3.1. 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 as a lifesaving mechanisms the bacteria could secrete different kinds’ high molecular weight molecules such as polysaccharides, polyamides, polyesters, and polyphosphates. These polymeric components can act as protective capsular layers for bacteria that survive under abiotic stress conditions (Moradali & Rehm, 2020). The synthesis and mobilization of PHA in bacteria have been directly related to environmental stress and fit for survival under abiotic stress (Niether et al., 2020). These metabolically adapted bacterial strains could produce fine quality and quantity of biopolymers.
3.2. Enumeration of biopolymer producing predominant isolate
About 17 bacterial isolates have been screened from a polluted soil sample collected from an industrial site. Different morphological characteristics 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 deliberated as positive for PHA producer (Mostafa et al., 2020). The Sudan black B can stain the lipid or lipid-associated polysaccharide material (polymeric) and produced deep blue-black color. On secondary screening in PHA selective media, deep bluish-black colonies of H09 was observed in the well-grown plate.
As a final confirmation, the TEM analysis was achieved to identify the PHA producing potential of isolate H09. The TEM images of test isolate H09 were showed that the presence of polymeric granules (PHA) located at the close region of the cytoplasmic membrane (Fig. 1). Most of the produced PHA granules were uniformly oval and elongated in shape with clear boundary line of each granule were seen in the cytoplasmic region of test isolate H09. Similarly, halophilic archaea isolated from polluted soil produces spherical, ovoid, and elongated shaped granules (Danis et al., 2015). This multi-shaped granules might cause little complications in the purification process. Fortunately, the granules produced by test isolate H09 showed identical in shape that could be considered as more advantage of this test isolate for mass production with fine quality polymeric substances.
3.3. Genomic identification of test isolate
The PHA was producing test isolate H09 was genotypically characterized using 16S rRNA analysis. The amplification characteristics of the 16S rRNA gene of isolate H09 were carried out by PCR and sequencing systems. The 1400 bp 16S rRNA genes sequence of isolate H09 was a blast and registered in GenBank http://www.ncbi.nlm.nih.gov/genbank 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 already existing genera 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 by using Neighbour-joining method (Fig. 2). It was revealed that the sequence of the isolate 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 (Charousová et al., 2018; Salgaonkar & Bragança, 2017). They isolated commercially valuable Streptomyces sp. from polluted soil and characterized through 16S rRNA sequencing and compared by phylogenetic analysis. The Minimum Free Energy (MFE) secondary structure of the 16S rRNA gene of S. toxytricini D2 (MT228958.1) showed 27 stems in their MFE structure (Fig. 3). However, S. toxytricini D2 has GC-59% and AT-41% (Fig. 3). These observations also agree with the results reported by researchers on Streptomyces sp. isolated from soil sample (Heinsch et al., 2019). The S. toxytricini D2 is a mesophilic soil bacteria and previously reported as a suitable candidate for commercially valuable enzyme production (Kumar et al., 2020). The genetic modification in S. toxytricini D2 is possible due to appropriate restriction sites and its fine GC content could balance the stability to broad growth conditions (Bhagowati et al., 2015). Thus, this genomic trait of S. toxytricini D2 could support the biopolymer commercial production.
3.4. Categorisation of PHA produced by S. toxytricini D2
The polymeric extracted from S. toxytricini D2 was successfully removed and confirmed as PHA by compared with reference PHA (commercially available) molecule by UV-Vis spectrophotometer with absorbance in the range of 200 to 360 nm (Sathiyanarayanan et al., 2013). The highest absorbance peak for PHA extract was recorded at 240 nm (OD value: 2.46) and it was almost identical to commercial PHA (OD value: 2.40) and it confirmed that the extracted polymer was PHA molecule (Fig. 4). Similarly, the polymeric granules are also removed from the Bacillus subtilis NCDC0671 and confirmed that molecule as PHA through UV-Vis spectrophotometer analysis (Umesh et al., 2018).
3.4.1. FT-IR
The figure. 5 displays the FTIR spectrum of the Polyhydroxyalkanoates as extracted from S. toxytricini D2. The absorption band appeared at around 3500 cm− 1 associated with the free-hydroxyl group's stretching frequency in the polymer chain (Alarfaj et al., 2015). The multiple peaks were observed between 2800 and 3100 cm− 1 corresponding to the symmetric and asymmetric stretching vibrations of -CH3 and -CH2-CH3 alkane groups. Furthermore, the low intensity of –-CH3 peak attributes the crystallisation process's conformational disorder (Biradar et al., 2018). Interestingly, the absorption band of carbonyl functional group 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, corroborates the stretching vibrations of carbonyl ester and intracellular amide of microbes respectively (López-Cuellar et al., 2011; Mostafa et al., 2020). The terminal methyl group (-CH3) was confirmed from the intense peak observed at 1379 cm− 1, and the cluster of absorption peaks appeared at below 1200 cm− 1 might be associated with the stretching frequency of –C-O-C-, -C-O and –C-C- functional groups (Getachew & Woldesenbet, 2016). The FTIR spectrum's designated absorption peaks (Fig. 5) reveals that the formation of PHA polymer in amorphous phase with a trace amount of impurities from the starting materials and well correlated with the reported literature (Bhatt et al., 2008; López-Cuellar et al., 2011).
