{"paper_id":"3f684472-7c92-4baa-a578-23ac150f2f5a","body_text":"Bioconversion of whey to Polyhydroxyalkanoate (PHA): Process Optimization and Yield Enhancement | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Bioconversion of whey to Polyhydroxyalkanoate (PHA): Process Optimization and Yield Enhancement Bhanu Pratap Singh, Satish Babu Rajulapati, Sridhar Pilli, R. D. Tyagi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7168043/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Polyhydroxyalkanoate (PHA) is a biodegradable biopolymer with significant potential as an eco-friendly substitute for conventional plastics. This study investigates microbial bio-transformation and enhanced biosynthesis of polyhydroxyalkanoate (PHA) using cheese whey (a dairy industry processing waste) as a substrate. The process employs Stutzerimonas stutzeri BPSNITW100893, a novel strain isolated from food waste generated at the Institute Food Court-C of NIT Warangal, which produces higher PHA, compared to six isolated strains from initial screening using mineral salt media (MSM) and characterized using FTIR and NMR. A rotatable Central Composite Design (rCCD) based optimization using four key factors remarkably enhanced production under optimal physiological conditions, i.e., C/P ratio (147.6 w/w), fermentation time (69.6 hours), inoculum to substrate ratio 8.83 (v/v) %, C/N ratio 45.9 (w/w). A high PHA mass fraction yield % of 66.51% was observed as compared to the predicted yield of 56.48% from cheese whey hydrolysate as feed. Scale-up studies were successfully conducted up to 3 L with optimized parameters confirmed by cell proliferation studies. These studies demonstrated high productivity with a maximum PHA mole fraction yield of 73.03 ± 4.92% and a productivity rate of 1.319 ± 0.089 g/L/h, highlighting the potential of dairy processing waste as a substrate for sustainable biopolymer production and waste valorization. Polyhydroxyalkanoate cheese whey hydrolysate polymers waste valorization Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Biodegradable polymers are defined by various standards, with institutions such as ASTM, ISO, and DIN offering slightly different criteria based on microbial action and degradation outcomes (ASTM Committee 2021 ; Hahn and Hennecke 2022 ). Polyhydroxybutyrate (PHA), a type of biodegradable thermoplastic, has garnered interest for its biocompatibility and environmental benefits (Amelia et al. 2019 ; Acharjee et al. 2023 ). However, high production cost, especially due to the use of expensive carbon sources and complex processing, limit its industrial viability. In contrast, dairy industry waste streams, such as cheese whey and associated wastewater, present a promising, nutrient-rich but underutilized resource for sustainable PHA production (Pires et al. 2021 ). Cheese whey (CW), a by-product of cheese production, is generated in massive quantities worldwide. Producing 1 kg of cheese requires approximately 10 kg of milk, resulting in about 9 kg of by-products, primarily whey (Prazeres et al. 2012 ). As of the FAO 2022 report, India’s cheese production reached approximately 23.1 million tonnes, generating an estimated 208 million tonnes of cheese whey annually (Food and Agriculture Organization of the United Nations, 2022). The environmental implications of such vast whey volumes are significant due to CW’s high biochemical oxygen demand (BOD), which ranges from 15,000 to 35,000 mg/L. This high organic load contributes to CO₂ emissions and presents serious risks to aquatic ecosystems (Gonzfilez Siso 1996 ; Carvalheira et al. 2022 ). Notably, the valorization of 10,000 litres of whey can offset about 0.10 to 0.24 tonnes of CO₂, demonstrating its potential environmental benefit when diverted from waste streams to useful applications. CW is a nutrient-rich substrate, typically containing around 44 g/L of lactose, 6 g/L of proteins, and 4 g/L of lipids, along with small amounts of poorly biodegradable proteins (Reddy et al. 2022 ). These characteristics make whey a promising feedstock for microbial fermentation processes, particularly in the production of biodegradable plastics such as polyhydroxybutyrate (PHA). However, its inherent limitations—like