Blend of Polyhydroxyalkanoates Synthesized By Lipase Positive Bacteria From Plant Oils

Abstract: A total of 5 biochemically characterized lipase positive bacterial strains were screened for Polyhydroxyalkanoates (PHA) production by Nile blue staining and confirmation was done by Sudan Black B. PHA production ability for all strains was optimized followed by time profiling calculation and comparison via using glucose and two plant oils i.e., canola and mustard oil. Quantitative analysis showed that glucose can serve as a carbon source for maximum biomass (2.5 g/L CDW for strain 5) and PHA production (70.3% for strain 2). PHA produced by strain 2 was further analyzed for its chemical composition and type via Fourier Transform Infrared (FT-IR) spectroscopy. It revealed homopolymer (PHB) and copolymer (PHB-co-PHV) production of PHA (peaks at 1743 cm-1 and 2861 cm-1, respectively) with both canola and mustard oil unlike glucose which produced only homopolymer one i.e., PHB (peaks at 1110 cm-1, 1411 cm-1 and 1650 cm-1). Crystallinity of FT-IR analyzed PHA was calculated using mathematical formulas which showed decrease from glucose to canola to mustard oil. This study revealed that plant oils can serve as better carbon source to produce better quality (ductile and copolymer) PHA. Moreover, 16S rRNA gene sequencing analysis showed that strain 1, strain 2, strain 3, strain 4 and strain 5 are Stenotrophomonas sp. N3, Exiguobacterium sp. N4, Exiguobacterium sp. Ch3, Cellulosimicrobium sp. A8 and Klebsiella sp. LFSM2, respectively.


Introduction
Biodegradable plastics are gaining importance; as the problems of using synthetic plastics like solid waste management, increment of CO2 in the atmosphere, being non-dismantled in soil for longer time and depletion of non-renewable resources could be controlled by using them [17]. This can be realized by the huge increase in bioplastic production, which is expected to touch 1.7 million tons by 2015 (http://www. chem.umn.edu/csp/pdfs/CWJune2012%20article.pdf). Wide varieties of microbes like bacteria, cyanobacteria, plants and yeasts have been reported to synthesize and accumulate PHA [7]. Low yield was observed from plant cells. They are able to produce <10% (w⁄w) of PHA while bacteria canproduce as high as 90% (w⁄w) of the dry cell mass [2]. It is believed that about 300 different prokaryotic microorganisms exhibit the phenomenon of intracellular PHA production and many of them, such as Ralstoniasp., Bacillus sp., Pseudomonas sp. and Alcaligenessp.etc. produce PHAs naturally [14]. All Gram positive and negative bacteria, Archaea, halophiles bacteria are able to produce PHA [28]. Out of these, Bacillus has been reported as the first genus to accumulate PHAs while Haloferaxmediterranei has the ability to accumulate PHA in large amounts.
Biosynthesis of PHA is activated when there is reduction nofnutrients essential for growth, such as magnesium, sulfur, nitrogen or phosphorus but additional accessibility of carbon source [20,30]. Under nutrient depletion conditions growth rate of bacteria slows down and they start storing their carbon reserves and energy as PHA granules, inside their cells [10] mainly in cytoplasm as water-insoluble granules [29]. More than 150 identified polyhydroxyalkanoates constitute short-chain length and mediumchain length monomers [9,11]. Several PHAs like PHB and Poly(3HB-co-3HV) are produced industrially now a days and one of the examples is KANEKA Biopolymer AONILEX [18]. By structure, PHA granules are subcellular complexes which consist of a polyester core, regulatory proteins and associated proteins such as phasins, PHA synthases and PHA depolymerases [11].
Several carbohydrates including different sugars like glucose, fructose, sucrose, maltose, cellobiose, arabinose, propionate, glycerol, and mannitol has been used as carbon source for PHA accumulation in Bacillus sp. [21]. Production cost of PHA remainshigher as compared to conventional plastics when these sugars are used as carbon source. PHA production was also checked by using fatty acids [23]. The production cost can bereduced by trying to produce PHAs from plant oils which are more proficient carbon source [4]. So, now days, plant oils are being used industrially as a potential feedstock for PHA accumulation e.g., Danimer Scientific, USA is using Cold pressed canola oil to produce mcl-PHA (Nodax® PHA) and Kaneka Corporation, Japan is using Plant oils to produce PHB-PHHx (AONILEX®).Different plant oils like linseed, soybean, cottonseed, oilseed radish, and peanut oils have been reported for bioplastic production [13]while this study deals with canola and mustard oil. Canola (Brassicanapus) is included in oil seed crop with protein contents of 30-45% (w/w) and oil content of 40-50% (w/w) in defatted meal [12] and considered to be a cheap carbon source for bioplastic production. Mustard oil is considered to be an antibacterial essential oil which inhibits bacterial growth by disrupting the membrane of bacterial cells [5].
In the present study, five bacterial strains were analyzed to produce PHA using three different carbon sources. The goal of this study was to evaluate a) how different carbon sources affect the quantity and quality of produced PHA helping to produce good quality PHA with cheap carbon source and b) how mustard oil affects bacteria in terms of PHA production despite its antibacterial nature.

