PHB and Nanocomposite Production By Coconut Oil Utilizing Native Pseudomonas Aeruginosa KSR3

Abstract

We describe the characterization of polyhydroxybutyrate (PHB)-producing bacteria isolated from an automobile industry effluent polluted soil. Out of the 25 isolates screened based on a colony staining method with sudan black using PHA detection agar (PDA), seven were found to produce PHB. The isolate showed maximum production, KSR3 was identified as Pseudomonas aeruginosa based on conventional microbiological methods. Gel permeation chromatography analysis

5revealed that the average molecular weights of PHB accumulated by Pseudomonas aeruginosa KSR3 are 2.85×10 . Thermoplastic nanocomposites were synthesised from these PHB using dioxane and the prepared nanocomposites were characterised using SEM, ATR and NMR. These studies revealed that dioxane PHB gel produced a more satisfactory nanostructure.

Keywords: Bioplastics, Fatty acids, Nanofiber, Polyhydroxyalkonates.

availability and costs of chemicals that serve as precursor substrates to the bacteria rather than the substrate range of PHA synthesing enzymes. Therefore, the chances of obtaining PHA with new HA or unusual combination of HA in future, will depends largely on the successful screening of bacteria that synthesize these precursor substrates endogenously from simple and cheap carbon sources. Complex carbon sources that occur in some environments provide the precursor substrates (Steinbüchel and Valentin, 1995). The fluorescent Pseudomonas belonging to the rRNA homology group I have been reported to synthesize and accumulate large amount of medium chain length polyhydroxyalkanoates (mcl-PHA) consisting of various saturated 3-hydroxy fatty acids with carbon ranging from 6 to 14 carbon atoms and they act as energy storage compound. The PHA comprising of medium chain monomers (6 or more carbon length) is more elastomeric and may contain unsaturated carbon bonds. They are more conducive for coating and film materials and offer greater possibilities for the chemical modifications (Nazia Jamil and Nuzhat Ahmed, 2008). Naturally, PHAs are synthesized from coenzyme A thioesters of the hydroxyalkanoic acids, which are synthesized during fatty acid metabolism (Thakor et al., 2005). Plant oils are desirable feed stocks for PHA production because in contrast to the other carbon sources, the theoretical coefficients of PHA production from plant oils are high as over 1.0 g-PHA per g-plant oils used, since they compose a much higher number of carbon atoms per weight (Akiyama et al., 2003). Pseudomonas species are important decomposers of organic matter in soil, water and food products, but are also pathogens in plants, animals and humans. Their biotechnological importance is partly due to their potential plant growth-promoting effects and application in biological control of fungal diseases in plants for example. Recent interest has also focused on the use of these bacteria in decontamination of soil (bioremediation) because of their capability to

1. PG and Research Department of Microbiology, Center for Biological Sciences, K.S. Rangasamy College of Arts and Science, Tiruchengode-637209, Tamilnadu, India 2. Department of Botany and Microbiology, King Saud University, P.O.Box 2455, Riyadh-11451, Saudi Arabia*Corresponding Author : E-mail: murutan@gmail.com

Introduction

Accumulation of plastic wastes due to population growth and changing lifestyles has become a major concern in terms of the environment. Conventional plastics not only take many decades to be decomposed in nature, but also produce toxins during the process of degradation. For this reason, there is special interest in producing plastics from materials that can be readily eliminated from our biosphere in an “environmentally friendly” fashion. The allure of bioplastic is also linked to diminishing petrochemical reserves (Suriyamongkol et al., 2007). In this context, family of polyhydroxyalkanoates (PHA) including poly[(R)-3-hydroxybutyrate] [P(3HB)] as mother polymer has been the focus of research. These biopolymers are considered environmental friendly their biodegradability, leaving no troublesome waste in time and production from renewable natural resources such as glucose, sucrose and vegetable-oil derivatives (Akiyama et al., 2003). These biodegradable thermoplastics are water insoluble, non-toxic, biocompatible, and are known their applications in the packaging industry, medicine, agriculture, and food industry (Mizuno et al., 2010). These natural thermoplastic polyesters – essentially carbon storage reserves are known to be synthesized and accumulated in the form of cytoplasmic granules over 300 different microbial species (Lee, 1996). Among them, the most studied Poly(3-hydroxybutyrate) (PHB) has been known to be synthesized by several microorganisms.