a low carbon-to-nitrogen (C/N) ratio and complex, variable composition—can complicate direct microbial utilization. Nonetheless, research has shown that certain microbial strains, particularly those from the Pseudomonas genus, and native consortia isolated from dairy effluents, are capable of efficiently converting CW into PHA. This not only addresses the waste management challenge but also aligns with the principles of a circular economy, turning dairy industry by-products into eco-friendly, high-value materials. To enhance PHA yields from CW, careful optimization of bioconversion conditions is critical. Important factors include maintaining ideal C/N and C/P ratios (Valentino et al. 2015 ), regulating pH, temperature, and aeration, and selecting effective cultivation strategies. Two-stage fermentation—where microbial growth precedes a nutrient-limitation phase—has proven effective in boosting PHA accumulation. Additionally, pre-treatment techniques improve substrate quality and fermentation efficiency. Fed-batch and semi-continuous fermentation methods help mitigate substrate inhibition, while recent advances in genetic engineering and omics technologies are enabling the development of highly efficient microbial strains. Through such integrated biotechnological innovations, cheese whey is increasingly being transformed from a significant pollutant into a valuable resource for sustainable bioplastic production. Stutzerimonas stutzeri is a bacterial domain, Proteobacteria phylum, Gammaproteobacteria class, a metabolically versatile, Gram-negative bacterium known for its ecological adaptability and broad biotechnological potential that was reclassified in 2021 from the genus Pseudomonas based on phylogenomics. S. stutzeri is widely distributed in soil, marine sediments, wastewater, and other contaminated environments. It exhibits notable capabilities in nitrogen cycling, particularly denitrification and, in some strains, nitrogen fixation. Owing to its ability to thrive under nutrient-limited and stress conditions, S. stutzeri has been employed in the bioremediation of hydrocarbons, heavy metals, and other industrial pollutants (Salvà-Serra et al. 2023 ; Zhang et al. 2025 ; Pérez-Padilla et al. 2025 ). Importantly, certain strains of the same genus are capable of accumulating polyhydroxybutyrate (PHA), a biodegradable polymer, under optimized carbon-rich and nitrogen-limited conditions. This makes it a promising strain for sustainable bioplastic production using low-cost substrates such as dairy wastewater or cheese whey. High genetic diversity, salt tolerance, and substrate flexibility of this novel strain further support its role in environmental biotechnology and circular waste valorization. As dairy wastewater volumes continue to rise alongside global dairy demand, efficient valorisation through optimized microbial fermentation and process innovation will play an increasingly important role in fostering a circular bioeconomy (Asiri and Chu 2022 ). This study aims to investigate the efficiency and underlying mechanisms of PHA production from dairy wastewater using a novel S. stutzeri strain, with the dual aim of mitigating environmental pollution and lowering the production cost of biodegradable plastics with high purity. 2. Materials and Methods 2.1 Isolation, Screening, and culture maintenance of Lipolytic microbes Food waste for microbial strain isolation used in this study was collected from the Institute Food Court - C at the National Institute of Technology (NIT), Warangal, India. The waste was composted aerobically through pit composting by burying it 25 cm below ground behind the mess facility for 45 days. Microbial isolation was carried out using serial dilution and spread plate techniques, followed by screening with Sudan Black B staining, Nile Blue A, and Nile Red assays to identify colonies capable of metabolizing complex organic substrates into polyhydroxyalkanoates (PHAs) (Godbole et al. 2016). Seven isolates were identified as responsible for producing PHA. Morphological characterization and biochemical assays were performed. Further identification of the isolate was confirmed through 16S rRNA gene sequencing. The selected strains were cultivated