Utilization of Various Carbon Sources for PHA Production by Bacteria
Reference carbon source i.e., glucose and experimental carbon sources i.e., canola (brand name CanOlive) and mustard oil (brand name Sarsona by Marhaba products) were diluted, adjusted to pH 7.0, filtered (0.45 µm) and added to PHA production medium broth (2.0 g/L (NH4)2SO4,13.3 g/L KH2PO4, 1.2 g/L MgSO4.7H2O, 1.7 g/L Citric acid, 10.0 mL/L trace element solution) in such a way that final concentration in solution was 2% i.e., 20 g/Lfor all three carbon sources while a lower concentration (1%) was also used in case of mustard oil due to its antibacterial activity so that bacteria not able to grow at higher concentration (2%) may get a chance to grow at lower concentration (1%). All five strains were inoculated and grown in nutrient rich medium (yeast extract 5 g/L, NaCl 2.5 g/L and trypton 8 g/L) to set up pre-culture via incubating at 200 rpm and 37°C. Once the optical density was reached to 0.5 at 600 nm (approx. within 8 h), 3 mL (3% v/v) pre-culture of each bacterial strain was inoculated into four flasks each containing 97 mL PHA production medium but with different carbon source (three containing glucose, canola and mustard oil at 2% final concentration and one containing mustard oil at 1% final concentration). All flasks were incubated at 37°C with 200 rpm speed and samples were collected after every 24 h from 0 to 96 h to analyze biomass and PHA.

PHA Extraction
Cells were harvested from collected samples by centrifugation (20 min at 6000 × g) followed by washing (with acetone) and drying of these cells under vacuum at room temperature. The dried biomass was used for PHA extraction using chloroform extraction method. In this method, 100 mL chloroform and 100 mL 6% sodium hypochlorite were used to homogenize about 12 g of biomass powder. The obtained mixture was shaken for 3 h at 300 rpm and 37°C followed by centrifugation for 20 min at 3000 × g. PHA containing bottom layer was collected. Ice cold methanol and chloroform (methanol: chloroform = 9:1) was used to precipitated PHA. Finally, the obtained precipitates of PHA were filtered by simple filtration followed by evaporative drying at 60°C [8]. PHA content was obtained by dividing weight of extracted PHA by cell dry weight (CDW) and was expressed in terms of percentage of CDW.

PHA Characterization by FTIR Spectroscopic Analysis
For FTIR analysis, strain 2 was grown with glucose (2%), canola (2%) and mustard oil (2%), each in three different flasks, and cells from each flask were collected after 72 h by centrifuging culture at 4000 × g for 20 min. Cell pellet was washed, lyophilized, mixed (5-10%) with KBr (90-95%) and grinded well to make a transparent pellet. The obtained pellet was processed in M-series MIDAC FT-IR processing system (Model-M2000, Serial-725) with frequency range of 500-5000 cm -1 . Different peaks were obtained after data analysis (GRAM/AI software) depicting relative absorbance intensities of ester carbonyl band at 1740 cm -1 and keto carbonyl band at 1715 cm -1 . Relative indices and crystallinity of PHA produced by strain 2 were determined by using following formulas [3]: Keto carbonyl bond index = I1715 / I1465 Ester carbonyl bond index = I1740 / I1465 Polymer material was also evaluated in terms of its percentage crystallinity based on method suggested by Zerbi [31] where Ia and Ib are the absorbance values determined from the bands at 1474 and 1464 cm -1 or from 730 and 720 cm -1 , respectively.

