehang T, Alissara R, Takaya H, Koichi Y, Masahiko S(2010) Screening of PHA-producing bacteria using biodiesel-derived wasteglycerol as a sole carbon source. J Water Environ Technol 8:371–381

30. Ramachandran H, Abdullah AA (2010) Isolation of PHA-producing bacteriafrom Malaysian Environment. Proceedings of the 7th IMT-GT UNINET and The3rd International PSUUNS Conferences on Bioscience 178–179

31. Kitamura S, Doi Y (1994) Staining method of poly (3-hydroxyalkanoic acid)producing bacteria by Nile Blue. Biotechnol Tech 8:345–350

32. Salehizadeh H, Van Loosdrecht MCM (2004) Production ofpolyhydroxyalkanoates by mixed culture: recent trends andbiotechnological importance. Biotechnol Adv 22:261–279

33. Dias JM, Lemos PC, Serafim LS, Oliveira EC, Albuquerque M, Ramos M,Oliveira R, Reis MA (2006) Recent advances in polyhydroxyalkanoateproduction by mixed aerobic cultures: from the substrate to the finalproduct. Macromol Biosci 6:885–906

34. Ganzeveld KJ, Van Hagen A, Van Agteren MH, Koning DW, Uiterkamp AMJS(1999) Upgrading of organic waste: production of the copolymerpoly-3-hydroxybutyrate-co-valerate by Ralstonia eutrophus with organicwaste as sole carbon source. J C lean Prod 7:413–420

35. Mockos G R, Loge FJ, Smith WA, and Thompson DN (2008). Selectiveenrichment of a methanol- utilizing consortium using pulp & paper mill wastestreams. http://www.ncbi.nlm.nih.gov/pubmed/18418753" \o "Appliedbiochemistry and biotechnology. Appl Biochem Biotechnol 148(1–3):211–26.

36. Rao MN, Datta AK (2007) Waste water treatment. 702 Co. Pvt. Ltd, NewDelhi. pp 203–208

37. Holt John G, Krieg NR, Peter S, Staley HA, Williams Satnley T, James T (2009)Bergey's manual of determinative bacteriology, 9th edn. Lippincott Williams& Wilkins, Baltimore, United States

38. Mirac Y, Haluk S, Yavuz B (2004) Determination of poly-β-hydroxybutyrate(PHB) production by some Bacillus spp. World J Microbiol Biotechnol21:565–566

39. Quagliano JCF, Amarilla G, Fernandes DM, Miyazaki SS (2001) Effect ofsimple and complex carbon sources, low temperature culture and complexcarbon feeding policies on poly-3-hydroxybutyric acid (PHB) content andmolecular weight (Mw) from Azotobacter chroococcum 6B. World J MicrobiolBiotechnol 17:9–14

40. Pozo C, Martı’nez-Toledo MV, Rodelas B, Gonza’lez-Lo’pez J (2002) Effects ofculture conditions on the production of polyhydroxyalkanoates byAzotobacter chroococcum H23 in media containing a high concentration ofalpechín (wastewater from olive oil mills) as primary carbon source. JBiotechnol 97:125–131

41. Yüksekdağ ZN, Aslim B, Beyatli Y, Mercan N (2004) Effect of carbon andnitrogen sources and incubation times on polybeta-hydroxybutyrate (PHB)synthesis by Bacillus subtilis 25 and Bacillus megaterium 12. African JBiotechnol 3(1):63–66

42. Khanna S, Srivastava A (2005) Recent advances in microbialpolyhydroxyalkanoates. Process Biochem 40:607–619

43. Page WJ, Manchak J, Rudy B (1992) Formation of poly(hydroxybutyrate-co-hydroxyvalerate) by Azotobacter vinelandii UWD.Appl Environ Microbiol 58:28–66

44. Klu¨ttermann K, Tauchert H, Kleber HP (2002) Synthesis of poly-beta-hydroxybutyrateby Agrobacterium radiobacter after growth on D-Carnitine. ActaBiotechnol 22:261–269

45. Singh G, Mittal A, Kumari A, Goyal V, Yadav A, Aggarwal NK (2013) Costeffective production of poly-β-hydroxybutyrate by Bacillus subtilis NG 05using sugar industry waste water. J Polym Environ 21:441–44

