A Novel Insight Into the Fabrication of Polyhydroxyalkanoates from Actinobacteria Streptomyces toxytricini D2: Screening, Optimization, and Biopolymer Characterization

The aim of this research was to identify a biopolymer producing potential actinobacteria candidate from polluted soil. The Streptomyces toxytricini D2 was enumerated as potential biopolymer producer, which was isolated and characterized using 16S rRNA sequencing. The S. toxytricini D2 produced polymeric granules were categorized through FTIR, 1H NMR, and 13C NMR analyses, which confirmed that the granules were Polyhydroxyalkanoates (PHAs). The S. toxytricini D2 has the competence to produce 86.56% of PHAs (23.64 g L−1 of PHAs from 27.31 g L−1 of biomass) under optimized growth conditions: 8% of tapioca molasses, 4% of (NH4)2SO4, 8% of inoculum, pH 6.5, 30 °C and 72 h of incubation. These findings concluded that S. toxytricini D2 could be used as an efficient candidate for the mass production of PHAs polymer under optimized conditions, with a low-cost carbon source and in a short time of production period.


Introduction
Traditional polymeric substances have been used in a variety of fields, including construction materials, equipment, packaging, and so on. Due to the nature of plastic, global demand is expanding on a daily basis [1,2]. The continuous use of non-renewable petroleum-based plastics would be depleted very rapidly due to overuse and lack of recycling management technology [3,4]. Furthermore, the widespread use and unscientific disposal of these traditional polymers are causing severe soil and water pollution [5]. Besides that, during accidental combustion, toxic greenhouse gases are released into the environment, causing severe air pollution [1]. The abandoned used plastic materials can persist in the environment for several years due to its less or non-degradability in nature [6,7]. However, complete elimination of traditional plastics is not possible until an eco-friendly alternative material for traditional plastic is discovered [8]. Nature, fortunately, has provided excellent renewable sources in the form of microbes and plants [3]. Among these, microbes, particularly bacteria, are a preferred source for environmentally friendly biodegradable plastic production [9,10]. The bacterial based biopolymer producing bacteria isolation and biopolymer production receiving more attention among researchers due to its multiple application as similar to traditional plastics and its biodegradable nature [11]. The potential outcome of biodegradation after use is a significant difference among both conventional polymers and biopolymers. Since the majority of biopolymers are composed of carbon and nitrogen, they can act as a nutrient for microbial growth and result in the reduction of carbon dioxide and water via microbial enzyme activity [12]. Among the numerous bacterial based polymers, polyhydroxyalkanoates (PHAs) are the most preferred because they can synthesized by a large number of bacteria as an energy backup nutrient components and protect the bacterial cell from adverse environmental stress conditions and a limited supply of essential elements such as nitrogen and phosphorous [13]. Numerous bacterial species including Bacillus sp., Alcaligenes eutrophus, A. latus, Protomonas extorquens, P. oleovorans, Azotobacter vinelandii, Escherichia coli, and others have been identified as viable candidates for biopolymer (PHA) production [3,14]. PHA is primarily composed of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-3-hydroxyvalerate (PHBV) [11]. The majority of bacterial species that accumulate these PHA molecules in the cell cytoplasm [15]. These mobile, amorphous, and liquid granules found in the cytoplasm of actinobacteria and may support bacterial growth under environmental stress conditions biopolymers allowing microbial survival under stress conditions [9]. However, due to the high cost of commercial production, the effective replacement of oil-based polymers with biopolymers is constrained [12]. To address these issues, a low-cost raw material (carbon and nitrogen sources) utilizing and biopolymer producing bacterial candidate is urgently required. Therefore in this study, in addition to common bacterial species, an actinobacterial species (S. toxytricini D2) isolated from polluted industrial effluents site was investigated to assess its PHA producing competence and it could be first report. As perfect PHA producing a limited number of Actinobacterial species such as Kineosphaera limosa, Rhodococcus sp., Nocardia sp., and others have been discovered [16]. The PHAs produced intracellularly and stored as carbon and energy reservoir by several bacteria. The PHA accumulated in the actinobacteria is found in the cell cytoplasm, where it is mobile, amorphous. These liquid granules allow the Actinobacterial to survive under abiotic stress conditions such as nutritional factors and the presence of excess carbon source [17]. Based on the foregoing considerations, the purpose of this study was to isolate the most active PHA producing Actinobacteria species from polluted soil, characterize the PHA, and optimize the growth parameters.

