Many biopolymers have been synthesized by the living matter. On the basis of their chemical structures, different classes of these biopolymers were distinguished such as nucleic acids, polyamides, polysaccharides, polyoxoesters, polythioesters, polyanhydrides, polyisoprenoides and polyphenols, etc. (Tan et al., 2014). PHAs (a type of natural polyoxoesters) have been produced by different bacteria as energy storage compounds and attracted much attention because of their structural diversity and close similarity to plastics. PHAs accumulation has been known as an inherent response to the stress conditions faced by bacterial cells. The in vitro studies to understand the controlled biosynthesis of PHA were carried out by exposing bacteria to nutrient limitations, due to which they switched their metabolic pathways and initiated PHA production as their carbon and energy reserves (Verlinden et al., 2011 and Borrero-de et al., 2014).
The trait of PHA accumulation has been widespread among the prokaryotic microorganisms including in around 70 bacterial and archaeal genera. The most commonly studied genera were Alcaligenes, Aeromonas, Bacillus, Burkholderia, Corynebacterium, Cupriavidus, Halomonas, Pseudomonas and Rhodopseudomonas. Cupriavidus necator was the most researched and most frequently used bacteria in PHA biosynthesis studies. Data published in most of the literature in the last 15-20 years referred to it as Ralstonia eutropha (Jendrossek et al., 2009).
Among bioplastics, PHAs were only bioplastics completely synthesized by microorganisms. These biopolymers have been synthesized by over 30% of soil-inhabiting bacteria (Wu et al. 2000). Different bacteria in activated sludge, deep seas and in extreme climates were also found to be capable of producing PHAs. Depending on the growth conditions and bacterial species, homopolymers, random copolymers and block copolymers of PHA have been produced at laboratory scale (Wang et al., 2015). Some of them have attracted industrial attention and were commercialized in the past few decades. Homo and copolymers of PHAs (Table1) have varied in the type and proportion of monomers and been typically random in sequence (Li et al., 2015).
Table 1: Homo and copolymers of PHAs (Li et al., 2015).
Conventional abbreviations (short) Full abbreviations Structures
PHB P(3HB) Homopolymer
PHV P(3HV) Homopolymer
PHBV P(3HB-co-3HV) Copolymer
PHBHx P(3HB-co-3HHx) Copolymer
PHBO P(3HB-co-3HO) Copolymer
PHBD P(3HB-co-3HD) Copolymer
PHBOd P(3HB-co-3HOd) Copolymer
Not only the type of PHA but also yield of biopolymers produced was found to be dependent on the type of microorganisms used, choice of carbon source and fermentation conditions. In previous studies it was suggested that under different carbon sources and fermentation conditions, the metabolism of bacteria not only maximized the utilization of various carbon substrates for PHA biosynthesis but also enhanced the robustness of bacterial strains providing nutritional advantages over standard carbon substrates (Jendrossek et al., 2009; Verlinden et al., 2011 and Wang et al., 2015).
Present work on biosynthesis of biopolymer has been initiated with the available information on PHA accumulating bacteria Cupriavidus necator. The Cupriavidus necator strain known as MTCC 1285 procured from IMTECH Chandigarh was compared with PHA-producing soil bacteria. Different waste carbon sources and fermentation condition were optimized for maximum biosynthesis of biopolymer taking economic considerations into the account.
2.1.1 BIOSYNTHESIS OF PHA
PHA has been synthesized by either chemical or biological methods (He et al. 1999). Biosynthesis of PHA in microorganisms has provided a tool to lead synthesis of much higher molecular weight biopolymers. Being a biological process biosynthesis of PHA did not allow significant control over the monomer structures in the PHA biopolymers; although the specificity of PHA polymerase (or PHA synthase) has been known to influence the monomers incorporated into the polymer chain (Chen et al., 2004). Since biosynthesis of PHA in the laboratories has been carried out by microorganisms grown in an aqueous solution known as fermentation media (containing sustainable carbon resources such as starch, glucose and even nutrients in waste material) under controlled temperature and atmosphere pressure, it has been considered as sustainable and environment friendly approach, especially when petroleum as a non-sustainable resource for plastics production has been depleting quickly (Vo et al., 2015).
Most PHAs have been synthesized by prokaryotic microorganisms, although transgenic plants also known to produce PHA (Bohmert et al., 2002). Oligomers of PHA were discovered in eukaryotes as well, such as tissues and blood of human and animals (Reusch, 1989). Optically active biological linear polyester polyhydroxy-3-butyrate [P(3HB) or (PHB)] was most common PHA found in nature and represented by model bacteria Cupriavidus necator. PHB was insoluble in water and exhibited a high degree of polymerization that ranges from 105 to approximately107. The biosynthesized PHB was therefore perfectly isotactic and upon extraction from the microorganisms showed a crystallinity of about 55-80% with a melting point at around 180Â°C.