in nitrogen-balanced Mineral Salt Medium (NB-MSM) supplemented with a 1 molar glucose solution (10 mL/L) as the primary carbon source, using a shake flask with an incubation speed of 150 rpm and a temperature of 30℃. NB-MSM composed of (per litre): 10 g C 6 H 12 O 6 , 2 g (NH 4 ) 2 SO 4 , 6.8 g Na 2 HPO 4 .7H 2 O, 1.5 g KH 2 PO 4 , 100 mg CaCl 2 .2H 2 O, 60 mg NH 4 -Fe-(III) citrate, 200 mg MgSO 4 .7H 2 O and 1 ml trace elements solution. The composition of trace elements in 1 L of solution is as follows: 100 mg ZnSO 4 ·7H 2 O, 30 mg MnCl 2 ·4H 2 O, 300 mg H 3 BO 3 , 200 mg CoCl 2 .6H 2 O, 20 mg CuSO 4 ·5H 2 O, 20 mg NiCl 2 .6H 2 O, and 30 mg NaMoO 4 (Bose et al. 2023 ). One high-yielding novel strain, Stutzerimonas stutzeri BPSNITW100893, was selected for better results in terms of a maximum 6.41 g/L PHA biomass produced (approximately 61.32% PHA biomass yield). The isolated strain of S. stutzeri was maintained on nutrient agar plates at 4°C for short-term storage and further experimental use, and subculturing was performed once every thirty days to keep them alive. 2.2 Pre-treatment of cheese whey as a carbon source Cheese whey was collected in a 5 L sterilized container from a domestic dairy near Warangal, India. In this study, the thermo-chemical method is used for pre-treatment to remove excess proteins before using whey lactose as a carbon source. CW is subjected to thermal pretreatment by boiling for 15 minutes at 100°C, followed by cooling to room temperature. It was then filtered, and the whey supernatant pH 7.3 ± 0.15 was adjusted to neutral pH 7.0 ± 0.1 using 1N (NaOH/ H₂SO₄) and used as a feed for microbial growth and PHA production. 2.3 PHA accumulation using submerged culture The organism was cultivated using a nitrogen-balanced (NB-MSM), to enhance the intracellular accumulation of PHA, bacterial cultures were grown in a nitrogen-deficient medium (ND-MSM), as previously described by (Brennan et al. 2021 ). PHA production using a two-stage batch cultivation strategy by S. stutzeri was studied with 250 mL Erlenmeyer flasks with 100 mL of media containing deproteinized cheese whey, which was replaced with glucose (10 g/L) used for control growth media. In the first stage, bacterial cells were cultivated in NB-MSM at 30°C with agitation at 150 rpm until they reached the exponential growth phase. For enhanced PHA accumulation, the culture was then transitioned to a nitrogen-limited (ND-MSM) medium in the second stage. The initial nitrogen concentration in the medium was maintained at 2.0 g/L using ammonium sulphate (NH 4 ) 2 SO 4 as the nitrogen source. To assess the influence of nitrogen availability on PHA accumulation, bacterial growth and PHA content were monitored across varying concentrations of the nitrogen source (0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0 g/L). 2.4 Extraction and Recovery of PHA PHA extraction was carried out following the dispersion method using sodium hypochlorite and chloroform (Pati et al. 2020 ; Bose et al. 2023 ). Cells were harvested by centrifugation at 10,000 rpm for 20 minutes at 4℃. The collected pellets were washed sequentially with deionized water and phosphate-buffered saline (PBS). After washing, the pellets were air-dried for 20 minutes, and the dry cell weight (DCW) was recorded using Eq. 1. $$\\:DCW(g/L)\\:=\\:(Final\\:dry\\:weight\\:-\\:Initial\\:weight)/Volume\\:of\\:culture\\:\\left(L\\right)$$ The extraction process involved adding chloroform and sodium hypochlorite (6%w/v) solution to washed cell pellets in a 1:1 volume ratio (12.5 µL (v/v) each) adjusted proportionally to the dry cell weight to ensure complete lysis and efficient PHA extraction (Ratnaningrum et al. 2020 ). The mixture was then incubated at 30°C for 12 hours in borosilicate glass reagent bottles with leak proof blue screw cap. Following incubation, the solution was centrifuged at 10,000 rpm for 10 minutes at room temperature, leading to the formation of three distinct layers. The bottom chloroform layer, containing the dissolved PHA, was carefully separated and transferred to a clean ampoule for volume measurement. To precipitate the PHA, a methanol and water mixture (7:3 v/v) was added at five times the volume of the chloroform