Collection and Processing of Soil Sample
The Hosur SIPCOT region was chosen for this study, since the bacteria that live in contaminated soil need to be able to produce biopolymeres like molecules in order to survive [12]. With this assumption, about 10 g of polluted soil from four different SIPCOT regions was collected individually from 5 to 10 cm depth in ethanol sterilized glass containers. The sample collection containers were immediately transferred to the laboratory, where the studies were carried out in real time.

Enumeration of Actinobacteria Culture
The standard serial dilution process was used to enumerate indigenous Actinobacteria species from polluted soil samples. By spread plate method, 0.1 ml of 10 -5 dilution from each (four) samples was inoculated on Actinobacteria isolating selective media (YIM 7 HV medium) [9]. The YIM 7 HV medium (pH 7.2) contains 0.5 g of Disodium hydrogen phosphate, 1.0 g of Humic acid, 1.7 g of potassium chloride, 0.05 g of Magnesium sulphate, 0.01 g of Ferrous sulfate heptahydrate, 1 g of Calcium dichloride 0.5 mg of each B-vitamins, and 20 g of agar for 1000 mL distilled water. The inoculated plates were labelled and incubated at 30 °C for 48 h.

Screening of Biopolymer Producing Actinobacteria
Primary Screening YIM 7 HV medium was used to isolate 17 active cultivable Actinobacteria isolates during the initial screening process. The ability of these isolates to produce biopolymers was investigated [18]. To summarize, the pure form of these 17 well grown colonies were individually treated (poured over the grown colonies) with 2.5 ml of 0.05% of Sudan Black B stain and kept undisturbed for 30 min at room temperature before being rinsed with 60% ethyl alcohol. The Sudan Black B stained plates were incubated at room temperature for 35 min and observed the formation as black greenishblue color, which was determined to be positive for PHAs biopolymer.

Secondary Screening
The PHA primary screening results revealed that, of the 17 predominant isolates, the isolate H09 was the only one that tested positive for PHA producer. This isolates PHA producing potential was determined by exposing it to the PHA selective media (0.25 g of potassium dideuterium phosphate, 2.5 g of di-sodium hydrogen phosphate dehydrate, 10 g of mannitol, 2 g of sodium chloride, 0.1 g of magnesium sulphate, 10 g of sodium pyruvate, 1 g of peptone, 0.12 g of bromothymol blue, and 2 g of agar) test. This PHA selective media containing plate was inoculated with a loop of test isolate (H09) and incubated at 30 °C for 48 h [19].

Tertiary Screening by Morphological Analysis
The Transmission Electron Microscope (TEM) (TEM-Talos F200i Thermo Fisher, Mumbai, India) was used to finalize the PHA producing potential of test isolate H09 by following the standard protocol [20]. In brief, about 2.5 mL of test (H09) isolate (1 × 10 2 cells mL −1 ) at exponential phase was fixed with glutaraldehyde (4%), subsequently with osmium tetroxide (1%) and desiccated with 80-100% of acetone by consecutive treatment. The dried and fixed cells were submerged in epoxy resin and kept at sample boat, and the resin was polymerized at 60 °C for 24 h. Sub sequentially, the resin-coated fixed cells were stained with uranyl acetate (22 min) and lead citrate (5 min) and observed under TEM.