3.4.2. 1H NMR and 13C NMR analysis
The structural specifics of S. toxytricini D2 produced PHAs were studied by 1H NMR and 13C NMR analyses. The figure. 6 shows the 1H NMR spectrum of the PHAs produced by S. toxytricini. The resonance signal observed at 5.25 ppm was due to methylene protons adjacent to carboxyl groups of HB (Linton et al., 2012). Whereas the multiplet resonance of protons of methylene and methane of α-carbon were observed at 2.5 ppm. Peaks at 1.45–1.52 ppm were observed concerning the methylene protons adjacent to the β-carbon of the saturated side chain (Bhuwal et al., 2013; Singh et al., 2011). The predominant peak observed at 1.23 ppm is ascertained to the presence of methyl protons of the side chain.
The figure. 7 shows the 13C NMR spectrum of PHAs produced by S. toxytricini D2. The carbon resonance peak observed at 22.72 ppm was due to the presence of methyl carbon. The peaks observed from 43.09 to 45.2 ppm were due to the saturated side chain's methylene carbon. Further peaks were observed at 138.5 ppm for methylene carbon attached to a carboxylic acid group. At 173.10 ppm carboxyl carbon peak was observed (Sabarinathan et al., 2018; Yalpani et al., 1991). Thus the results of 1H and 13C NMR spectra indicated that the intracellular molecules produced by S. toxytricini D2 were mostly identical with the PHAs.
3.5. The growth parameters optimization for PHA production by S. toxytricini D2
The favorable growth conditions are the most significant factor in attaining the expression of the maximum potential of all organisms (Charousová et al., 2018). Similarly, the bacteria also required the most suitable growth conditions for more human and environmental welfare products such as PHA (Krishnan et al., 2017). In this study, the optimal growth requirements such as the various concentration of various low-cost carbons sources (tapioca molasses, sugarcane molasses, and pulverized rice bran), nitrogen sources ((NH4)2SO4, NH4NO3, 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.
3.5.1. Suitable carbon and nitrogen sources
For the effective and quality microbial production process, generally, the microbes require carbon and nitrogen sources as most significant factor and it determines the quantity and quality of microbial products such as biopolymers (PHA) (Krishnan et al., 2017). The carbon and nitrogen sources are the most significant microbial product quality and quantity determining factors (Mohapatra et al., 2017). The S. toxytricini D2 effectively utilized and produced PHA (86.65%) at 8% concentration of tapioca molasses as 17.34 g L− 1 of PHA from 20.01 g L− 1 of cell biomass. It was statistically significant (p > 0.03) to other concentration and carbon source. The pulverized rice bran, and sugarcane molasses were acted as a fine carbon source for S. toxytricini D2 and produced 80.18% and 69.81% of PHA at 10% concentration of both carbon sources, respectively (Fig. 8a to 8c). 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 (NH4)2SO4 was served as a most suitable 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 to 8f). The S. toxytricini D2 produced 51.47 and 61.85% of PHA from 4% of NH4NO3 and yeast extract correspondingly. The statistical significance of these values among the remaining concentrations were p > 0.05. These results indicate that even though the carbon and nitrogen sources are essential factor, when their concentrations increase, the bacteria could not utilize them all. Furthermore, the presence of excess concentration might reduce cell viability and activity (Pérez et al., 2020). Thus the sufficient quantity of suitable nitrogen and carbon sources could enhance bacterial metabolic activity and growth. It leads to a reasonable quantity of bacterial products (PHA) with fine quality (Rendón-Villalobos et al., 2016). Besides that, 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 (Shanmugam & Abirami, 2019). The MCL PHAs possess more elasticity and low melting temperature with least degree of crystallinity in nature (Zikmanis et al., 2020). The SCL PHAs are termed as polyhydroxybutyrate. It has a high degree of crystallinity (> 50%) with thermoplastic in nature (high melting temperature: 180°C). Some other low-cost materials also previously reported such palm (Gabr, 2018; Khiyami et al., 2011) 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 (Patel et al., 2017). It was correlated with this study's findings since more quantity of PHAs produced at 4% concentration of (NH4)2SO4 than 5%. The limited nitrogen sources with readymade form could improve the biopolymer yield (Kourmentza et al., 2017).