Biosynthesis of PHA has required three essential enzymes, Î²-ketothiolase (phaA), acetoacetyl-CoA reductase (phaB) and PHA synthase (PhaC). These enzymes have been involved in the biosynthesis of pre-cursor molecules (R)-3-hydroxyacyl-CoA and subsequent enzymatic conversion into PHA (Jain et al., 2014).
Figure 1: Metabolic pathway involved in the synthesis and breakdown of PHB in Cupriavidus necator (Madisonet al., 1999).
The central molecule for P(3HB) synthesis has been known as acetyl-CoA. Generally, carbohydrates have catabolized to pyruvate which then been converted to acetyl-CoA through dehydrogenation. During the balanced growth, acetyl-CoA has been terminally oxidized to CO2 through tricarboxilic cycle (TCA) generating ATP and reducing equivalents such as NADH, NADPH and FADH2. In the TCA cycle, biosynthetic precursors have been made necessary, e.g. protein synthesis. Though, when deficiency in nutrients occurred, NADH and NADPH has been started to accumulate resulting in protein synthesis inhibition. At this point, acetyl-CoA has been channelled for P(3HB) synthesis as shown in Figure 1 (Lee et al., 1994 and Madisonet al., 1999). Enzymes and steps involved in the synthesis of biopolymer have already been discussed in chapter 1.
2.1.2 MODELS FOR THE BIOSYNTHESIS OF PHA INCLUSIONS
The type of PHA synthesized by bacteria was primarily dependent on the carbon substrate utilized and type of organism (Tan et al., 2014). Biosynthesis of PHA inclusions has been mediated by the presence of (R)-3-hydroxyacyl-CoA thioesters and PHA synthase upon availability of carbon substrates. During the polymerisation process the PHA synthase has been found covalently attached to the growing polymer chain until metabolic polymerisation has terminated (Grage et al., 2009). Though the precise mechanism of PHA inclusion biosynthesis was unknown, but currently two in vivo models for the biosynthesis of PHA inclusions were described: (a) Micelle model and (b) Budding model (Figure 2).
The âmicelleâ model was based on the hypothesis that the initially soluble enzyme was believed to dimerise when polymerisation was initiated by the presence of substrate and transformed into an amphipathic molecule. This molecule underwent a self-assemblization and therefore the growing hydrophobic PHA chain was accumulated into a micelle-like structure (Grage et al., 2009 and Rehm, 2007). Phospholipids and other inclusion associated proteins then gradually incorporated with the growing micelle-like structure (Grage et al., 2009). However, this model was supported by PHA inclusion formation in vitro only (Rehm, 2006).
Figure 2: Models for biopolymer inclusion self-assembly. 1 and 2 indicate possible routes during in vivo assembly (Rehm, 2006)
The âbuddingâ model on the other side suggested that soluble enzyme localized to the inner surface of the cytoplasmic membrane either innately or as soon as PHA chain occurred from the enzyme (Thomson et al., 2010). In this case, the growing polyester chain was synthesized into the inner membrane space and continued to grow into a PHA inclusion surrounded by a mono-phospholipid layer (Rehm, 2007). Inclusion associated proteins were also thought to be incorporated into the phospholipid monolayer until it was finally budded off the membrane into the cytoplasm (Rehm, 2007 and Thomson et al., 2010). Recently evidences were in the favour of the budding model (Rehm, 2006 and Rehm, 2007).
A third model was also suggested based on PHB inclusion biosynthesis in Cupriavidus necator (Tian et al., 2005). Interestingly Tian et al. have found newly emerging inclusion only from the centre of the cell and at unknown dark staining mediation elements (Tian et al., 2005). These emerging beads were found to be localized at the mediation element and therefore were not seen randomly distributed. This model was correlated with the other two models mentioned above. These mediation elements were suggested to act as scaffolds and provided a site for initiation of inclusion biosynthesis. This proposed model was not well supported (Rehm, 2007).
2.1.3 PREVIOUS DEVELOPMENTS IN THE FIELD AND PRESENT STUDY
Most of the PHA-producing bacteria were Gram-negative. Scl-PHA has generally been produced by bacteria from Azohydromonas, Burkholderia and Cupriavidus species (Tan et al., 2014). These bacterial species have been reported to produce up to 88% cell dry mass (%CDM) of PHB. Cupriavidus necator H16 have been suggested to exhibit well characterized heterotrophic and autotrophic PHA biosynthesis. This bacterium was able to use diverse carbon substrates such as CO2, glucose, fructose, hydroxyhexanoic acid, corn and other vegetable oil for PHB production. Gram-negative methylotrophs such as Methylobacterium extorquens and Paracoccus denitrificans were also found to produce biopolymer using different carbon substrate. The biosynthesis of mcl-PHA was usually reported in Pseudomonas sp., such as Pseudomonas putida, Pseudomonas marginalis Pseudomonas mendocina and Pseudomonas oleovorans (Sun et al., 2007). Pseudomonads have also known for their bioremediation properties and been successfully applied in the treatment of polluted effluents, exhaust gases and soil. Other Gram-negative extremophilic bacteria such as halophilic Halomonas boliviensis and thermophilic Thermus thermophilus were also found to produce biopolymers using waste effluents in high salt concentrations or temperatures (Pantazaki et al., 2009).