More than 140 different hydroxyalkanoic acids have been identified as constituents of microbial PHAs when bacteria were cultivated under conditions of nitrogen limitation and surfeit carbon source. The impending for the production of new PHA seems to be limited by the

2 1 l 2* Kasi Murugan , J Pandiarajan, Prabakaran , Saleh A A and Sohaibani

Full Length Article

The PHA producing colonies were grown on Bushnell Haas mineral salts medium (BH medium) (Hi-media, India). The composition of the medium used is (per litre of distilled water): 0.2 g of MgSO , 0.02 g of 4

CaCl , 1.0 g of KH PO , K HPO and NH NO each and 0.05 g of FeCl . 2 2 4 2 4 4 3 3

Coconut oil purchased from the local market of Tiruchengodu, Tamilnadu, India were supplemented at 1% (wt/vol) in BH medium and

oincubated overnight on a rotary shaker at 200 rpm at 30 C.for growth and PHAs accumulation (Thakor et al., 2005).The grown cells were observed for the presence of PHA granules by staining with Sudan black (Schlegel et al., 1970). Sudan black stained bacterial smears of 48 h grown cells was observed under phase contrast microscope (Nikkon Eclipse TE300, Tokyo, Japan) to visualize the PHA granule. Identification of the PHA producing native culture (KSR 03) was carried out as described in the Bergey's Manual of Systematic Bacteriology (Doudoroff et al., 1974).

Fatty acids profile analysis of ground nut oil by gas chromatography (Folch et al. 1957)

The free fatty acid composition of the substrates used was determined by GC after methanolysis with NaOH solution of sodium methoxid by standard techniques. 100mg of the oil was taken in a Teflon lined screw caped vial. Then 1ml of methanol and 3ml of acetyl chloride mixture was

o oadded. The caps were air tighted and heated at 85 C to 90 C in water bath for one hour. The tubes were cooled and the contents were transferred to centrifuge tube containing 8ml of 0.88% NaCl and 3ml of n- hexane and centrifuged at 2000 pm for 10minutes. The anhydrous NaSO was added, 4

moisture was removed and then it was filtered through 0.22 µm membrane filter. The hexane layer was condensed by passing N gas. 2

0.1µl of the condensed hexane layer was taken in a GC syringe and TMinjected into the SP – 2380 capillary column. The chromatograph

operating parameters were maintained as described (30mx0.25mm film othickness, Oven temperature was maintained at 160 C for 3 minutes

o o ofollowed by a ramp of 5 C/ minutes upto 220 C and held at 220 C for 7 ominutes. Samples were injected splitless mode at 230 C, detector

otemperature was set at 220 C.The carrier gas Helium flow rate: N -4-5 2

ml, 2. H – 30ml/min, Zero air – 300ml/, 4. Spilt: 50:1)2

PHB production, extraction and quantification

Intracellular PHA polymers were isolated from freeze dried cells grown on BH medium, treated with acetone as described by Sujatha et al. (2005). The acetone dried cell mass (2.0g) was treated with a dispersion

oof chloroform (25 ml) and 30% sodium hypochlorite(25 ml) at 37 c for o90 min. The dispersion was centrifuged at 8000 g for 20 min at 30 c.The

bottom chloroform phase was filtered through a Whatman No 1 filter paper, the PHA was then purified using ice cold methanol. The amount of PHA was expressed as a percentage of the cellular dry weight (% CDW). Biomass was estimated after centrifugation of culture broth at 8000 rpm

0for 15 min followed by washing and drying of the cell pellet at 50 C to a constant weight.