Molecular Characterization of H09
The screening studies revealed that only one isolate (H09) out of 17 have the potential to produce PHA. The test culture was suspected of belonging to the actinobacteria group based on its fundamental cultural and morphological characteristics. As a result, 16S rRNA sequencing was completed in order to identify the genus and species of the test isolate (H09). To summarise, the RNA was extracted using a standard RNA extraction kit from SRL Chemicals, Pvt. Ltd, Mumbai, India, and amplified using a standard universal primer (8F (5'-AGA GTT TGA TCC TGG CTC AG-3'). The PCR amplification conditions were optimized as follows: denaturation: 95 °C (2 min), annealing: 55 °C (30 s), and extension was at 72 °C (10 min) with 30 cycles. The amplified PCR component was decontaminated using a PCR product purification kit (SRL Chemicals, Pvt. Ltd, Mumbai, India) and sequencing system (518F/800R) was used for sequencing the purified PCR product [20]. The aligned sequences were found using nucleotide BLAST at the National Center for Biotechnology Knowledge in regions of local similarity with the Genbank database. The isolated species were described using the Neighbour-joining method with a maximum sequence difference of 0.75 distance using BLAST based on similar scores with registered species. Furthermore, the sequence was submitted to NCBI and assigned the accession number MT228958.1 [21]. The MFE secondary structures of Streptomyces toxytricini D2 was predicted using the available online RNAWebSuite/ RNAfold bioinformatics tools. The following link was used http:// rna. tbi. univie. ac. at/ cgi-bin/ RNAWe bSuite/ RNAfo ld. cgi [22]. To assess the possibilities of genetic modification in this attained S. toxytricini D2 was studied by bioinformatics tool. The restriction sites in 16S rRNA of S. toxytricini D2 was analysed using NEB cutter programme version 2.0. The following links were used http:// nc2. neb. com/ NEBcu tter2/ cutsh ow. php? name= 6e610 b1b-MT228 957.1 [23].

Extraction of PHA from Streptomyces toxytricini D2
The PHA fabricating test isolate was identified as Streptomyces toxytricini D2 by 16S rRNA sequencing. For the characterization of PHA molecule synthesised by S. toxytricini D2 was extracted [24]. Concisely, the biomass of S. toxytricini D2 (earlier or middle stationary phase) from YIM 7 HV broth medium was extracted by spun at 10,000 rpm for 8 min. Then pellet was treated 1 min with acetone and ethanol (1:1 ratio) to break the cell wall and to extract the PHAs granules from S. toxytricini D2 and subsequently centrifuged at 10,000 for 3 min and discarded the supernatant. About 1 mL of 4% NaOCI was added to dissolve the pellet and kept undisturbed for 30 min at room temperature and centrifuged at 10,000 rpm for 5 min. around 2:1 ratio of acetone and ethanol was used to wash the pellet. The polymers containing pellet was then dissolved in a sufficient amount of chloroform and filtered through filter paper (Whatman No. 1). The polymers containing filtrate were treated with 5 mL of concentrated sulfuric acid to reduce the polymeric granules into crotonic acid. The optical absorbance value of this sample was measured at 240 nm using UV-vis spectrophotometer (UV/Vis/NIR-LAMBDA 1050+ , PerkinElmer, USA), and with various concentration of commercial PHA (SRL, Chemicals Pvt. Ltd., Mumbai, India) molecule was used as a reference.

Fourier Transform Infrared Spectroscopy Study
FTIR analysis was performed on the polymeric granules produced and extracted from S. toxytricini D2 [24]. In brief, a portion of crude PHA (1 mg) extracted using the above procedure as dissolved with chloroform (5 mL) and mixed gently for few minutes. A few drops of the sample were smeared on FTIR KBr disk (PerkinElmer, spectrum 3™ Tri-Range FTIR). The spectra of solvent evaporated PHA extract were then read using an IR double beam spectrophotometer at a reasonable resolution (4 cm −1 ) and a vacuum pressure of 400-4000 cm −1 .

H NMR Analysis
The monomers ( 1 H) contents of PHA molecules produced by S. toxytricini D2 was determined using 1 H-NMR analysed [24]. Tetramethyl saline was used as an internal reference, and 10 mg of crude PHA granules were dissolved in 1 mL of chloroform. The spectra of liquefied PHA molecules were recorded in DMSO on Bruker ACF300 spectrophotometer at 300.53 MHz frequency with the resolution of > 0.1 deg and > 0.1 Hz respectively.

