isolation, screening and characterization of polyhydroxyalkanoates ...

Journal of Environmental Research And Development

Vol. 5 No. 3A, January-March 2011

ISOLATION, SCREENING AND CHARACTERIZATION OF POLYHYDROXYALKANOATES PRODUCING BACTERIA UTILIZING EDIBLE OIL AS CARBON SOURCE Darshan Marjadi*1 and Nishith Dharaiya2 1. Department of Biotechnology, Sheth M. N. Science College, Patan, Gujarat (INDIA) 2. Department of Life Sciences, HNG University, Patan, Gujarat (INDIA)

Received October 28, 2010

Accepted February 16, 2011

ABSTRACT Environmental biotechnology has the intention of increasing sustainability of production processes by employing biological systems and thereby benefiting the environment. Microorganisms are a biological system which is generally used for the reduction of pollution from air, aquatic or terrestrial systems. Edible oil and fats are utilized by microorganisms and produces new product such as lipase and biodiesel was investigated. In present study the microorganisms utilizing edible oil as carbon source were isolated and investigation of their characteristics towards the production of Polyhydroxyalkanoates (PHA), which is now a days well known as Biodegradable polymer. Sixteen bacterial colonies were isolated, screened by providing various edible oils as carbon source and preserved using glycerol. The microorganism then stained for PHA with Sudan Black B stain. We have found that nine out of sixteen strains exhibited PHA producing ability. The organisms were identified through several biochemical morphological, culture and physiological characteristics and showed affiliation to Bacillus, Citrobacter, Enterobacter, Escherichia, Pseudomonas and Staphylococcus. With the positive isolates, an attempt was made for the production and extraction of Biodegradable polymer. Finally, the biomass and extracted PHA content was determined where high level of production of PHA was observed under aerobic condition using sesame oil (4.5%) by Staphyococcus (DSM-IV), palm oil (3.7%) by Pseudomonas (DSM-V), Soya bin oil (2.7%) by Pseudomonas (DSM-IX.).

Key Words : Pollution, Plastic, Edible oil, Polyhydroxyalkanoates (PHA), Biological system

INTRODUCTION The world population is expanding at an alarming rate. The current worldwide demand for plastics has increased to more than 100 million tones per year 1 . Unfortunately these petroleum based plastics remain in the environment for the longer period of time in harsh condition and remain protected from attack of microorganisms and chemicals. Therefore, they result in environmental pollution by buried in landfills everyday and take up space. Thus, various investigations have focused on the biosyntheses of a class of polyesters, known as poly-(b-hydroxyalkanoates) (PHAs). PHAs are naturally occurring, optically *Author for correspondence

active polyesters that are produced metabolically from the bioconversion of alkanes and alkanoic acids by a number of bacterial strains.2-4

Biodegradable plastics are special type of biological material which is degradable and eco-friendly in their chemical nature. Polyhydroxyalkanoates (PHAs) are biodegradable polyesters and elastomers, which gets accumulated as cytoplasmic inclusion in certain bacteria during unbalanced growth condition as an intracellular storage material of carbon and energy5-7, usually characterized by an excess carbon supply and the lack of one or more essential nutrients8. About 150 different hydroxyalkanoic acids have been identified as constituents of 764

Journal of Environmental Research And Development 9

bacterial polyesters . PHAs have recently attracted industrial attention because of their potential use as practical biodegradable and biocompatible ther moplastics 10. The first determination of the composition of PHA was discovered by Lemoigne in 192611. Polyhydroxyl butyrate (PHB) is a biodegradable thermoplastic polymer that have many advantages similar to that of many conventional petrochemical derived plastics 11 . The main advantage is that, the biodegradable polymers are completely degraded to water, carbon dioxide and methane by anaerobic microorganisms in various environments such as soil, sea, lake water and sewage and hence, is easily disposable without harm to the environment. Poly 3-hydroxy butyrate (PHB) belongs to the PHA and is used widely as a storage compound produced by the bacteria. This is observed as hydrophobic inclusions in the cytoplasm of Bacillus megaterium and in many gram negative and gram-positive bacteria12. A long list of bacterial strains capable of producing PHA has been reported 9.These includes Grampositive and Gram-negative species and cyanobacteria. Recently, much efforts 13 have been given to produce PHA in pilot scale of continuous mode from waste water systems to make this production feasible. One of the major stumbling blocks in the large scale synthesis is the high production cost. Studies have shown that, the raw material costs (mainly carbon source) contribute most significantly to the overall production cost 14. By changing the carbon source and bacterial strains used in the fermentation process, it is possible to produce related biopolymers having properties ranging from stiff and brittle plastics to rubbery polymers 5. The previous study had demonstrated that inexpensive renewable plant oils are excellent carbon sources for the efficient production of PHA15 as it has been calculated that 2.5 kg of glucose must be used for each kilogram of polymer produced. Plant oils may be a better carbon source whereby a kilogram of oil can give rise to a kilogram of polymer16. As with most microbial products, the search for new producer organisms is a continuous process, necessitated by the desire for higher product yield, more efficient utilization and conversion of specific raw materials, tolerance