Gel-permeation chromatography (GPC) analysis

The molecular weight of the obtained PHB polymers were determined as described (Luo et al., 2006). The molecular mass was obtained by GPC

0at 40 C using a Spectra System P2000 equipped with Shimadzu HSG 60 column. Chloroform was used as eluent at a flow rate of 1ml min- 1, and sample concentrations of 1mgml- 1 were applied. Polystyrene standards with low polydispersity were used to construct a calibration curve.

Fabrication of PHB matrices

oPHB was dissolved in chloroform at 60 C to become a clear solution; 1.0–6.0 mL dioxane was added in the 50 mL beaker with 10 mL PHB

degrade xenobiotic compounds (Andersen et al., 2000). Pseudomonas species growing on hydrocarbon contaminated environments are known to produce biosurfactants which helps easy uptake of hydrophobic substances. PHA formation appeared to be a common trait in a number of fluorescent pseudomonads. Already many workers reported the ability of biodegradable polymer PHA production by various members of the genera Pseudomonas (Taylor and Antony, 1976; Suzuki et al., 1986). Out of the 29 phenanthrene-degrading bacteria isolated from a coal gasification site in Frederiksberg, Copenhagen, Denmark, belonged to the genus Pseudomonas, only two were found to accumulate PHB (Andersen et al., 2000). They synthesize PHAs during cultivation on fatty acids and related compounds (Huisman et al., 1989).

While minimizing or mitigating environmental impacts is often cited as a driving factor in improving commodity manufacturing, ultimately cost competitiveness controls practice. Only through addressing the two factors together will we make significant strides toward minimizing anthropogenic impacts of manufacturing processes on the natural environment (Coats et al., 2008). PHB exhibits similar characteristics to polypropylene, including melting temperature and crystallinity, but the polymer is brittle upon crystallization and thus exhibits little stress resistance (Madison and Huisman, 1999). Research efforts are currently being harnessed in developing a new class of fully biodegradable 'green' composites by combining (natural/bio) fibres with biodegradable resins (Netravali and Chabba, 2003). The use of nanopolymer compounds in commercial applications has opened new areas of research worldwide. The use of most studied biodegradable polymer PHB is limited due to its characteristics like molecular weight; structural crystallinity and amorphous phase which promote bad mechanical properties. Polymer nanocomposites have been widely studied to make use of the sole properties of nanoparticles. Hence generation of nanocomposite based on these biodegradable polymers has been considered one of the best routes to improve the mechanical properties of them (Bruno et al., 2008). The main applications of PHB are in biomedical, agricultural, and packaging products (Scott et al., 2002), being the last industry one of the most interested in the use of PHB in form of nanocomposites (Botana et al., 2010).

Hence in the present work, an attempt has been made isolate PHA producing native Pseudomonas sp utilizing vegetable oil as a substrate from automobile industry effluent contaminated soil and the characteristics and suitability of the produced PHA for nanocomposites production were determined.

Materials and Method

Isolation and screening of PHB producing Pseudomonas.

Bacterial isolates were isolated from local automobile industry wastewater contaminated soil by spreading serially diluted samples on nutrient agar (HiMedia, Mumbai, India).The obtained isolated colonies were streaked on Kings B medium (HiMedia, Mumbai, India) and

oincubated at 36 c for 24 hours. Yellow green pigmented colonies were purified and strains inoculated on PHA – Detection broth (PDB) consisted (2 g/L disodium sulphate, 1.33 g/L dihydrogen potassium phosphate, 13.3 g/L magnesium sulphate, 1.7 g/L citric acid, 2 g/L carbon source, trace element solution 10 mL/L (10 g/L FeSO . 7H O , 4 2 4

2.25 g/L, ZnSo . 7H O, 1g/L CuSo . 5H O, 0.5 g/L MnSo , 2 g/L CaCl . 4 2 4 2 4 2

2H O, 0.23 g/L Na B 07. 10H O, 0.1 g/L (NH ) MO O , 10 mL HCl). 2 2 4 2 4 6 7 24

pH of the medium was adjusted to 7.0. Glucose was used as a carbon source and PHA production was detected macroscopically by observing the turbidity on PDA (Arnold et al., 1999). Colonies which are positive for PHA production were selected and used for further study.