Among Gram-positive genera Bacillus, Clostridium, Caryophanon, Corynebacterium, Micrococcus, Microcystis, Microlunatus, Rhodococcus, Streptomyces and Staphylococcus were known for PHA biosynthesis (Lu et al., 2009). Scientists have also discovered PHA accumulation in haloarchaeal species, especially in Haloferax, Halalkalicoccus, Halobacterium, Haloarcula, Halobiforma, Halopiger, Halococcus, Haloquadratum, Halorhabdus, Halostagnicola, Halorubrum, Haloterrigena, Natrinema, Natrialba, Natronobacterium, Natronomonas, Natronococcus and Natronorubrum (Han et al., 2010 and Danis et al., 2015). These microorganisms were reported to produce scl-PHA homopolymer comprising either 3HB or 3HV monomers, or scl-PHA heteropolymer comprising both 3HB and 3HV monomers from glucose, volatile fatty acids and other carbon sources such as starch, whey hydrolysate and crude glycerol. Most of haloarchaea were found to accumulate PHA at low cellular contents ranging from 0.8 to 22.9 % of cell dry weight only (Han et al., 2010).
Among different microbially biosynthesized homopolymer PHA, the first biopolymer discovered was PHB. Few studies related to other non-PHB homopolymers such as poly(4-hydroxybutyrate) (P4HB) (SteinbÃ¼chel et al., 1994), poly[(R)-3-hydroxyvalerate)] (PHV) (SteinbÃ¼chel et al., 1995), poly[(R)-3-hydroxy-co-(R)-5-phenylvaleric acid] (Anderson et al., 1990), poly[(R)-3-hydroxyhexanoate] (Anderson et al., 1990), poly[(R)-3-hydroxyheptanoate] (Anderson et al., 1990; Chung et al., 1999 and Chen et al., 2009), poly[(R)-3-hydroxyoctanoate] (PHO) (Anderson et al., 1990) and poly[(R)-3-hydroxynonanoate] (Anderson et al. 1990 and Chung et al. 1999) were also reported (Table 2). Many of these homopolymers have not yet been fully characterized.
Table 2: PHAs homopolymers (Anderson et al. 1990 and Chung et al. 1999)
Chemical name Abbreviation R group
Poly(3-hydroxypropionate) P(3HP) Hydrogen
Poly(3-hydroxybutyrate) P(3HB) Methyl
Poly(3-hydroxyvalerate) P(3HV) Ethyl
Poly(3-hydroxyhexanoate) or poly (3-hydroxycaproate) P(3HHx) or P(3HC) Propyl
Poly(3-hydroxyhexanoate) P(3HH) Butyl
Poly (3-hydroxyoctanoate) P(3HO) Pentyl
Poly (3-hydroxynonanoate) P(3HN) Hexyl
Poly(3-hydroxydecanoate) P(3HD) Heptyl
Poly(3-hydroxyundecanoate) or P(3HUD)or P(3HUd) Octyl
Poly(3-hydroxydodecanoate) P(3HDD) or P(3HDd) Nonyl
Poly(3-hydroxyoctadecanoate) P(3HOD) or P(3HOd) Pentadecanoyl
Poly(4-hydroxybutyrate) P(4HB) Hydrogen
Poly(5-hydroxybutyrate) P(5HB) Methyl
Poly(5-hydroxyvalerate) P(5HV) Hydrogen
In many cases, short-chain-length (scl) PHA copolymers were also reported consisting of C3 and C5, that included poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyvalerate] (PHBV) (Alderete et al., 1993), poly[(R)-3-hydroxybutyrate-co-4-hydroxybutyrate] (Saito et al., 1996), poly[(R)-3-hydroxypropionate-co-(R)-3-hydroxybutyrate] (Shimamura et al., 1994) and poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyvalerate-co-4-hydroxybutyrate] (Chen et al., 2007). Many Pseudomonas sp. were found to accumulate mcl PHA copolymers containing C6 to C12 monomers as well. Typical mcl PHA have been known as poly(hydroxybutyrate-co-hydroxy valerate) (PHBV), poly(hydroxybutyrate-co-hydroxyhexanoate) (PHBHx), poly(hydroxybutyrate-co-hydroxyoctadecanoate) (PHBOd) and poly(hydroxybutyrate-co-hydroxyoctanoate) (PHBO) (Lageveen et al., 1988).