to environmental conditions, and novel end products.

AIMS AND OBJECTIVES The present study is focused on the isolation of bacterial strains, screening the strains for PHAproducing ability using different edible oil as carbon source.

MATERIAL AND METHODS Sampling The soil samples were collected from the sites near the industries producing edible oil. The soil samples were also characterized by some primary physicochemical tests such as pH, texture, organic carbon and organic nitrogen using standard methods17 . Isolation of Bacteria The Isolation of oil degraders were made by serial dilution and enrichment isolation technique. Here, one gram of soil samples was added in to 10 ml of distilled water and homogenized for 1 min with a blender. This homogenized suspension is serially diluted and spreded on Nutrient Agar (Hi-Media) and incubated for 24 h at37 oC. Screening of soil bacteria for PHA production Each of the bacterial colony obtained on the nutrient agar medium plates were picked up; examined microscopically and purified by streaking on N-Agar medium. All the colonies were grown in 5 ml of nutrient broth with shaking for 24 h at 370C.These culture were then used as inoculums (1% v/v) for a 50 ml nitrogen free, carbon – rich MSB medium18 containing sesame oil (1%w/v) as the sole carbon source for 24-48 h at 370C with shaking at 150 rpm. As turbidity increase in the culture medium, a loopful of each of these cultures was smeared on glass slide, heatfixed and stained with Sudan Black B (Loba)19 to detect the presence of Intracellular granules. At the same time cultures was spread on tributyrine agar plate for the detection of lipase producer. Only those isolates which could grow on tributyrine agar plate and synthesize PHA were selected as PHA producers and further identified by morphological and biochemical characteristics.

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Culture condition All the isolate were first grown for 72 h at 370 C with shaking at 150 rpm in a carbon – rich MSB medium containing sesame oil (1%w/v) as the sole carbon source. To study the effect of various edible oil as carbon source on the PHB accumulation cells were cultivated in 250 ml of modified mineral salts basal medium (MMSB) medium contain: (NH4)2HPO4 (l.lg), K2 HPO4 (3.7g), 10 ml of 0.1M MgSO4 and 1.0 ml of a microelement solution (this microelement solution contained FeSO 4 -7H 2 O(2.78g), MnCl 2 4H 2 O(1.98g), CoSO 4 -7H 2 O(2.81g), CaCl 2 2H2O(1.67g), CuCl2-2H2O(0.17g), and ZnSO4 7H2O (0.29g) in 1-l of IN HC1) the pH was adjusted to 7.00. supplemented with individual 1% (v/v); four edible oils i.e. Sesame, Soya bin, Palm and Coconut, in different 1l of Erlenmeyer flasks and incubated for 72 h on rotary shaker at 150 rpm, at 37 0C .At the end of the cultivation (72 h), cells were harvested by centrifugation at 10,000g for 10 min. The cell pellet was then resuspended in 100 ml of hexane by vortexing and then centrifuged again at 10,000g for 5 min to remove the remaining oils. Samples were harvested at different time intervals: the OD at 600 nm and dry weight was recorded and determine for the growth. Biomass measurement Cell dry weight (CDW) was obtained after centrifugation of 5mL of culture at 8000xg for 15min at 4 oC (Remi,India)and then the harvested cells were washed twice with distilled water and dried at 100 oC to a constant weight. Extraction of Polymer from cells 5 ml of culture was centrifuged at 10, 000 g for 10 minutes and supernatant was discarded. The pellet should be suspended in 2.5 ml of 4 % sodium hypochlorite for digestion and 2.5 ml of hot chloroform and was incubated at 37°C for 1 hour. The suspension was centrifuged at 1500 g for 10 minutes. (The upper phase contains hypochlorite solution and the middle phase contains chloroform with cell debris).The bottom phase containing PHA with chloroform was collected and further was followed by extraction with hot chloroform and precipitated with ethanol and acetone (1:1).The


precipitate was allowed to evaporate for dryness at 30oC to obtain PHA crystals and weighted. The polymer content (w/w) was defined as the percentage of PHA in dry cell mass.