Pseudomonas PHB and nanocomposites, Kasi Murugan et al., Full Length Article

ochloroform solution. The mixture was incubated in the refrigerator (4 C) for a 10 min to allow gel formations in the beaker. The gels were immersed into water for 1 day with twice replacements of the distilled water. Subsequently, the gels were placed in a freeze-dryer (iLSHIN,Model TFD 5505, Kryptonstraat,The Netherlands) for 48 h

oafter 1 h incubation at _80 C. On the other hand, some PHB solutions added with dioxane were incubated in liquid nitrogen for 15 min, or sonicated for 20 min before the gel formation process. For a comparison of cell growth behaviour, ordinary solid-walled matrices were prepared as described previously (Zheng et al., 2005). Briefly, 1.0 g of PHB was

odissolved in 50 mL of chloroform at 60 C. The solution was poured into 120-mm Petri dishes. The dishes were then maintained at room temperature to allow evaporation of chloroform for 1 day. Subsequently, the dishes with casting matrices were placed in a freeze-dryer (iLSHIN, Model TFD 5505, Kryptonstraat, The Netherlands) for 48 h. The evaporation of solvent resulted in the formation of solid-walled matrices with approximately 180 mm in thickness measured by Vernier calliper. Prior to application in cell cultures, all resulting matrices including solid-walled and nanofibrous matrices were cut uniformly into disks of 1.5 cm in diameter and sterilized by immersion in 75% (v/v) ethanol for 2 h and ultraviolet radiation for 1 h. Then, all matrices were immersed in PBS overnight (Li et al., 2008).

Scanning Electron Microscope (SEM)

The surface of the sample (PHB matrices) was mounted on the alluminium stumps that were followed by coating with gold. Scanning Electron Microscope (Jeol. JSM – 5600 LV ,Sastra university, Thanjore, Tamilnadu, India) was used for analysing the prepared nanocomposites. The sputtering device is used for 1.5min at 15 mA. The operating conditions included a working distance of 15mm and an acceleration voltage of 3.0 KV (Xie et al., 2009). The average diameter of the individual fibers was measured from multiple SEM images at the magnification of X 1000, X 2000, X 5000, X 10000, X 20000 and X 30000.

1 13H and C NMR analysis

The identity of the individual monomer units was confirmed by proton and carbon nuclear magnetic resonance (NMR). Freeze dried cells were

orefluxed with chloroform at 50 C for 5–6 h for 1H NMR spectroscopy. The polymer was recovered from the chloroform layer using hexane (1:2). The product was air dried to a constant weight. 1H NMR spectra were recorded using purified PHA samples (5 mg) in deuterated chloroform at 400 MHz. The spectra were recorded on a Bruker AMX 500 MHz instrument (Sastra University, Thanjore, India).

Attenuated Total Reflectance IR spectroscopy

For monitoring the stretching and bending of PHB, Bruker IFS 88 spectrometer equipped with three reflectors diamond ATR is used. The PHB mixed chloroform sample was made into two fractions 3mg of PHA sample were pumped onto the ATR cell with a beam condenser of 5000-

-1330cm was used. The ATR flow cell was flushed with 5% NaHCo solution for 15min. followed by rinsing with distilled water for 25min (Schmitt et al., 2002).

Results and Discussion

Research on various substrates for PHA production, especially plant oils, e.g. soybean oil, palm oil, olive oil, etc been evaluated and found to be excellent carbon source for PHA production (Ng et al., 2010). P (3HB) yields from plant oils are almost two-fold higher than that from glucose due to their high carbon content per weight (Akiyama et al., 2003). As vegetable oils differ characteristically in carbon chain composition, for

