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Abstract

Polyhydroxyalkanoates (PHA) accumulating bacteria were isolated from Rubber plant growing areas of Kerala. They were isolated by PHA production medium with sucrose as a sole carbon source. PHA producing bacteria were screened by staining with Sudan black B and PHA from the same bacteria was extracted by sodium hypochlorite. An efficient PHA producing isolate (PHA7) was identified and subjected for its morphological, biochemical properties, and molecular characterization. The identified Bacillus cereus yielded PHA of 0.436 g/l, amounting to 13.77% (w/w) of dry cell weight. The extracted polymer was analysed for oil absorbing, oil retention and biodegradability of PHA. Further, characterization of this polymer using Gas chromatography-Mass spectrometry (GC-MS) confirmed it as polyhydroxyhexonate.

Introduction

Global dependence on petroleum derived plastics has increased considerably over a decade (Philip et al., 2007). Plastics have versatile qualities of strength, lightness, durability and resistance to biodegradation (Khanna and Srivastava, 2005). They are produced from non-renewable resources such as petro-chemicals and are not compatible with natural carbon cycle, because of their non-degradable characteristics. They pose serious threat to environment and wild life due to their persistence (Chua et al., 2003). Accumulation of recalcitrant plastics in the environment has become a worldwide problem. Recycling of plastics can be done but it is a tedious time consuming process. In such cases, biodegradable plastics offer the best solution to the environmental hazard posed by conventional plastics as they are ‘eco-friendly’ in nature. Biodegradable plastics can be divided into four categories, such as polyhydroxyalkanoates (PHAs), polylactides, aliphatic polyesters and polysaccharides and in those, PHAs are the only 100% biodegradable polymer.

PHAs are optically active biopolyoxoesters and composed of (R) 3-hydroxy fatty acids, which represent a complex class of storage polyesters. They are synthesized by some Archaea and wide range of gram-positive and gram-negative bacteria in aerobic and anaerobic environments. These biopolymers are accumulated as inclusions (PHA granules) in the bacterial cytoplasm in response to organic nutrient limitation, generally, when the microbes are cultured in the presence of an excess carbon source. At present, PHAs are classified in two major classes: short-chain-length PHAs (scl-PHAs) with C4-C5 monomers and medium-chain-length PHAs (mcl-PHAs) with C6-C14 monomers. Mcl-PHAs are mainly produced by Pseudomonas sp. Because of structural differences, the physical properties of mcl-PHAs are generally quite different from the archetypal polyhydroxybutyrate (PHB) and other scl-PHAs (Matrinez et al., 2011).

In this study, an attempt has been made to explore the polyhydroxyalkanoates production from rhizosphere soil bacteria of rubber plant. The extracted PHA was subjected to structural characterization by GC-MS analysis and the rate of biodegradation of the PHA film was done by open windrow composting method.

Materials and Methods Sample collection

Isolates were obtained from rhizosphere soil of Rubber plantation areas located at different places in the state of Kerala such as Guruvayur, Trivandrum, and Sabarimala. Collected soil samples were kept in plastic bags and stored at 4ºC for further use. Each sample was homogenized by sieving (2.0 mm pores); dry weight equivalents were established by treating three samples having 20 g of fresh soil (Moreno et al., 2007).

 

Isolation of bacteria

One gram of rhizosphere soil was serially diluted in sterile distilled water and plated on nutrient agar plates and incubated at 30ºC for 24 hours. Various colonies of different morphologies including branched, circular and rhizoidal forms were individually picked and subcultured 3-4 times on nutrient agar plates to obtain pure culture (Aarthi and Ramana, 2011). The bacterial isolates were maintained as pure culture on nutrient agar slants and stored at 4ºC for further use.

Screening of PHA producing bacteria

Sudan Black B staining was used for detecting the presence of PHA in cytoplasm of bacterial cells by Moreno et al. (2007). However, before screening, the isolates were induced to accumulate PHA by growing in PHA production liquid medium; a nitrogen-limiting medium containing 2% (w/v) glucose for 24 hours. Smear was made on a clean glass slide. After drying, 0.3% of Sudan black was added and kept for 10 minutes. Later, the slide was washed gently with distilled water and allowed to air dry. The dried slides were then immersed in xylene for few seconds and allowed to dry. Then 0.5% of safranin was added to the slides for 30 sec, the slide were washed gently, allowed to air dry and observed under light microscope.