Time Profiling of Biomass and PHA Production
Time profiling for PHA production was done for all strains with all three carbon sources by analyzing samples at equal intervals of 24 h up to 96 h (Figs. [1][2][3][4][5]. Maximum biomass was 2.5 g/L produced by strain 5 with 2% glucose at 72 h ( Fig. 5(A)) while maximum PHA was 70.3% produced by strain 2 with 2% glucose at 48 h ( Fig. 2(A)).  Fig. 2) to be shifted with the shift of carbon source (Fig. 6, Tab. 1). Shifting was also observed with crystallinity with following trend; Glucose > Canola oil > Mustard oil (Fig. 7).

Discussion
Bioplastics, in comparison with synthetic plastics, have renewability, sustainability, biodegradability and eco-friendly behavior, because of which these naturally occurring plastics are becoming popular day by day [25]. Till now many different carbon sources have been utilized to get best yield of PHA. Some carbon sources are supposed to be good for PHA production but are much expensive e.g., ethanol which is expensive due to its volatility [25]. Plant oil for PHA production is said to be an economically viable source due to cheapness of plant fatty acid mixtures rather than oily purified acids.
For the estimation of PHA production, PHA detection agar supplemented with Nile blue A dye was used. Nile blue A is responsible to give fluorescence to PHA producing strains under UV illuminator (302-365 nm). It is difficult to discriminate between PHA negative and PHA positive strains of some Gram positive bacteria such as Bacillus megateriumor Rhodococcusruber [24] so Sudan Black B staining [22] was also carried out. Moreover, granules were also seen by fluorescent microscopy with acridine orange. All the experimental strains were positive for PHA production due to the presence of PHA granules in them, seen by microscope.
Five genetically identified strains (Stenotrophomonassp.N3, Exiguobacteriumsp. N4, Exiguobacteriumsp. Ch3, Cellulosimicrobiumsp. A8 and Klebsiellasp. LFSM2) were used in this research work. Their microscopic analysis revealed that Stenotrophomonassp.N3 and Klebsiellasp. LFSM2 are Gramnegative while all others are Gram-positive due to variable composition of cell wall. These bacterial strains were further analyzed for their biochemical characterization. All of the strains were lipase positive i.e., they catalyze the hydrolysis or breakdown of the fats or lipids due to the presence of "lipase" [26]. Different microorganisms showed different growth patterns with different carbon sources depending upon ease of utilization. In this research work, glucose (2%) was used as a standard while canola oil (2%), mustard oil (2%) and mustard oil (1%) were used as experimental carbon sources to evaluate their utilization by bacterial strains. From all five strains, Stenotrophomonassp.N3, Exiguobacterium sp. Ch3 and Klebsiellasp. LFSM2 showed maximum growth with glucose, 1.91, 2.34 and 2.07 O. D at 600 nm respectively, while Exiguobacteriumsp. N4 and Cellulosimicrobium sp.A8 showed maximum growth with canola oil, 2.02 and 2.01 O. D at 600 nm respectively (data not shown). With mustard oil, all strains showed lesser growth with 2% concentration of it as compared to that of 1% due to more inhibitory effect with more concentration of mustard oil. This attribute of mustard oil can be used to control many pathogenic organisms [5].The purpose of using mustard oil was to check that either mustard oil triggers the bacterial organism to produce PHA or not along with growth inhibition as PHA is produced under harsh environment [15].
Among different carbon sources, it was observed that glucose (2%) produced maximum 70.3% PHA with Exiguobacteriumsp. N4 and 2.5 g/L biomass with Klebsiellasp. LFSM2; canola oil (2%) produced maximum 62.4% PHA with Exiguobacteriumsp. Ch3 and biomass 1.82 g/L with same strain; mustard oil (1%) produced maximum 50.7% PHA with Cellulosimicrobiumsp.  [27]. Our strains can touch these figures by further optimizing the conditions and putting some genetic modifications in them. Most organisms grow happily in glucose unlike other carbon sources so the maximum growth obtained was that with glucose. When there will be high number of cells then the overall percentage of PHA will also be higher which is obvious from our results.