e.g. the saturated to unsaturated fatty acid content in coconut oil is 91:9; Jatropha oil 21:79; palm oil 45:55; soybean oil 16:84;rapeseed oil5:95 the fatty acid composition of substrate coconut oil was determined. The fatty acid content analysis of the substrate coconut oil showed that it contains saturated Palmitic acid (16.12-17.01%), Steric acid (3.01-3.88%), Oleic acid (18.88%), Arachidic acid (1.89%), Lauric acid (30.09%) and unsaturated (Linoleic 3.04%, Linolenic -1.20). The medium chain fattyacid lauric acid is found to be high. The medium chain fatty acids and their esters are shown to be antibacterial in nature for Gram negative bacteria (Petschow et al., 1998; Hismiogullari et al., 2008) particularly lauric acid is known to potentially inhibiting growth of number of bacteria. In this study, twenty five Pseudomonas sp. isolates were isolated from a automobile industry effluent polluted soil using a selective media. Among all the isolates obtained and examined

1

2

3

4

5

6

7

8

KSR 1

KSR2

KSR3

KSR4

KSR5

KSR6

KSR7

KSR8

Medium

Low

High

Medium

Low

Medium

Low

Absent

4.8

1.5

5.7

5.3

2.4

4.6

1.7

ND

17

ND

28

16

ND

15

ND

ND

2.35

ND

2.85

2.32

ND

1.8

ND

ND

S. No Strain name Turbidity Dry cell weight (g/l)

PHAContent(wt%)

Molecular Weight(105)

Table 1: Showing PHA producing Pseudomonas aeruginosa strains,their PHA accumulation and molecular weight of the accumulated PHA

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

Grams staining

Motility

Growth at 37 °C

Oxidase

Catalase

Gelatinase

Indole

Methyl red

Vogus-Prouskauer

Arginine dihydrolase

Denitrification

Citrate utilisation

Starch

Triple sugar iron agar

Urease

Glucose

Lactose

Sucrose

Gluconate

Malate

L-Tyrosine

D-Mannitol

Grams negative, rod

Motile

+

+

+

+

-

-

-

+

+

+

-

+(k/k)

+

+

-

+

+

+

+

-

S. No Characteristics Results

Table 2 Taxonomic characteristics of Pseudomonas aeruginosa KSR 3

Pseudomonas PHB and nanocomposites, Kasi Murugan et al.,Full Length Article

for their ability to utilize coconut oil as a carbon source and produce PHBs, only a few were found to produce them (Table 1). Seven PHA-producing strains exhibiting a significantly high intensity of fluorescence were selected, and their products were further quantified (Table.1). The microorganisms accumulate PHB as a polymeric material for preserving energy in their cells (Anderson and Dawes, 1990).

Biodegradable polymer extraction with chloroform from bacterial biomass was also reported by many other researchers (Schlegel et al., 1961; Braunegg, 1978). PHA granules were observed in all those strains which showed turbid growth in PDA broth. Cells stained with Sudan black B, a lipophilic dye gives purple colour to PHA granules with pink back ground (Smibert and Krieg, 1981). The strain (KSR3) exhibited best production was identified to the species level as Pseudomonas aeruginosa (Table 2) and selected for further study. At the exponential growth phase Pseudomonas aeruginosa accumulates maximum percentage of PHA up to 5.6% cell dry mass, while comparing to other Pseudomonas (Durner et al., 2001). Synthesis of PHA by P. aeruginosa when glucose is incorporated as a carbon source in the medium yields up to 60% of the cell dry mass (Haywood et al., 1990). Chlorination of the PHA-rich biomass, a process also referred to as alkaline hydrolysis, was utilized principally as a means to arrest bacterial metabolic activity and prevent depolymerisation of cellular PHA, and not for polymer purification. However, the treatment is also a common first step in commercial purification of PHA (Middelberg, 1995). When PHB dissolved in chloroform was added with 1,4-dioxane (Diox) PHB gel formation takes place within 30 minutes and is turned to nanofiber matrix with diameters around 250 nm. Fabrication of nanofiber matrices made of PHA when studied under different conditions, PHA nanofiber

Fig. 1 a-d: SEM micrographs of the matrix prepared from 2% (wt/v) PHB chloroform; e-h: PHB nanocomposites from 2% (wt/v) PHB chloroform with Dioxane

Fig.(2) Attenuated Total Reflectance IR spectra of synthesised PHB nanocomposites showing increase in OH stretching bonds

Fig.(3a). 13C NMR spectrum showed the carbon composition (methyl, methylene, methane, and carbonyl)of the monomers belonging to the polymer extracted with chloroform from Pseudomonas aeruginosa KSR 3 cells.