Those PHA producing bacteria screened by Sudan Black B staining were subjected to sodium hypochlorite (Moreno et al., 2007). The screened bacterial isolate was grown in conical flask containing PHA production medium on a shaker at 30ºC for 72 h with an agitation rate of 125 rpm. After 72 h, the bacterial culture was centrifuged at 6500×g, the supernatant was discarded and the pellet was transferred into pre-weighed petriplates by dissolving it in distilled water. The plates were dried by keeping it in hotplate at 80ºC. The dried weight of the pellet was taken in order to know the biomass weight. Sodium hypochlorite solution was added to the dried pellet and transferred to the centrifuge tubes and kept in shaker for 30 minutes at 37ºC. After incubation, the samples were centrifuged at 6500×g for 15 minutes. The supernatant was discarded and the pellet was washed with the distilled water and acetone was added to remove the hypochlorite solution by centrifugation. 10 ml of chloroform was added to the pellet and filtered into the pre-weighed petriplates. The chloroform gets evaporated which leaves the PHA film in the petriplates. The weight of the PHA film was observed. The PHA production was calculated by following formula, % of PHA production = (Weight of PHA/ Weight of biomass) × 100.

Identification of efficient PHA producing bacteria by 16S rRNA sequencing

The efficient PHA accumulating bacterium PHA7 was characterized by 16S rRNA gene sequencing. The genomic DNA from the PHA7 was isolated as per the standard protocol described by Sambrook et al. (1989). Amplification of bacterial 16S rRNA gene from the extracted genomic DNA was performed using the universal 16S rRNA gene primers: 16S rRNA forward primer 5’ AgAgTTTgA TCM TGG CTC Ag3’ (27f) and the reverse primer 5’ TAC ggY TAC CTT gTTACg ACT T3’ (1492r). The amplification of 16S rRNA gene was confirmed by 1.5% agarose gel electrophoresis in 1X Tris-acetate-EDTA buffer. The amplified product was further resolved and amplicon size corresponding to 1400bp was purified from agarose gels using QIA gel extraction kit following the manufacturer’s protocol. The amplified 16S rRNA gene after gel elution was sequenced using forward and reverse, about 1400bp were carried out in Applied Biosystems^TM by Life Technologies^TM ABI Prism^R 3730, Ocimum Bio solutions, Hyderabad, India.

Determination of oil-absorbing capacity

PHA film was weighed prior to immersion into mineral oil. After 1 min, the film was removed and excess oil was allowed to drip and PHA film weight was measured. The oil-absorption capacity is expressed as mass of oil per unit mass of initial film (Sudesh et al., 2007) according to the formula: Oil absorbing property (g/g) = [(Film weight after dipping in oil – Initial film weight) / Initial film weight)].

Determination of oil-retention capacity

After determining the oil-absorption capacity, the PHA film was placed under a Petriplate wrapped with aluminium foil. Weight was placed on the plate for 1 min and then the film was weighed again (Sudesh et al., 2007). The percentage oil retention was calculated as : Oil retention (%) = {[1-[(Film weight after dipping in oil - Film weight after pressing) / Film weight after dipping in oil] × 100]}.

GC- MS analysis
Sample preparation

Five milligrams of the sample was taken and added to dry test tubes. One milliliter of chloroform, 850 µl of methanol, and 150 µl of sulphuric acid were added and mixed well. The tubes were sealed and allowed for methanolysis. For methanolysis, the sample containing test tubes were kept in a beaker containing glycerine and boiled up to 100ºC. During methanolysis, the sample gets converted into methyl esters (HB/HV) and dissolved in chloroform. The sample was then heated to 2 h and 15 min. After cooling, the seal was broken and 1 ml of distilled water was added and shaken well. Bilayer formation occurred. These tubes were centrifuged at 9000×g for 5 min. The bottom layer, which contains chloroform with methyl esters was taken and transferred to Eppendorf tubes. The tubes were kept at - 40ºC (Chia et al., 2010).

Conditions of GC-MS

The capillary column was used with the following conditions: Capillary Column Elite - 5MS (5% Phenyl, 95% dimethlpolysiloxane); Dimensions (L × OD): 30 m × 0.25 mm; Carrier gas: He at flow rate 1ml/minute; Injector temperature: 290ºC; Column temperature: 200ºC; Detector temperature: 250ºC; and Temperature programming:60ºC (1min) at 6ºC/min 220ºC (3min) @ 8ºC/min 290ºC (5 min).

Biodegradability of PHA in soil

The PHA film produced from the fermented broth of PHA7 isolate was subjected to biodegradability test. The PHA film was buried into the soil in plastic tray to check its biodegradation rate and was maintained for 54 days in open windrow composting method. The film was visually inspected for changes in their morphology at different time interval (1, 14, 28, and 54^th days).

Results and Discussion
Isolation and screening of PHA producing bacteria

Morphologically different types of bacteria were isolated from rhizosphere soil of rubber plantation collected from three different places of Kerala. The total number of 152×10⁶ CFU/g of rhizosphere soil in Trivandrum area, 148×10⁶ CFU from Sabarimala area, and 83×10⁶ CFU for Guruvayur were recorded. The bacterial population varied significantly among all these three areas; probably due to the temperature, pH, and fertility of the soil.