Copolymers of scl and mcl PHA have showed flexible mechanical properties and now considered as the preferred materials for different application development. A successful example was the PHBHHx and produced of an industrial scale (Lomas et al., 2012). US based company Procter & Gamble (P & G) has trademarked their scl and mcl PHA copolymers of C4 and C6 to C12 as NodaxTM (Noda et al. 2009). Different small companies have currently been producing bacterial PHA. For example, PHB Industrial (Brazil) have been making PHB and PHBV (HV = 12 %) with 45 % crystallinity, using sugar cane molasses as a carbon source (Lomas et al., 2012).
It was already proven by the industrial PHA production that the most efficient processes were never able to compete with petroleum-based polymers on the basis of price. Around 50% or more of PHA production cost was estimated to carbon sources. Therefore, it was necessary to substitute pure substrates with cheaper carbon sources such as waste and surplus materials. This has encouraged the present study of PHA biosynthesis from cheaper carbon sources and to lower PHA cost. In this study utilization of waste products as carbon sources has presented the advantage of both management of waste products and the production of value-added biopolymer.
Soil has been known as one of the richest source of common and unknown, both microorganisms (Schallmey et al., 2013). A continuous search of microorganism and their potential to produce products for human welfare has been required, therefore in the present investigation bacteria from soil were isolated and compared with well characterized and known PHA producer Cupriavidus necator MTCC 1285. The various stress conditions were known to trigger PHA production; therefore, the main challenge was the optimization of fermentation parameters for efficient and effective bioconversion of carbon substrates into PHA (Vo et al., 2015). Parameters such as pH, temperature and agitation rate were required to be fine-tuned in order to maximize the bioconversion process and therefore were studied in the present investigation.
The crystalline nature of PHB, its brittleness and low flexibility, long degradation rate under physiological conditions and poor processability has limited its potential in different applications. Blending of PHB with other polymers was reported to improve the inherent brittleness and to reduce the high production cost of these microbial polyesters as well. Crystallization, physical properties and biodegradation behaviour of the microbial polyester has been signiï¬cantly affected by the nature of the blend partner component (Jain et al., 2015); therefore in this research work the effect of FDA approved blending agents on biopolymer films were also studied.
The work carried out has been outlined as mentioned below:
o Isolation of bacteria and comparison of isolates with known Cupriavidus necator MTCC 1285 strain for production of bioplastics
o Optimization of the waste based medium and fermentation conditions for biopolymer production
o Preparation of the bioplastic film by taking different concentrations of blending agent (ethyl cellulose and cellulose acetate butyrate)
The study was an attempt to study soil bacteria for their potential of biosynthesis of PHB and to check the waste carbon substrates such as kitchenâs waste edible oils, by-products from pulp & juice industry like apple pomace and agro-allied wastes as analogous carbon substrate to the standard carbon source, i.e. glucose in fermentation. The present investigation has helped to overcome the conundrum of cost-effectiveness leading to an effective commercial biosynthesis of biopolymer.
2.2 MATERIALS AND METHODS
188.8.131.52 SOIL SAMPLES
In the present investigation, soil samples from 3 different locations (Table 3) were collected and transported to the Lab into sterile plastic containers. All three samples were processed immediately for isolation of bacteria (Willey et al., 2008).
Table 3: Sample collection sites
Sample A Sample B Sample C
RGPV medicinal garden Agricultural soil Sludge area
184.108.40.206 WASTE FRYING OIL (SOYBEAN OIL)
Waste frying oil was collected from local snacks vendors / hotels near railway station, Bhopal.
220.127.116.11 APPLE POMACE
Apple pomace was prepared using fresh apples purchased from local market.
18.104.22.168 AGRICULTURAL WASTE
The sample was provided by School of Biotechnology, RGPV, Bhopal.
22.214.171.124 MODEL BACTERIAL STRAIN
Model bacterial strain Cupriavidus necator MTCC 1285 (former Ralstonia eutropha) was obtained from MTCC, IMTECH, Chandigarh.
2.3.1 MEDIA PREPARATION
Principle: The media preparation has been carried out based on the requirements of different microorganisms. From isolation to product extraction the microorganisms have required different environment (called culture media) to act upon. The culture media has been varied in form and composition depending on the species to be cultured and known to provide nutritional support for their growth (Willey et al., 2008).
Procedure: Media components used in the isolation of microorganisms and fermentation studies were purchased from HiMedia Laboratories, India. Different media components for nutrient broth (NB), nutrient agar (NA), tryptone soya broth (TSB), tryptone soya agar (TSA), basal salts medium (BSM), trace element solution were weighted according to the compositions given in annexure I and sterilized by autoclaving at 15 lbs pressure. Sterilized media then poured aseptically into sterile glass wares (petri dish/test tubes/flasks) and allowed to solidify/cool. These media were used for isolation, growth and fermentation of microorganisms (Verlinden et al., 2011).