C NMR Analysis
The carbon skeleton of 20 mg of concentrated PHA (CDCl 3 dissolved) molecules extracted from S. toxytricini D2 was recorded at 30 °C by 13 C NMR [25] using a 500 MHz NMR spectrometer (Bruker ACF300 spectrophotometer: has 1 H and 13 C dual probe) with the MAS frequency of 170 MHz and 10 kHz correspondingly.

Growth Parameters Optimization for PHA Synthesise
To evaluate the PHA secreting extent potential of S. toxytricini D2, the basic growth parameters and find low cost nutritional factors were investigated in a one-factor-at-atime mode [26]. The parameters include different carbon (w/v) concentrations (2, 4, 6, 8, and 10%) sources such as pre-treated tapioca molasses, sugarcane molasses, and pulverized rice bran. The different concentrations (1, 2, 3, 4, and 5%) of various nitrogen sources such as (NH 4 ) 2 SO 4 , NH 4 NO 3 , and yeast extract were added in minimal medium (contain: 0. 5 g of MgSO 4 ⋅7H 2 O, 0.5 g of NaCl, 1 g L −1 of K 2 HPO 4 ). Various percentages (2, 6, 8, 10, and 12%) of inoculum of S. toxytricini D2 (1 × 10 6 CFU mL) were investigated. The various degrees of temperature (25,30,35, and 40 °C), different pH (6.5, 7.5, 8.5, and 9.5) and various incubation time (24,48,72, and 96 h) were evaluated. To ensure the accuracy and reproducibility of the results, each parameter was completed in triplicate. The bacteria inoculated with the above-mentioned parameters and media were incubated on a shaker incubator with 150 rpm for up to 96 h. The amount of PHA produced (extracted using the previous method), and growth of S. toxytricini D2 from each parameter were measured at 240 nm and 600 nm respectively using UV-Vis spectrophotometer on a day-to-day basis [27].

Growth Kinetics and Production of PHA Under Optimized Conditions
The optimal growth conditions for the maximum PHA production by S. toxytricini D2 were 8% of tapioca molasses, and 4% of NH 4 NO 3 were served as suitable carbon and nitrogen sources respectively. Furthermore, 8% of inoculum, 30 °C, pH 6.5, and 72 h of incubation were optimized as suitable growth conditions for PHA fabrication by S. toxytricini D2. The maximum PHA producing potential of S. toxytricini D2 was studied by inoculating 6% of S. toxytricini D2 (1 × 10 6 CFU mL) on minimal broth medium compiled with optimized parameters (8% of tapioca molasses, 4% of NH 4 NO 3, 30 °C, pH 6.5) and incubated in a shaker incubator for 72 h with 120 rpm. The growth kinetics and PHA production were recorded by method as mentioned earlier [19].

Statistical Analysis
To achieve accuracy and reproducibility, all experiments were carried out in triplicate. SPSS was also used to compute the mean, standard error, and one-way ANOVA Version 13.

Brief Profile of Soil Sample Collected Site
The soil samples collected sites have been polluted by various industrial activities, since it is an industrial area. Hence the bacteria that survive in this abiotic stress environment may secrete various types of high molecular weight molecules such as polysaccharides, polyamides, polyesters, and polyphosphates. These polymeric components can serve as protective capsular layers for bacteria that are exposed to abiotic stress [28]. PHA synthesis and mobilisation in bacteria have been linked to environmental stress and adaptation to abiotic stress [29]. These metabolically adapted bacterial strains were able to produce biopolymers of high quality and quantity.

Enumeration of Biopolymer Producing Predominant Isolate
A polluted soil sample collected from an industrial site yielded approximately 17 bacterial isolates. Despite the fact that morphological characteristics of bacteria were observed, nevertheless in the initial Sudan Black B stain screening study stated that only one bacterial strain was showed dark blueish black color colonies and this was designated as positive for PHA producer [30]. The Sudan black B has the ability to stain lipids or lipid-associated polysaccharide materials (polymeric) and produced deep blue-black color. Secondary screening in PHA selective media revealed deep bluish-black colonies of H09 was observed in the wellgrown plate.
The TEM analysis was performed as a final confirmation to determine the PHA producing potential of isolate H09. The presence of polymeric granules (PHA) in the close region of the cytoplasmic membrane was revealed by TEM images of test isolate H09 (Fig. 1). In the cytoplasmic region of test isolate H09, the majority of the produced PHA granules were uniformly oval and elongated in shape, with distinct boundary lines between each granule. Similarly, halophilic archaea isolated from polluted soil produces granules with spherical, ovoid, or elongated shapes [31]. The presence of these multi-shaped granules may cause minor complications in the purification process. Fortunately, the granules produced by test isolate H09 were identical in shape, which could be considered as additional benefit of this test isolate for mass production of high quality polymeric substances.