Fig.(3b). 13C NMR spectrum showed the carbon composition of the monomers belonging to the PHB nanofibres from Pseudomonas aeruginosa KSR 3 showing chemical shift assignments of all carbon resonances

Pseudomonas PHB and nanocomposites, Kasi Murugan et al., Full Length Article

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A sufficiently high molecular weight of the polymer matrix, weak interfacial adhesion and a good dispersion of the nanofillers are necessary to improve toughness and stiffness simultaneously in nanocomposites (Xie et al., 2009). Scanning electron microscopy observation also showed that the crystallization rate and mechanical properties could be further improved by optimizing the interfacial interaction and compatibility between the PHB and Dioxane. The surface layer of a polymer often exhibits different properties, e.g. morphology, composition, and structure, from the bulk material and it sometimes determines the overall characteristic of the polymer (Fig.1). Crystallization behavior of macromolecules at the surface and interface regions plays an important role in numerous properties, e.g. mechanical strength, chemical compatibility, and biocompatibility of polymeric materials (Strobl 1996). Attenuated total reflection (ATR) infrared (IR) spectroscopy is known as one of powerful surface characterization techniques that can provide information, e.g. chemical reactions, functional groups, and molecular orientation, at the surface and interface regions of polymers (Padermshoke et al., 2004). ATR revealed the characteristic absorption band of the poly (β- hydroxyalkaonates) (PHA). The PHA alcohol stretching was characterized by the adsorption band at wave number 3270.23 cm-1 for alcohol, 2922.33 cm-1 for alkanes and 1634.88 cm-1 for alkanes. The amount of OH stretching bands increased due to the intermolecular hydrogen bonding between dioxane and was showed in (fig. 2) PHA. ATR analysis showed molecular interaction between chloroform and PHA and crystallinity changed.

The 1H-NMR spectrum (Fig.3a&3b) showed the presence of signals characteristic group of the homopolymer PHB, and the 13C-NMR spectrum showed four narrow lines appeared at very strong intensities in addition to the weak lines associated with bacterial cellular material. These four peaks were assignable to the methyl, methylene, methane, and carbonyl carbon resonances of PHB. The chemical shift assignments of all carbon resonances were entirely consistent with those results in previous publications. Poly-3-hydroxybutyrate (PHB) is the most abundant of a wide range of high-molecular-mass microbial polyhydroxyalkanoates. These polyesters comprise repeating hydroxyacyl monomers of general formula (- 0 - CH(R) - CH, - CO -) n, where R = CH, in PHB, and are of considerable commercial importance due to their thermoplastic properties and biodegradability (Henderson and Jones, 1997).

Conclusion

Till date, many species of PHA-producing bacilli have been isolated from various environments and characterized. Some of these are able to produce PHA copolymers from inexpensive and structurally unrelated carbon sources. Still the search continues for obtaining efficient organisms utilising unfamiliar low cost substrates. The automobile effluent isolate P.aeruginosa KSR 3 has been found to produce PHB from coconut oil and the same can be used to synthesis nanocomposites. These nanocomposites may also have the advantage of faster decomposition in the environments which can also serve as a platform for new greener products demanded by environmentally conscious consumers and regulatory authorities. Alternatively, the use of food-grade coconut oil to produce these bioplastics can be prevented by searching this potential isolates ability to use non-food grade oils like Jatropha oil. This study also revealed that the less studied, unexplored hydrocarbon contaminated environments can be explored for isolation of PHB producing low cost vegetable oil using organisms. This organism's ability of using inexpensive carbon source which is important in industrial-scale production can be further explored.

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