46. Tamdoğan N, Sidal U (2011) Investigation of poly-β-Hydroxybutyrate (PHB)production by Bacillus subtilis ATCC 6633 under different conditions. KafkasUniv Vet Fak Derg 17(Suppl A):173–176

47. Aslim B, Yüksekdağ ZN, Beyatli Y (2001) Determination of growth quantitiesof certain Bacillus species isolated from soil. Turk Electr J Biotechnol(Sp. Issue):24–30

48. Hamieh A, Olama Z, Holail H (2013) Microbial production ofpolyhydroxybutyrate, a biodegradable plastic using agro-industrial wasteproducts. G Adv Res J Microbiol 2(3):054–064

49. Sindhu R, Ammu B, Parameswaran B, Deepthi SK, Ramachandran KB, SoccolCR, Pandey A (2011) Improving its thermal properties by blending withother polymers. Brazilian J Microbiol 54(4):783–794

50. Flora G, Bhatt K, Tuteja U (2010) Optimization of culture conditions forpoly-β-hydroxybutyrate production from isolated Bacillus species. J CellTissue Res 10:2235–2242

51. Shivakumar S (2009) Optimization of process parameters for maximumpoly-β-hydroxybutyrate production by Bacillus thuringiensis IAM 12077. Pol JMicrobiol 58(2):149–154

52. Tanamool V, Danvirutai P, Thanonkeo P, Imai T, Kaewkannetra P (2009)Production of poly-β-hydroxybutyric acid (PHB) from sweet sorghum juiceby Alcaligenes eutrophus TISTR 1095 and Alcaligenes latus ATCC 29714 viabatch fermentation. The 3th International Conference on FermentationTechnology for Value Added Agroculture Products 1–6

53. El-Sayed AA, Abdelhady HM, Abdel Hafez AM, Khodair TA (2009) Batchproduction of polyhydroxybutyrate (PHB) by Ralstonia eutropha andAlcaligenes latus using bioreactor different culture strategies. J Appl Sci Res5(5):556–564

54. Khanna S, Srivastava A (2006) Optimization of nutrient feed concentrationand addition time for production of poly(β-hydroxybutyrate). EnzymeMicrobiol Technol 39:1145–1151

55. Tanamool V, Imai T, Danvirutai P, Kaewkannetra P (2011) Biosynthesis ofpolyhydroxyalkanoate (PHA) by Hydrogenophaga sp. isolated from soilenvironment during batch fermentation. J Life Sci 5:1003–1012

56. Yezza A, Halasz A, Levadoux W, Hawari J (2007) Production ofpoly-β-hydroxybutyrate (PHB) by Alcaligenes latus from maple sap.Apply Microbiol Biotechnol 77:269–274

57. Pachekoski WM, Agnelli JAM, Belem LP (2009) Thermal, mechanical andmorphological properties of poly (hydroxybutyrate) and polypropyleneblends after processing. Material Res 12:159–164

58. He W, Tian W, Zhang G, Chen GQ, Zhang Z (1998) Production of novelpolyhydroxyalkanoates by Pseudomonas stutzeri 1317 from glucose andsoybean oil. FEMS Microbiol Lett 169:45–49

59. Galego N, Rozsa C, Sánchez R, Fung J, Vázquez A, Tomás JS (2000)Characterization and application of poly (β-hydroxyalkanoates) family ascomposite biomaterials. Polym Test 19:485–492

60. Choi JY, Lee JK, You Y, Park WH (2003) Epoxidized polybutadiene as athermal stabilizer for poly(3-hydroxybutyrate). II. Thermal stabilization of poly(3-hydroxybutyrate) by epoxidized polybutadiene. Fiber Polym 4:195–198

61. Parra D, Rosa F,D, Rezende SP, Ponce J, Luga˜o AB (2011) Biodegradation ofirradiated poly-3-hydroxybutyrate (PHB) films blended with poly(ethyleneglycol). J Polym Environ 19:918–925

doi:10.1186/s40643-014-0009-5Cite this article as: Bhuwal et al.: Poly-β-hydroxybutyrate productionand management of cardboard industry effluent by new Bacillus sp.NA10. Bioresources and Bioprocessing 2014 1:9.

isolation and screening of polyhydroxyalkanoates...

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