RESULTS AND DISCUSSION The soil has been classified as Typical Haplaquet, containing 90% silt and 10% sand, with 3% organic carbon, 0.3% organic nitrogen and all the isolates are capable to grow luxuriously at optimum pH 5.7 and at the temperature up to 30-400 C. Total 16 bacterial strains were isolated form various oil industries soil samples when stained with Sudan Black and viewed under the Olympus BX-40 phase contrast microscope for taking photographs. Nine bacterial strains (DSM-I, DSM-II, DSM-IV, DSM-V, DSM-VI, DSM-VII, DSM-VIII, DSM-IX and DSM-XIV) showed characteristic granules, indicating the possible presence of PHA(s) in the cells and all the nine strains were able to secrete the lipase and detected by zone of hydrolysis on Tributyrine agar plates (Table 1). With regard on biochemical characterization edible oil degrading strain showed resemblance to Pseudomonas, Enterobacter, Citrobacter, Bacillus, Escherichia and Staphylococcus (Table 2). To analyze the substrate specificity of all the isolate, in the present study, four different edible oils with all nine isolates, used for PHA accumulation added in MMSB. PHA production was also found to be influenced by the utilization of various edible oil. Of the different edible oil used (as sole carbon source with MMSB) out of nine strain three strain of Pseudomonas (DSM-V, DSM-VII, DSM-IX) were able to utilize all the different types of edible oil; where as the strain of Enterobacter (DSM-I) and Citrobacter (DSM-VIII) were not able to grow in production medium and are not accumulating PHA which can observe by increase in turbidity of the medium as well as Sudan black staining at the regular interval. Escherichia (DSM-II) able to utilize only soya bin oil. Strain of Staphylococcus (DSM-IV) was able to utilize sesame oil in highest amount (Fig. 1). In all the nine isolates high level of production of PHA was observed under aerobic condition using sesame oil (4.5%) by Staphyococcus (DSM-IV) followed by palm oil (3.7%) by Pseudomonas (DSM-V).

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Table 1: Screening for potential PHA producersa Strain

Sudan Black stain

Tributyrine Hydrolysis

DSM-I

++ ve

++ve

DSM-II

++ ve

++ve

DSM-III

–ve

–ve

DSM-IV

++ ve

++ve

DSM-V

++ ve

++ve

DSM-VI

++ ve

++ve

DSM-VII

++ ve

++ve

DSM-VIII

++ ve

++ve

DSM-IX

++ ve

++ve

DSM-X

+ ve

+ve

DSM-XI

+ ve

+ve

DSM-XII

+ ve

+ve

DSM-XIII

–ve

–ve

DSM-XVI

++ ve

++ve

DSM-XV

– ve

–ve

DSM-XVI

– ve

–ve

a Legend: ++ ve = granules fully observed, + ve = granules slightly observed, –ve = no granules observed,++ ve = Zone fully observed, + ve = Zone slightly observed, –ve = no Zone observed

767

+ve/-ve

-ve/-ve

-ve/+ve

DSM-VIII

DSM-IX

DSM-XIV

-ve/-ve

DSM-V

-ve/-ve

+ve/+ve

DSM-IV

DSM-VII

+ve/+ve

DSM-II

-ve/-ve

-ve/+ve

DSM-I

DSM-VI

MR/VP

Strain No.