2.3.2 SERIAL DILUTION OF SOIL SAMPLES
Principle: Serial dilution has been used to determine number of bacteria per unit volume in original culture (determination of culture density in cells per ml). Once culture has been diluted, it was spreaded on the agar plates. Agar plates have been allowed for individual bacterial cells to be separated spatially with low probability of having two cells very close to each other. Number of colonies observed has been therefore a direct measure of number of bacteria spreaded on the surface of plate (Willey et al., 2008).
Procedure: 1gm of soil was taken and mixed with 10 ml of distilled water. And then 9 sterile test tubes were taken with 9 ml of distilled water in each tube. 1ml of soil sample was transfer serially to all tubes to make a dilution 10-1 to 10-9. 0.1ml of solution from each tube was plated with a glass spreader on the petri plates containing nutrient agar medium. Plates were incubated for 2-days at 30oC and then checked for microbial colonies (Redzwan et al., 1997 and Bhuwal et al., 2013).
2.3.3 PURE CULTURE
Principle: Pure culture has been described as a method of multiplying microbial organisms by letting them reproduce in predetermined culture media under controlled laboratory conditions. Culture medium has been inoculated with desired bacteria and then incubated at best suitable temperature for their growth (Ienczak et al., 2011 and Willey et al., 2008).
Procedure: Well-separated single colonies, from each serially diluted spread plates were picked up and re-streaked onto fresh nutrient agar plates. Plates were incubated at 30oC for 2 days. After incubation grown culture were cheeked under microscope for purity at 1000x. This procedure was repeated until pure cultures were obtained. Pure cultures were sub-cultured in every 15 days (Redzwan et al., 1997 and Verlinden et al., 2011).
2.3.4 MORPHOLOGICAL AND BIOCHEMICAL CHARACTERIZATION
a) Morphological characterization
Principle: In order to obtain the pure culture of organism, the isolated colonies has been aseptically studied for the characteristics features such as shape, size, elevation, surface, edges, colour, structure, degree of growth, etc. (Ienczak et al., 2011 and Willey et al., 2013).
Procedure: From the incubated plates, bacterial isolates were selected by considering their morphology & pigmentation and studied under a microscope at 100Ã for their characterization. Selected isolates were sub-cultured in every 15 days and stored at 4ï,°C for further use (Willey et al., 2008 and Verlinden et al., 2011).
b) Biochemical characterization
Principle: Biochemical characterization was carried out with HiMViC Biochemical Test Kit (HiMedia). HiMViC Biochemical Test Kit has been designed for four conventional biochemical tests (Indole, Methyl Red, Voges Proskauerâs Test and Citrate utilization test). These tests were based on the principles of pH change and substrate utilization. On incubation, the organism underwent metabolic changes, which have been indicated as a color change in the media. All necessary reagents like Kovacâs Reagent, Methyl Red and Barritt Reagent A & B were provided with Kit (HiMedia).
Procedure: Loopful of the culture was inoculated into nutrient broth and incubated overnight at 37ï,°C, and then the density of culture was adjusted to 0.1 O.D. at 620 nm. Each well (oval compartments) of HiMViC strip was inoculated with 50 Âµl of the culture inoculum and incubated at 37ï,°C for 24hrs. After incubation of 24 hrs 2drops of Kovacâs Reagent, Methyl Red and Barritt Reagent A & B were added into well no. 1, 2 and 3 respectively. Well no 4 was observed directly without addition of any reagent (HiMedia- HiMViC kit protocol).
c) Gram staining
Principle: Gram staining has been used to differentiate bacteria by the chemical and physical properties of their cell walls by detecting peptidoglycan (present in a thick layer in gram-positive bacteria). In a Gram stain test, gram-positive bacteria has been retained the crystal violet dye, while a counterstain (commonly safranin) added after the crystal violet has been known to give all gram-negative bacteria a red or pink color (Willey et al., 2008).
Procedure: A smear of test bacteria was prepared on glass slide. The slide with the bacterial smear was heat fixed by passing it over flame several times using a forceps and then placed on the staining tray. Fixed smear was flooded with crystal violet and allowed remaining for 1 minute. Crystal violet was rinsed off with tap water and then slide was flooded with iodine solution and allowed remaining for 1 minute. Iodine solution was rinsed off with tap water and slide was flooded with decolorizer (95% alcohol) for five seconds. Decolorizer was rinsed off with tap water and slide was flooded with safranin and allowed remaining for 30 seconds. Safranin was rinsed off with tap water. Slide was dried on absorbent paper and placed in an upright position. Stained slide was then microscopically examined for categorization of bacteria under a 100x objective (Verlinden et al., 2007).
2.3.5 PHYLOGENETIC ANALYSIS OF BACTERIAL ISOLATES
a) DNA isolation of bacterial isolates
Principle: Genomic DNA isolation has been described as a DNA purification process that involved physical and chemical methods. The process has involved steps starting from cell lysis to removal of membrane lipids, proteins, RNA and finally purification of lysate to obtain DNA. After purification DNA has been stored in TE buffer or in ultra-pure water (Willey et al., 2013).