Genomic Identification of Test Isolate
The PHA was producing test isolate H09 was genotypically characterized using 16S rRNA analysis. PCR and sequencing systems were used to examine the amplification characteristics of isolate H09's 16S rRNA gene. The 1400 bp 16S rRNA genes sequence of isolate H09 was a blast and registered in GenBank http:// www. ncbi. nlm. nih. gov/ genba nk and acquired the accession number from NCBI as Streptomyces toxytricini D2 (MT228958.1). The partial sequence of the S. toxytricini D2 (MT228958.1) was compared with sequences of actinomycetes from NCBI database to determine the phylogenetic relatedness clustered together as one clade segments corresponding to an evolutionary distance of 0.002 and 0.009 as shown with bars using Neighbour-joining method (Fig. 2). It was revealed that the sequence of S. toxytricini D2 found 99% similarity with the existing species of S. toxytricini strain HBUM174624 (Fig. 2). The results were directly compared with the previous findings [32,33]. They isolated commercially valuable Streptomyces sp. from polluted soil and characterized it using 16S rRNA sequencing and compared it to other strains using phylogenetic analysis. The Minimum Free Energy (MFE) secondary structure of the 16S rRNA gene of S. toxytricini D2 (MT228958.1) revealed 27 stems in their MFE structure (Fig. 3). However, S. toxytricini D2, on the other hand, has GC-59% and AT-41% (Fig. 3). These findings are also consistent with the findings of researchers on Streptomyces sp. isolated from soil samples [34]. The S. toxytricini D2 is a mesophilic soil bacteria that has previously been identified as a promising candidate for commercially valuable enzyme production [21]. The genetic modification in S. toxytricini D2 is possible due to appropriate restriction sites and its fine GC content may balance the stability to broad growth conditions [35]. Thus, this genomic trait of S. toxytricini D2 could aid in the commercial production of biopolymers.

Categorisation of PHA Produced by S. toxytricini D2
The polymeric extracted from S. toxytricini D2 was successfully removed and confirmed as PHA by comparing it to a commercially available reference PHA molecule using UV-Vis spectrophotometer with absorbance in the range of 200 to 360 nm [17]. The highest absorbance peak for PHA extract was recorded at 240 nm (OD value: 2.46) and was nearly identical to commercial PHA (OD value: 2.40) and it confirming that the extracted polymer was PHA molecule (Fig. 4). Similarly, polymeric granules were extracted from the Bacillus subtilis NCDC0671 and identified as PHA using UV-vis spectrophotometer analysis [36].

FT-IR
The FTIR spectrum of Polyhydroxyalkanoates extracted from S. toxytricini D2 is shown in Fig. 5. The absorption band appeared at around 3500 cm −1 and was associated with the stretching frequency of the free-hydroxyl group's in the polymer chain [24]. The multiple peaks were observed between 2800 and 3100 cm −1 corresponding to the symmetric and asymmetric stretching vibrations of -CH 3 and -CH 2 -CH 3 alkane groups. Furthermore, the low intensity of -CH 3 peak reflects the conformational disorder of the crystallisation process [37]. Interestingly, the carbonyl functional group absorption band was observed as the doublet of ketone group (C=O) at nearly 1742 cm −1 and amide group (N-C=O) at 1660 cm −1 , corroborating the stretching vibrations of carbonyl ester and intracellular amide of microbes respectively [30,38]. The terminal methyl group (-CH 3 ) was confirmed by the intense peak observed at 1379 cm −1 , and the cluster of absorption peaks seen below 1200 cm −1 could be related to associated with the stretching frequency of -C-O-C-, -C-O and -C-C-functional groups [27]. The FTIR spectrum's designated absorption peaks (Fig. 5) show that PHA polymer is formed in amorphous phase with a trace amount of impurities from the starting materials and is well correlated with the reported literature [38,39].