extracted. Further centrifugation at 10,000 rpm for 20 minutes at 4℃ enhanced the precipitation of PHA and calculated using Eq. 2 denoting the PHA content in biomass (PHA mass fraction or yield %). $$\\:PHB\\:content\\:in\\:biomass=\\frac{Weight\\:of\\:PHB\\:\\left(powder\\right)\\:obtained\\left(in\\:grams\\right)}{Dry\\:cell\\:weight\\:\\left(in\\:grams\\right)}*100$$ The resulting pellets were separated, and 0.1 mL of concentrated sulfuric acid (H₂SO₄) was added to the pellets. The mixture was then boiled at 100°C for 25 minutes and allowed to cool. A standard calibration curve for crotonic acid was prepared using known concentrations, and the PHA content was quantified by referencing a standard calibration curve with the standard graph and using the purity formula as in Eq. 3 (Mamat et al. 2014 ; Farid et al. 2015 ; Parodi et al. 2021 ; Bose et al. 2023 ). $$\\:PHB\\:Purity\\left(\\%\\right)=\\frac{Expected\\:Crotonic\\:Acid\\:\\left(\\mu\\:g/mL\\right)from\\:pure\\:PHB}{Measured\\:Crotonic\\:Acid\\:(\\mu\\:g/mL))}\\times\\:100$$ 2.5 Statistical optimization of culture media In this study, a rotatable Central Composite Design (rCCD) was employed to optimize the production of high cell density biomass using deproteinized cheese whey as a carbon source. The optimization focused on four key factors: carbon to nitrogen ratio (C/N), carbon to phosphorus ratio (C/P), fermentation time, and inoculum to substrate ratio. The C/N and C/P ratios were varied by adjusting the concentrations of ammonium sulphate and potassium dihydrogen phosphate, while keeping the carbon input (from cheese whey) constant. To fix the desired carbon-to-nitrogen (C/N) and carbon-to-phosphorus (C/P) ratios in the cheese whey-based medium, the total carbon content was first estimated based on the lactose concentration in whey, assuming that lactose comprises approximately 40% carbon by weight. For instance, with 20 g/L lactose, the carbon content was calculated as 16 g/L. Based on the selected C/N and C/P ratios in the experimental design, the required nitrogen and phosphorus concentrations were determined by using the formulas: \\(\\:\\:\\text{R}\\text{e}\\text{q}\\text{u}\\text{i}\\text{r}\\text{e}\\text{d}\\:\\text{N}=\\text{C}\\text{a}\\text{r}\\text{b}\\text{o}\\text{n}\\:(\\text{g}/\\text{L})/(\\text{C}/\\text{N}\\:\\text{r}\\text{a}\\text{t}\\text{i}\\text{o})\\:\\) and \\(\\:\\text{R}\\text{e}\\text{q}\\text{u}\\text{i}\\text{r}\\text{e}\\text{d}\\:\\text{P}=\\:\\text{C}\\text{a}\\text{r}\\text{b}\\text{o}\\text{n}\\:(\\text{g}/\\text{L})/(\\text{C}/\\text{P}\\:\\text{r}\\text{a}\\text{t}\\text{i}\\text{o})\\) . External sources of nitrogen and phosphorus—ammonium sulphate and potassium dihydrogen phosphate, respectively—were added to the medium in amounts calculated to meet these ratios. The required mass of each compound was calculated using their respective nitrogen and phosphorus content by weight (approximately 21.2% for (NH 4 ) 2 SO 4 and 22.8% for KH₂PO₄), ensuring precise control over nutrient levels in the medium while maintaining a constant carbon input from whey. This approach allowed systematic evaluation of the effects of nutrient balance on biomass accumulation. Four independent variables were selected based on their critical role in microbial growth and fermentation efficiency and were designated as: X 1 , X 2 , X 3 and X 4 . The CCD generated a total of 30 experimental runs, which included factorial points, axial points, and 6 center points to ensure robustness and to detect any curvature in the response surface. The results from experimental designs using a shake flask were analyzed and interpreted using Design Expert software V 13.0.5.0. Based on the cell density response and the interaction effects among the studied variables, multiple regression analysis was performed to model the relationship between the independent variables and the response. A second-order polynomial equation was fitted to the experimental data obtained from the Central Composite Design (CCD). This model allowed for the evaluation of both linear and quadratic effects, as well as two-way interactions between the variables. The general form of the second-order polynomial model is given as: Y = β₀ + Σβ i X i + Σβ i i X i ² + ΣΣβ i ⱼX i Xⱼ + ε where Y is the predicted response, X i and Xⱼ represent the coded levels of the independent variables (C/N ratio, C/P ratio, fermentation time, and inoculum to substrate ratio), β₀ is the intercept, β i are the linear coefficients, β i i are the quadratic coefficients, β i ⱼ are the interaction coefficients for i ≠ j, ε is the random error. This model was used to analyze the influence of each factor and their interactions, and to predict optimal conditions for maximum biomass production, omitting statistically not significant parameters (where p > 0.05). 