Procedure: Cells were grown in 2-5 ml nutrient broth till late log phase at 30Â°C and pelleted in microfuge (Remi, India) at 8000 rpm for 10 min. The obtained pellet was resuspended in 200 Âµl Tris- EDTA buffer containing 50 ng of RNase (Fermantas) and 400 Âµl of Sarkosyl solution (1% Sarkosyl, 0.5M NaCl and 1% SDS) and incubated at 37ÂºC with intermittent shaking after every 5 min until the solution became clear. DNA was purified by using equal volume of phenol: chloroform: isoamyl alcohol (25:24:1) and precipitated with 0.1 volume of sodium acetate (3M) and 0.6 volume of isopropanol in microfuge at 8000 rpm for 10 min. The obtained chromosomal DNA was washed with 500 Âµl of 70% ethanol for 10 min by spinning at 8000 rpm and then dried by placing open tube on bench top for 20 min. The resulting dried DNA was resuspended in 50 Âµl TE buffer. A Band for the corresponding DNA was observed on Agarose gel (Pednekar et al., 2010).
b) Agarose gel electrophoresis
Principle: The technique used for separation of proteins, DNA, RNA, etc. has been called Gel Electrophoresis. Agarose has been known as a matrix that used to trap the molecules based on their size and helped in separation. The length of DNA has been measured through comparison with a marker of known length (Willey et al., 2013).
Procedure: The first evaluation of the isolated DNA was done by agarose gel electrophoresis. 1.5% agarose gel was prepared in 0.5X TBE buffer by boiling the solution in a microwave oven. To avoid degradation of the DNA-coloring dye, ethidium bromide (Sigma) was added to a final concentration of 0.5 mg/ml after the agarose solution cooled down to less than 50oC. Warm agarose was then casted on gel plate and a comb was positioned into the solution. After the solution jellied, the plate was placed into an electrophoresis tank containing 0.5X TBE buffer and the comb was gently removed from the gel to form the slots for loading the DNA samples. DNA samples were mixed with the DNA loading dye (Fermentas) to a final concentration of 1X and loaded into the slots using a micropipette. DNA marker (Fermentas) was also loaded (1kb). Gel was run @ 120V for 90 min and then gel viewed under UV (in Gel Doc); bands were compared with the marker.
DNA concentration and purity was estimated from the optical density (OD) ratio (OD260nm/OD280nm) with a reading of between 1.7 and 1.9 using spectrophotometer (Smart Spec Plus, Biorad, USA). Concentration in spectrophotometer showed the purity and accuracy of the nucleic acids (Willey et al., 2008 and Schallmey et al., 2011).
Concentration of Double Stranded DNA ng/ÂµL =
OD Ã Dilution factor (100) Ã Conversion factor(50)
c) Polymerase chain reaction (PCR) for 16S rRNA gene amplification
Principle: The PCR has been described as a temperature based scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of enormity, generating thousands to millions of copies of a particular DNA sequence in very short duration. Principle of PCR was based on the mechanism of DNA replication in vivo. Double stranded DNA has been denatured to single stranded DNA and duplicated with the help of DNA polymerase enzyme. This process has been repeated along the reaction according to the number of cycles (Saiki et al., 1988).
Procedure: PCR was performed by DNA EngineÂ® Peltier Thermal Cycler manufactured by Bio Rad Labs. The gene amplification was accomplished in a total volume of 50 Âµl containing 25 Âµl master mix (fermantas), 10 ng DNA, 10 pmol bacteria specific primers (purchases from Bangalore Genei, India) 27F (5âGAGTTTGATCCTGGCTCA-3â) and 1385r (5â-CGGTGTGT(A/G)CAAGGCCC-3â), corresponding to Escherichia coli 16S rRNA numbering. The PCR conditions were as followed: initial denaturation (2 min at 95 ÂºC), followed by 30 cycles of denaturation (1 min at 95 ÂºC), primer annealing (1 min at 52 ÂºC) and primer extension (1.5 min at 72 ÂºC). Amplified gene products were visualized on 1% Agarose gel under UV at 254 nm (Pednekar et al., 2010).
d) Blast and phylogeny
Principle: An algorithm called âBasic Local Alignment Search Toolâ (BLAST) has been known to compare primary biological sequence information, such as the amino-acid sequences of different proteins or the nucleotides of DNA sequences (also called query sequences) with a library or database of sequences and to identify library sequences that resembled the query sequences (Thakur et al., 2008).
Procedure: The PCR amplified product was purified using a Qiagen kit (No. 28104). The recovered fragment was sequenced using ABI 3700 sequencer. The obtained sequences were entered into the ânucleotide-nucleotide BLASTâ (Algorithms: blastn, see http://www.ncbi.nlm.nih .gov/BLAST/) and phylogenetic neighbors were studied. Sequences were aligned in clustalW (1.8) and unrooted tree was prepared using Phylip (3.6). Tree was visualized with Tree View (Pednekar et al., 2010).
e) RFLP (Restriction fragment length polymorphism)
Principle: Restriction fragment length polymorphism (RFLP) has been used to differentiate variations in homologous DNA sequences. In RFLP analysis, the DNA has digested into pieces by restriction enzymes and the resulting restriction fragments been separated by gel electrophoresis according to their lengths. The fragment pattern generated has been used to differentiate species (and even strains) from one another (Willey et al., 2008).