H NMR and 13 C NMR Analysis
The structural characteristics of S. toxytricini D2 produced PHAs were investigated using 1 H NMR and 13 C NMR analyses. Figure 6 depicts the 1 H NMR spectrum of the PHAs produced by S. toxytricini. The resonance signal observed at 5.25 ppm was caused by methylene protons adjacent to  carboxyl groups of HB [40]. At 2.5 ppm, the multiplet resonance of protons of methylene and methane of carbon was observed. Peaks at 1.45-1.52 ppm were observed for the methylene protons adjacent to the carbon of the saturated side chain [41,42]. The presence of methyl protons in the side chain is responsible for the dominant peak at 1.23 ppm.
The Fig. 7 depicts the 13 C NMR spectrum of PHAs produced by S. toxytricini D2. The presence of methyl carbon caused the carbon resonance peak at 22.72 ppm to be observed. The saturated side chain's methylene carbon was responsible for the peaks observed between 43.09 and 45.2 ppm. Further peaks for methylene carbon attached to a carboxylic acid group were observed at 138.5 ppm. A carboxyl carbon peak was observed at 173.10 ppm [43,44]. Thus the 1 H and 13 C NMR spectra revealed that the intracellular molecules produced by S. toxytricini D2 were nearly identical with the PHAs.

The Growth Parameters Optimization for PHA Production by S. toxytricini D2
The most important factor in achieving the expression of all organisms' maximum potential is favourable growth conditions [32]. Similarly, the bacteria also required the best possible growth conditions in order to produce more human and environmental welfare products such as PHA [19]. The optimal growth requirements in this study included the various concentrations of various low-cost carbons sources (tapioca molasses, sugarcane molasses, and pulverized rice bran), nitrogen sources ((NH 4 ) 2 SO 4 , NH 4 NO 3 , and yeast extract), different percentage of inoculum, temperature, pH, and different incubation time (one factor at one time) and triplicates were performed for each parameter analysis.

Suitable Carbon and Nitrogen Sources
Microbes require carbon and nitrogen sources as the most important factor in the effective and quality microbial production process, which determines the quantity and quality of microbial products such as biopolymers (PHA)  [19]. Carbon and nitrogen sources are the most important determinants of microbial product quality and quantity [1]. At 8% tapioca molasses concentration, S. toxytricini D2 efficiently utilized and produced PHA (86.65%) as 17.34 g L −1 of PHA from 20.01 g L −1 of cell biomass. Other concentrations and carbon sources were statistically significant (P > 0.03). Pulverized rice bran, and sugarcane molasses were used as fine carbon source for S. toxytricini D2 and produced 80.18% and 69.81% of PHA at 10% concentrations of both carbon sources, respectively (Fig. 8a-c). At 10% concentration, these acquired value were statistically significant at P > 0.05 than other concentrations (2 to 8%) of pulverized rice bran and sugarcane molasses. About 4% of (NH 4 ) 2 SO 4 was used as the best nitrogen source for PHA (66.02%) production in S. toxytricini D2 as produced 8.2 g L −1 of PHA from 12.42 g L −1 of cell biomass, and it was statistically significant (P > 0.03) to other concentration and nitrogen source (Fig. 8d-f). The S. toxytricini D2 produced 51.47 and 61.85% of PHA from 4% of NH 4 NO 3 and yeast extract correspondingly. These values had a statistical significance of P > 0.05among the remaining concentrations were. These results suggest that, while carbon and nitrogen sources are essential factor, when their concentrations increase, the bacteria are unable utilize them all. Furthermore, high concentrations may reduce cell viability and activity [3]. As a result, a sufficient supply of suitable nitrogen and carbon sources may boost bacterial metabolic activity and growth. It results in a sufficient quantity of high-quality bacterial products (PHA) [18]. Furthermore, the carbon and nitrogen-based substrate determine the type of polymeric component (PHAs and PHBs), since the quantity of carbon atoms exist in the biopolymers are typed into two as 3-5 carbon atoms based PHAs (Short Chain Length PHAs: SCL PHAs) and 6-14 carbon atoms based PHAs (medium-chain length: MCL PHAs), due to the activity of substrate specific PHA synthases that can recognize 3-hydroxyalkanoic acid with assured range of carbon length [15]. The MCL PHAs possess more elasticity and low melting temperature with least degree of crystallinity in nature [12]. The SCL PHAs are termed as polyhydroxybutyrate. It has a high degree of crystallinity (> 50%) with thermoplastic in nature (high melting temperature: 180 °C). Other low-cost materials also previously reported such palm [45,46] had been recorded as suitable carbon source for bacterial (Bacillus sp.) based biopolymer (PHA and PHB) production (58%). The maximum volume of PHAs could be synthesized under the limited dosage of nitrogen contents [47]. It was correlated with this study's findings since more quantity of PHAs produced at 4% concentration of (NH 4 ) 2 SO 4 than 5%. The limited nitrogen sources with readymade form could improve the biopolymer yield [48].