The PHA of all strains was extracted in little glass vials and macroscopic analysis was done. Exiguobacterium sp. N4 produced both main types of PHA i.e., thread-like and granular, with different carbon sources unlike other strains (data not shown). Once high yield and seemingly better quality of PHA was noted with Exiguobacterium sp. N4, the next aim was to do detailed chemical analysis of PHA produced by this strain. For this purpose, FT-IR analysis of PHA, produced by using 2% glucose, canola and mustard oil, was performed. FT-IR spectra of biomass ( Supplementary Fig. 2) have bands which characterize cellular macromolecules like lipids, proteins, carbohydrates, nucleic acids and PHAs. Spectrum of FTIR in actual, is a chemical profile of a sample. With glucose, FTIR spectra of Exiguobacteriumsp. N4 revealed sharp absorption bands at 1110 cm -1 , 1411 cm -1 and 1650 cm -1 which correspond to ester carbonyl group (C=O), ester group stretching C-O and amine carbonyl group (C=O) respectively and correspond to the exact peak positions for PHBs. With canola oil, it showed peaks at 1172 cm -1 (C=O), 1465 cm -1 (aromatic ring stretch), 1643 cm -1 (C=O), 1743 cm -1 , (C=O stretch) and 2861 cm -1 (-CH2) wavenumber and each peak represents particular PHB functional group (showed within respective brackets). Peaks at 1743 cm -1 and 2861 cm -1 also showed that Exiguobacteriumsp. N4 can accumulate exact PHB polymer as well as PHB copolymer (PHB-co-PHV) by using canola oil. Particular peaks at 2931.1 cm -1 and 1279.5 cm -1 shown by poly(3HB-co-3HV), corresponds to typical poly(3HB-co-3HV) which is very close to the value shown by our strain. With mustard oil (2%), it showed peaks at 1180 cm -1 , 1743 cm -1 and 2861 cm -1 which correspond to ester carbonyl group (C=O), exact PHB ester carbonyl group (C=O) and aliphatic compound group (CH2) respectively. While shifting of functional group has also been noticed as the carbon source changes from glucose to plant oils. Production of poly (3HB-co-3HV) was evident by the presence of peaks at 2861 cm -1 positions in our strains (Tab. 1). These observations due to the complex nature of plant oils which make bacteria to switch another metabolic pathway as PHA synthesis is directly associated with the carbon source [23].
Evaluation of diverse production of PHA can be done by comparing the FT-IR results by Exiguobacteriumsp. N4. Our results evaluate that canola oil is responsible for producing the PHA with wide variety of functional groups i.e., PHB and PHB-co-PHV. Although, glucose produced maximum amount of PHA but that plastic was containing just homopolymer (PHBs) but not any copolymer (PHB-co-PHV).
It has been reported that a polymeric material which is a blend of PHBs and PHVs is less crystalline, stronger and flexible than that containing just PHBs [19]. Hence, copolymer material (like PHB-co-PHV) is considered to be more suitable for practical applications. In our study, only PHB was produced with glucose while co-polymer was produced with both canola and mustard oil but ratio of PHB to PHB-co-PHV is higher with canola unlike mustard oil so crystallinity of mustard oil is lower as compared to canola. Change in indices and crystallinity of bio-plastic with change of carbon source was also observed ( Fig. 6 and Fig. 7). This change is considered to be due to the difference in composition of produced bioplastics. Maximum crystallinity is with glucose (88.7%), then canola (88%) and finally with mustard oil (84%). This change in crystallinity tells about the nature of bioplastic produced by using these carbon sources which can be explained on the basis of FT-IR results. Although, crystallinity difference among carbon sources is not so much high but it could be made considerable via changing the growth conditions like temperature, pH, nutrients, etc.

Conclusion
Although maximum amount of PHA was produced by utilizing glucose but the type of PHA was not good. It means that it was highly brittle with maximum crystallinity. Moreover, bacteria under research not only produced PHBs but also produced PHBs and PHVs copolymer by utilizing oils which more strongly states the potential of these bacteria to produce PHA of interested quality. It also showed that utilization of oils not only makes bioplastic material (PHA) economical but also improves its market. The findings of this research work recommend the use of canola and mustard oil as a cheap carbon source for bioplastic production.Future prospects include the optimization of these bacterial strains, either by genetic or growth environment modification, for large scale volumetric production of this Eco-Green PHA with less crystallinity using canola and mustard oil as cheap carbon sources.