Procedure: RFLP was performed in a total volume of 25 Âµl containing 2 Âµl of restriction enzyme (Hae III) purchased from Himedia, 6 Âµl of enzyme buffer and 10 ng of amplified DNA. Reaction mixtures were kept for overnight incubation at 37ï,°C to ensure complete digestion. Later, the DNA restriction digests (24 Âµl restriction digests + 6 Âµl Fermentas 6X loading dye) were electrophoresed using 2.4% horizontal agarose gel stained with ethidium bromide (0.5 mg/ml). The electrophoresis was carried out at 100 V for 30 minutes. 10kb DNA ladder (Fermentas) was used as molecular weight marker (Willey et al., 2013).
2.3.6 CULTURING OF CUPRIAVIDUS NECATOR MTCC 1285
Cupriavidus necator MTCC 1285 was obtained as lyophilized culture from IMTECH, Chandigarh and was revived for its future use in the present study.
Procedure: Freeze-dried Cupriavidus necator MTCC 1285 was provided in a sealed depressurized ampoule. The ampoule was break-opened carefully marking near the middle of cotton wool with the help of sharp file. Disinfection of the surface was done around the mark with alcohol. Thick cotton wool (or gauge) was wrapped around the ampoule and it was carefully broken at the marked area. Pointed top of the ampoule was removed gently to avoid release of fine particles of dried organisms into air of laboratory. Nutrient broth, tryptone soya broth and tryptone soya agar were sterilized by autoclaving at 121Â°C and 15 psi pressure for 15 min. The cotton plug was removed carefully and about 0.3 to 0.4 ml of specified medium (nutrient broth) was added to make a suspension of the culture. Frothing or creation of aerosols was avoided. The prepared suspension was added to 250 ml Erlenmeyer flasks each containing 100 ml tryptone soya broth. The flasks were incubated at 30Â°C for 24 hrs in the shaking incubator (Brunswick Scientific, USA) at 200 rpm. The suspension was also streaked on tryptone soya agar plates and kept in incubator at 30Â°C for 24 hrs. Cultures were stored at 4Â°C after achieving optimal growth and revived on tryptone soya agar after every 15 days (Verlinden et al., 2011).
Figure 3: MTCC instructions
2.3.7 RAPID SCREENING OF ISOLATE FOR PHB PRODUCTION
a) Screening using Sudan Black B stain
Principle: Sudan Black B (C29H24N6), a nonfluorescent, relatively thermostable lysochrome (fat-soluble dye) diazo dye has been used for staining of neutral triglycerides and lipids. This dye showed lipophilic nature; therefore it has been accumulated in fat globules within a cell and known to stain granules dark brown (Liu et al., 1998).
Procedure: Nutrient agar medium was sterilized by autoclaving at 121áµ’C for 15 minutes then cooled to 45áµ’C and was supplemented with 1 percent glucose. The medium was poured into sterile petriplate and allowed for solidification. The plate was divided into 4 equal parts and in each part, a bacterial isolate was spotted and incubated at 30áµ’C for 24 hours. Ethanolic solution of (0.02%) Sudan Black B (Sigma) was spreaded over the incubated colonies and petriplate was kept undisturbed for 30 minutes. Dye was washed with 96% ethanol to remove the excess stain from the colonies. The dark blue coloured colonies were taken as positive for PHB production (Legat et al., 2010).
b) Fluorescent staining with Nile Red and Acridine Orange
Principle: Nile Red, a (9-diethylamino-5H-benzo[alpha]phenoxazine-5-one) lipophilic stain has been found to exhibits properties of a near-ideal lysochrome. It was reported strongly fluorescent, but only in the presence of a hydrophobic environment and did not interact with any tissue constituent except by solution (Matias et al., 2011).
Acridine Orange has been known as a metachromatic fluorescent cationic dye. This dye has been diffused spontaneously into the membrane surrounding the microorganisms and allowed for visual detection of lipids granules (Siew et al., 2007).
Procedure for Nile Red: 1 % aqueous solution of Nile Red (Himedia) was prepared and filtered before use. Heat fixed smear of bacterial cells was stained with Nile Red solution at 55Â°C for 10 minutes in coplin staining jar. After being stained, slide was washed with tap water (to remove excess of stain) and then with 8% aqueous acetic acid for 1 minute. Stained smear was again washed with tap water and then covered with glass cover slip. Nile Red stained colonies were examined with microscope with episcopic fluorescent attachment at 460 nm excitation wavelength (Matias et al., 2011).