Percentage of Inoculum
The percentage of inoculum used in the microbiological fermentation process has received more attention because it determines the volume of yield and time duration of production process [32]. In this study, about 8% of inoculum of S. toxytricini D2 produced a reasonable quantity (66.7%) of PHAs (12.34 g L −1 of PHAs from 18.5 g L −1 of cell biomass) in a short duration of incubation than other dosages (Fig. 8g). It was statistically significant (P > 0.03) in comparison to the other concentrations. It was followed by 10% of inoculums, which resulted 62.74% of PHAs (Fig. 8g). The high inoculum dosage may reduce the bacterium lag phase and increase its ability to reach the log phase. There the PHAs production gets initiated; thus the short duration of the cell biomass could produce more volume of PHAs [39]. Nonetheless, less PHAs yield was observed in the 10% inoculum since the cell may use more nutrients during the lag phase due to inoculums concentration (batch typed fermentation). At the same time, it has reached the log phase and is experiencing nutrient depletion, which leads to decreased production [38]. Similarly, the low PHAs yield was observed at a low inoculum concentration; it is possible that lag phase requires more time to reach the log phase with average cell biomass, resulting in low PHAs yield [30]. The inoculum dosage for each bacteria used in production [5]. Accordingly, about 12.5% of inoculum of Bacillus sp. was found as an optimized dosage for biopolymer (PHAs and PHBs) production [27].

The Suitable Temperature
Temperature is one of the most important physical parameters in the active growth of microbes [9]. It determines the yield of biomass and microbial products since, under the optimized temperature, only the bacteria can continue their metabolic process [49]. In this study, the optimal temperature for significant growth and PHAs producing competence of S. toxytricini D2 was found as 30 °C. The PHAs yield was observed as 72.95% (10.36 g L −1 of PHAs from 14.2 g L −1 of cell biomass) at 30 °C and followed by 35 °C (Fig. 8h), and it was statistically significant (P > 0.03) when compared to other temperatures. Low PHAs and biomass yield were recorded at 25 °C (37.06%) and 40 °C (62.74%). Several studies have found that the optimal temperature for PHAs production by various bacterial species is between 25 and 35 °C [50]. Extreme and low temperatures may reduce the feast phase as well as their rate of nutrient uptake. Furthermore, because of the higher volume of dissolved oxygen at average temperatures (25-35 °C), O 2 transmission is more efficient. In terms of mass production and economics, the average temperature for the manufacturing process could reduce production costs [51]. Similarly, species of Nacardiopsis and Vibrio have effectively produced biopolymers at 30 °C [52]. The optimal temperature may differ between species. According to this, the Bacillus sp. [5], E. aquimaris [30], S. thermophilus [53], and produce a fine quantity of PHAs at 30 °C, 35 °C, and 50 °C respectively. This temperature differentiation might be related to the temperature of bacteria isolated sites, since the enzymatic and metabolic activity could be significant [3].