3.5.2. Percentage of inoculum
The percentage of inoculum applied on the microbiological fermentation process received more attention as it determine the volume of yield and time duration of production process (Charousová et al., 2018). 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) to other concentration. It followed by 10% of inoculum produced 62.74% of PHAs (Fig. 8g). The high dosage of inoculum might minimize the lag phase of the bacterium and enhance it 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 (Bhatt et al., 2008). Nevertheless, less PHAs yield was noted in 10% inoculum since at the inoculum concentration the cell might utilize more volume of nutrients during the lag phase itself (batch typed fermentation). At the same time, it reached to the log phase it faces nutrient depletion leads to less production (López-Cuellar et al., 2011). Similarly, the low PHAs yield was recorded at a low concentration of inoculum; it could be lag phase might take some more time to reach the log phase with average cell biomass and it leads to low PHAs yield (Mostafa et al., 2020). The dosage of inoculum for each bacteria for the production (fermentation) process should differ (Alshehrei, 2019). Accordingly, about 12.5% of inoculum of Bacillus sp. was found as an optimized dosage for biopolymer (PHAs and PHBs) production (Getachew & Woldesenbet, 2016).
3.5.3. The suitable temperature
Among the various physical parameters, the temperature plays a most significant role in the active growth of microbes (Girão et al., 2019). It determines the yield of biomass and microbial products since, under the optimized temperature, only the bacteria can continue their metabolic process (Johnson et al., 2010). 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) to other temperature. Low PHAs and biomass yield were recorded at 25°C (37.06%) and 40°C (62.74%). Several researchers have been reported that the optimal temperature for PHAs production through various bacterial species fell within the range of 25–35°C (De Grazia et al., 2017). The extreme and low temperatures might reduce the feast phase of bacteria along with swift nutrients uptake rates. Besides that, the O2 transmission is more proficient at average temperatures since the higher volume of dissolved oxygen persists at average temperature (25–35°C). In mass production and economic aspects, the average temperature for the production process could minimize the cost of production (Chan et al., 2017). Similarly, species of Nacardiopsis and Vibrio have effectively produced biopolymers at 30 ºC (Mahitha & Madhuri, 2015). The optimal temperature might vary from species to species. According to this, the Bacillus sp. (Alshehrei, 2019), E. aquimaris (Mostafa et al., 2020), S. thermophilus (Kalaivani & Sukumaran, 2013), and produce a fine quantity of PHAs at 30 ºC, 35 ºC, and 50 ºC respectively. This temperature differentiation might be related to the temperature of bacteria isolated sites, since the enzymatic and metabolic activity could be significant (Pérez et al., 2020).
3.5.4. Optimal pH
The pH is another most essential factor that regulates the cell metabolic process. Since each enzymatic mechanisms in the cell has been directly related to the pH of the medium. More microbial products is obtainable under the optimal pH condition (Sasidharan et al., 2015). 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 might be the PHAs syntheses enzymes of S. toxytricini D2 effectively produce and accumulate the PHAs at slightly acidic pH; it chelates the further metabolic process and finally yielded a reasonable volume of PHAs. Similarly, Ralstonia solanacearum produces more biopolymer (PHBs and PHAS) acidic conditions (Macagnan et al., 2017). The optimal pH for bacteria might differed for each for PHA production. Similarly, B. cereus produce high yield of polymeric molecules at pH 7.5 (Macagnan et al., 2017), and for Bacillus sp (F15) pH 7 (Alshehrei, 2019). 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 (Bhatt et al., 2008), PHAs synthase (Baran et al., 2018) etc. These adaptation strategies permit the bacteria to balance the pH of cytoplasmic content and regulate optimal cell functioning.
3.5.5. Suitable incubation time
To attain the extent beneficial products from microbes, it need to be maintained for sufficient incubation time. 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 (Bhagowati et al., 2015), Bacillus sp. INT005 (35.30% in 48 h) (Tajima et al., 2003), Bacillus cereus SPV (38.0 % in 48h) (Valappil et al., 2008), Bacillus mycoides (50% in 72 h) (Soam et al., 2012), Pseudomonas sp, Bacillus sp, and Rhizobium alti, 40 h (Sathiyanarayanan et al., 2013). The optimized incubation time increasing 1.82 fold of biopolymer production by Bacillus mycoides DFC1 at 72 h of incubation (Narayanan & Ramana, 2012).
3.6 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 (NH4)2SO4, 8% of inoculum, pH 6.5, incubated at 30 ºC 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 figure. 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.
Moreover, several numbers of bacterial species have been reported about the PHAs production under optimized conditions, the bacteria such as Ralstonia eutropha JMP 134 (50% of PHAs), Bacillus cereus FA11 (48.43%), Rhodococcus aetherivorans IAR1 (58.9%), Haloferax mediterranei (65%), Halomonas boliviensis LC1 (56%), H. boliviensis LC1 (88%), Synechococcus sp. MA19 (55%) etc. (Maskow & Babel, 2000; Ni et al., 2010; Singh Saharan et al., 2014). Very few reports are available in the Streptomyces sp. as biopolymer producers such as S. griseorubiginosus, S. coelicolor, S. antibioticus, S. venezuelae, S. hygroscopicus (Villano et al., 2010), S. aureofaciens, S. griseus, S. parvus, S. albus, etc. (Singh Saharan et al., 2014) all of this have reported as PHB producer. No previous reports are available about the production of PHAs from S. toxytricini D2. Thus this is the foremost study and reported about the PHAs producing potential of S. toxytricini D2 isolated from polluted soil.