Procedure for Acridine Orange:
10 Âµl of 48 hr old culture of the isolate was transferred to an eppendorf tube containing 50 Âµl of Acridine Orange (Himedia) and incubated for 30 minutes at 30Â°C. After the incubation period, the culture was centrifuged at 4000 rpm, for 5 min. The pellet was collected and resuspended in distilled water. A smear was prepared on a clean microscopic slide and observed in a fluorescent microscope at 460 nm (Siew et al., 2007).
2.3.8 APPLE POMACE PROCESSING
Principle: Apple pomace, the solid remains of grapes, olives, or other fruit after pressing for juice or oil has been mainly composed of carbohydrates and dietary fibre, small amounts of protein, fat and ash and exhibited good nutrition value for different uses (Sindhu et al., 2013).
Procedure: The whole apples were cleaned, washed and churned in the grinder; from which the juice was removed and the remaining waste called apple pomace was collected. The water was added in the pomace obtained in ratio 1:2 and was heated to separate adhered pulp. The separated pomace pulp was passed through stainless steel sieve of 20 mesh. The obtained filtrate as Pulp was spreaded in the tray for drying at 55-60Â°C for 24 hrs. The dried mats obtained were further dried by scrapping and then grinding of the dried matter resulted in the pomace powder formation. Pomace powder was treated with sulfuric acid solution (0.75 wt%, pH=1.1) for 15 minutes and then was neutralized with 0.5 N NaOH solution adjusting the pH to 7.0. The solution was then filtered and autoclaved at 121ËC for 15 minutes. Sugar concentration was measured in ËBrix by refractometer and the recorded data was converted to grams of sugars, through the following equation:
SCg = SC X SG X 10
Where, SC was sugar concentration in ËBrix and SCg represented sugar concentration in gL-1, SG was specific gravity of filtrate solution and 10 was the conversion factor (Wei et al., 2011).
2.3.9 OPTIMIZATION OF MEDIA AND FERMENTATION CONDITIONS
Principle: The media optimization has been carried out by conducting experiments with varying concentration of nutrients and applying different growth conditions like temperature, pH and agitation speed. This has been necessary for obtaining a good microorganism growth i.e. biomass production and also for by-product accumulation in biomass produced (Willey et al., 2008 and Willey et al., 2013).
Procedure: Basal salt medium (BSM) was used for batch fermentation and optimized by varying the concentration (1% to 3%) of different substrates as a sole carbon source. The substrates used were apple pomace hydrolysate (APH), waste frying oil (WFO), agricultural waste (AW) and glucose as control. Fermentation parameters such as pH, temperature and agitations rates were also optimized. Varying range used for fermentation parameters were pH (6.0 to 7.5), temperature (24Â°C to 37Â°C) and agitation rate (150 to 250 rpm).
Tryptone soya broth (TSB) was inoculated from a single colony of Cupriavidus necator MTCC 1285 and incubated for approximately 24 hours at 30Â°C. Cultures were checked for purity by Gram staining and observed under a microscope at 100Ã. 25ml TSB inoculum was added to 250 ml of BSM resulting in an inoculation ratio of 10% (v/v). Incubation was carried out for 48-72 hours and the growth of bacteria was checked intermittently after 24 hrs, 48 hrs and 72 hrs. All experiments were performed in triplicate. The biomass was separated by centrifugation at 10,000 rpm for 10 minutes at 4Â°C. Selection of the optimized carbon source concentration and fermentation conditions were carried out on the basis of cell dry weight and produced PHB (Verlinden et al., 2011).
2.3.10 RECOVERY OF PHB
Principle: The modification of cell membrane permeability has allowed release and solubilization of lysate. Lysate has been treated with different solvent to extract purified material (Madkour et al., 2013).
1. Cell dry weight estimation
Fermented broth was centrifuged at 10,000 rpm for 10 min. to sediment the cells. Supernatant was discarded, washed with distilled water and recentrifuged. The washed cell pellet was dried in the oven at 40Â°C for 24 h to obtain the cell dry weight (Shamala et al., 2012).
2. Polymer recovery
The cell pellet obtained after centrifugation was ruptured by sodium hypochlorite (5%) by keeping the cells for incubation in the solution for 90 minutes at 37Â°C on a shaker to achive complete digestion of cell components except PHAs (bi-lipids and proteins were degraded). Cells were first washed with distilled water by centrifugation at 8,000 rpm at 4Â°C for 20 minutes and then with acetone: methanol (1:1) mixture to remove the impurities. Finally cells were washed again with distilled water and centrifuged to get the PHA pellet. Obtained PHA pellet was dissolved in hot chloroform (60Â°C) and transferred to petriplate. Dry powder of PHA was yielded by evaporating the chloroform by air drying at room temperature for 24 hrs. PHB yield obtained was quantified by UV spectrophotometer and confirmed by FTIR analysis (Verlinden et al., 2011 and Heinrich et al., 2012).