Optimal pH
Another critical factor that regulates the cell metabolic process is pH. Since the pH of the medium has a direct relationship with each enzymatic mechanism in the cell. Under optimal pH conditions, more microbial products are available [54]. Accordingly, S. toxytricini D2 produced 67.42% (8.26 g L −1 of PHAs from 12.25 g L −1 of cell biomass) of PHAs at pH 6.5, and it was statistically significant (P > 0.03) to other pH (Fig. 8i). It is possible that the PHAs syntheses enzymes of S. toxytricini D2 effectively produce and accumulate PHAs at slightly acidic pH; it chelates the subsequent metabolic process, yielding a reasonable volume of PHAs. Similarly, Ralstonia solanacearum produces more biopolymer (PHBs and PHAS) acidic conditions [55]. The optimal pH for bacteria might differ for each for PHA production. Similarly, B. cereus produce high yield of polymeric molecules at pH 7.5 [55], and for Bacillus sp. (F15) pH 7 [5]. Moreover, some bacterial species might has the potential to grow under wide range of pH as acidic to alkaline, for that they developed particular adaptation strategy according to the pH and balancing the activity of enzymes such as ATP synthase, terminaloxidase [39], PHAs synthase [25] etc. These adaptation strategies permit the bacteria to balance the pH of cytoplasmic content and regulate optimal cell functioning.

Suitable Incubation Time
To obtain the maximum amount of beneficial products from microbes, sufficient incubation time is required. In this study, the isolated actinobacteria S. toxytricini D2 produce the maximum yield of PHAs on 72 h of incubation. The PHAs yield was recorded as 57.58% (7.21 g L −1 of PHAs from 12.52 g L −1 of cell biomass), and it was statistically significant (p > 0.03) to other incubation time (Fig. 8j). The optimal incubation time for the growth of bacteria might differ from each other. According to this, the Bacillus sp. Ti3 produced 51.6% of PHAs at 24 h of incubation time [35], Bacillus sp. INT005 (35.30% in 48 h) [56], Bacillus cereus SPV (38.0% in 48 h) [57], Bacillus mycoides (50% in 72 h) [58], Pseudomonas sp., Bacillus sp., and Rhizobium alti, 40 h [17]. The optimized incubation time increasing 1.82 fold of biopolymer production by Bacillus mycoides DFC1 at 72 h of incubation [59].

Production of PHA Under Optimized Conditions
The growth kinetics and extent PHAs producing potential of S. toxytricini D2 were studied with optimized growth conditions such as 8% of tapioca molasses, 4% of (NH 4 ) 2 SO 4, 8% of inoculum, pH 6.5, incubated at 30 °C for 72 h of incubation period. Under these optimized conditions, the S. toxytricini D2 yielded 86.56% of PHAs as 23.64 g L −1 of PHAs from 27.31 g L −1 of cell biomass on 72 h of incubation (Fig. 9). The quantity of PHAs and cell biomass (growth kinetic) were similarly increasing from 24 h onwards and it was continued up to 72 h of incubation. Furthermore, the growth of S. toxytricini D2 was get decreased from 72 h on; it indicated that the lag phase of this actinobacteria might take around 24 h, and noticed absence or least amount of PHAs (Fig. 9). The Fig. 9 revealed that on after 24 h of incubation the PHAs was gradually increasing along with cell growth and it declared that these actinobacteria reached the log phase at after 24 h of incubation and concurrently PHAs get produced on the same time onwards. The growth was reduced on 72 h of incubation and the PHAs production also halted on the same time of incubation, it confirmed that the cell had reached lag stationary and decline phase. The attained yield of PHAs from S. toxytricini D2 was statistically significant with the cell biomass as P < 0.003.

Conclusions
The bacteria isolated from polluted soil were identified as actinobacteria with the potential to produce PHAs, and the exact genus and species of this isolate were determined using 16S rRNA analysis as S. toxytricini D2. Successive screening studies confirmed this isolate's PHAs producing potential, with TEM analysis confirming that S. toxytricini D2 has the potential to produce PHAs. The results show that S. toxytricini D2 has a high potential for producing PHA molecules while using a low-cost carbon source and in a short incubation period. Thus, it could be a viable candidate for producing large quantities of PHAs molecules under optimal conditions.