768 -ve

-ve

-ve

-ve

-ve

-ve

-ve

-ve

-ve

+ve

+ve

+ve

+ve

+ve

+ve

+ve

+ve

+ve

+ve

+ve

-ve

+ve

-ve

+ve

-ve

-ve

-ve

K/K/-/+

K/K/-/-

A/A/-/-

K/K/-/-

A/A/-/+

K/K/-/-

A/A/-/-

A/A/-/-

A/A/-/+

TSI

-ve

-ve

-ve

-ve

-ve

-ve

-ve

-ve

-ve

Urease

-ve

-ve

+ve

-ve

+ve

-ve

+ve

+ve

+ve

Glu

-ve

-ve

-ve

-ve

-ve

-ve

+ve

-ve

-ve

Suc

-ve

-ve

+ve

-ve

+ve

-ve

+ve

+ve

+ve

Lac

Sugar fermentation

a : Legend, A=acidic, K=alkaline,Glu=glucose, Suc=Sucrose, Lac=lactose, Mal=maltose

+ve

+ve

+ve

+ve

+ve

+ve

-ve

-ve

-ve

Indole Citrate Catalase Oxidase

Table 2 : Biochemical properties of oil degraders and PHA producersa

-ve

-ve

-ve

-ve

-ve

-ve

-ve

+ve

-ve

Mal

Pseudomonas

Pseudomonas

Citrobacter

Pseudomonas

Bacillus

Pseudomonas

Staphylococcus

Escherichia

Enterobacter

Probable Genera

Journal of Environmental Research And Development Vol. 5 No. 3A, January-March 2011


(Fig. 2), Soya bin oil (2.7%) by Pseudomonas (DSM-IX) (Fig. 3), by and lowest PHA production was observed with coconut oil (0.13%) in Bacillus (DSM-VI)(Fig. 4). Maximum PHA production by Staphylococcus observed was in sesame oil medium under aerobic condition.

Sesame oil

Soyabin oil


Besides Staphylococcus (DSM-IV) strain of Pseudomonas (DSM-V, VII,IX, XIV) (Fig. 5 and Fig. 6) and also shows the PHA production. But when utilization observe for the stain of Enterobacter(DSM-I),Citrobacter (DSM-VIII) and Escherichia(DSM-II) very less or minute growth was observed.

Palm oil

Coconut oil

Fig. 1 : Production of PHA by strain of Staphylococcus (DSM-IV) various edible oil

Sesame oil

Soyabin oil

Palm oil

Coconut oil

Fig. 2 : Production of PHA by strain of Pseudomonas (DSM-V) on various edible oil

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Sesame oil

Soyabin oil


Palm oil

Coconut oil

Fig. 3 : Production of PHA by strain of Pseudomonas (DSM-IX ) on various edible oil

Sesame oil

Soyabin oil

Palm oil

Coconut oil

Fig. 4 : Production of PHA by strain of Bacillus (DSM-VI) on various edible oil

Sesame oil

Soyabin oil

Palm oil

Coconut oil

Fig. 5 : Production of PHA by strain of Pseudomonas (DSM-VII) oil on various edible oil 770


Sesame oil

Soyabin oil


Palm oil

Coconut oil

Fig. 6 : Production of PHA by strain of Pseudomonas (DSM- XIV) on various edible As far as the environmental and energetically issues are considered, triglyceride oils are expected to play a key role during the 21st century as enabling to synthesize polymers from renewable sources. In this study, different types of microorganisms were studied to evaluate their PHA production under different types of carbon source. Polyhydroxy alkanote obtained in this study were found to be sudanophilic, as confirmed by Sudan black-B staining procedures. Previous reports20 have confirmed the sudanophilic nature of PHA granules. A conventional method developed for the analysis of PHA by Kim et al.21 revealed that the polymer could be converted quantitatively to crotonic acid by heating in concentrated sulphuric acid and the ultra violet absorption was shifted to 235 nm. So crotonic acid was used as the standard in this study to quantify PHA. The aim of this study is to show that bacteria are able to use edible oil for growth and for conversion in to an interesting biotechnological product. Therefore here, it was demonstrated that the edible oils could be converted in to PHA.

CONCLUSION

Sesame oil was considered as ideal source for PHA production followed by soyabin and coconut oil and Staphylococcus is considered as ideal organisms to produce highest amount of PHA, followed by Pseudomonas and Bacillus. The usage of various edible oil in fermentation process of PHA production, tremendously reduce the cost of the bioplastic production. This preliminary comparative analysis of four types of edible oil has led to the novel invention of a biodegradable environment friendly polymer with a high potential for regular use.

ACKNOWLEDGEMENT We wish to express our sincere gratitude to University Grants Commission (UGC), Delhi, India for financial assistance in terms of Minor Research Project.

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