The bacteria were initially screened by Sudan black- B staining method, in order to find out the PHA granules present in the cytoplasm of bacteria. Among all the isolates, 4 isolates from Trivandrum, 3 from Sabarimala, 1 from Guruvayur were screened as PHA producers.  On microscopic observation, the 24 hours grown culture had showed the bluish black granules of PHA, which were ovoid or spherical in shape (Fig.1). The PHA producing isolates were designated as PHA1, PHA5, PHA7, PHA9, and PHA10.

In order to find out the efficient PHA producer from 8 isolates the sodium hypochlorite extraction method was performed. Based on the results, PHA7 isolates were found as more efficient than other PHA producing isolates. The film produced by the PHA 7 was found to be 0.028 g/l.

Fig. 1: Photomicrograph showing the PHA granules present in B. cereus (400X)

Table 1: Screening of efficient PHA producing bacteria

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S. No	Bacterial isolate	Biomass (g/l)	PHA film weight (g/l)	PHA (%)
1	PHA1	0.211	0.017	7.65
2	PHA5	0.254	0.02	21.90
3	PHA7	0.465	0.028	13.77
4	PHA10	0.006	0.006	2.08

Identification of efficient bacterial isolate (PHA7)

Amplification of 16S rRNA gene was confirmed by 1.5% agarose gel electrophoresis in 1X Tris - acetate - EDTA buffer. The amplified product was further resolved and amplicon size corresponding to 1400bp was purified from agarose gels using QIA gel extraction kit following the manufacture’s protocol. The PCR product was sequenced. The PHA7 was identified as Bacillus cereus.

Determination of oil - absorption and retention capacity of PHA

PHA films have the good capacity to absorb oil. The amount of oil absorbed and retained depends upon the efficiency of the organisms which produce PHA. Pre-weighed PHA film extracted form B. cereus was immersed in oil and reweighed for oil absorption (Fig. 2). PHAs with good commercial applications are often compared with PP (polypropylene) because of their similar characters in some respects. So, that PHA cast from the solution has an extraordinary ability to absorb oil. The oil rapidly absorbed into the film and caused obvious change to the film appearance, which was indicated by greater transparency.

The initial film weight of PHA film without oil was 0.036 g, after immersing in oil the weight of the PHA drastically increased up to 0.103 g. The difference after and before immersing in oil was found to be nearly up to 0.79 g (Fig. 3). Applying the respective values in the formula, the results were found to be 1.861 g. The value obtained was high when compared to the PHBHx oil retention capacity which was found to be around 1. The plant - fiber based facial blots absorb around 1.4 (Sudesh et al., 2007). The results showed that the PHA film produced by B. cereus had the ability to absorb oil.

Fig. 2: Appearance of PHA film produced by B. cereus (a) before and (b) after immersed in mineral oil

After absorbing, the oil in the film was then pressed in between the petriplates in order to find out the oil retention capacity. The PHA film weight after pressing was found to be 0.066 g, the weight of PHA film decreased. On by applying the respective values in the formula the result obtained was found to be 34.92% (Fig. 2). The oil retention capacity of PHA film was found to be 80% (Sudesh et al., 2007), which was higher than the PHA produced by B. cereus.

Characterization of PHA film

B. cereus was grown on production media amended with glucose as carbon source was used for direct chloroform extraction procedure to obtain PHA product, and was subjected to GC-MS analysis. 3HHx (3-hydroxyhexonate) was found at 28.66 RT and the molecular weight of the compound was 270 Da (Fig. 3) by MS library search. The compound was referred with MS library and identified as hexonate. GC–MS revealed that the B. cereus was mainly composed of 3HD (3- hydroxyl hexonate).

Fig. 3: GC–MS analysis of PHA produced by B. cereus

Biodegradability of PHA in soil

Degradability is the main difference between the synthetic plastics and bioplastics. Hence, the degradability of the PHA films produced by the B. cereus was tested in soil. The PHA film of B. cereus had degraded within 54 days. Biodegradation of PHA film on soil surface using open windrow composting method was examined (Fig. 4). The PHA film has the greatest affinity towards the depolymerase enzyme. The polymer film P (3HB-co-1 mol% 3 HV-co-3 mol% 3H4MV-co-18 mol% 3HHx) has been degraded on 54^th day. P(3HB-co-5mol% 3HHx) did not get degraded even after 60 days (Chia et al., 2010). This result showed that PHBHHx degrades faster than the PHBV.

Fig. 4: Biodegradation of PHA film produced from B. cereus. (a) 1^st day; (b) 14^th day; (c) 28^th day; (d) 54^th day of incubation

Conclusion

PHAs are found to be promising biodegradable plastics which show materials similar to that of synthetic plastics. The produced PHA was characterized as 3-hydroxyl hexonate by GC-MS.  It showed oil absorption and retention capacity. Due to these properties, it may have commercial application value in oil absorbing films in the cosmetics and skin care industries. Further studies on the increased production of PHAs are required for commercial application

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