2.6 Data analysis and Model validation of CCD The regression model is used to predict optimal conditions and evaluate the importance of various factors. ANOVA (Analysis of Variance) helps determine the statistical relevance of the regression model and its components by breaking down total data variation into contributions from the model (regression), lack of fit, and pure error. The F-test compares the mean square of the regression with that of the residual error to assess model significance. A high F-value and a low p-value (usually below 0.05) indicate a statistically significant model (Gutt et al. 2016 ) Residual analysis is conducted to check the model's suitability by examining the differences between observed and predicted values. Common diagnostic tools include the normal probability plot, which confirms if residuals follow a normal distribution; the residuals vs. fitted values plot, which identifies non-random patterns that may signal issues with the model; and Cook’s distance and leverage plots, which detect influential points that may unduly affect model outcomes. The Lack-of-Fit test distinguishes variation due to model inadequacy from variation due to pure error (repeated measurements). A significant result (p-value < 0.05) indicates that the model may not fit the data well and might need to be improved, such as by including interaction terms or transforming variables. After validating the model, visual tools like 3D response surface plots and 2D contour plots are employed to explore factor interactions and determine optimal response conditions. These visualizations help clarify how input variables influence outcomes, aiding in the identification of ideal settings and further aiding validation feasibility (Naik and Ranade 2020 ) 2.7 PHA characterization Characterization of the biopolymer sample is essential to evaluate its structural, thermal, morphological, and mechanical properties, ensuring its suitability for intended applications. Structural analysis is typically performed using Fourier-transform infrared spectroscopy (FTIR) to identify functional groups and confirm the presence of characteristic PHA peaks, such as the carbonyl (C = O) stretch around 1725 cm⁻¹. Nuclear magnetic resonance (NMR) spectroscopy, particularly ¹H and ¹³C NMR, further confirms the molecular structure and monomer composition. 3. Results and Discussions 3.1 Molecular Identification of the novel isolated strain The strain isolated was examined for the sequence similarities of the 16S rRNA gene sequence using the BLASTN 2.3.1 + programme and NCBI sequence viewer 3.51.1. The database alignments of BLAST analysis showed 99.87% similarity with the 16S rRNA gene sequence of Pseudomonas stutzeri A1501(GenBank: Accession no. CP000304.1). Figure 1 represents the isolated strain cultures on an agar plate, a dendrogram illustrating the relationship among eleven isolates belonging to the genus Stutzerimonas , bar 0.003 represents substitution. 3.2 PHA production using cheese whey hydrolysate Whey composition analysis reveals that it contains approximately 7.3% of total sugars, 0.65% proteins, 0.41% fats, including 0.57% minerals before pre-treatment. After pre-treatment, total sugars (7.1%) and minerals (0.54%) are mostly retained while proteins (< 0.1%) are precipitated and removed, and fats (< 0.1%) are reduced. Pure sugars such as glucose, fructose, sucrose, xylose, and mannose are frequently used as model substrates in research and scale-up studies because they are easily metabolizable by many PHA-producing bacteria and allow controlled fermentation conditions due to their consistent composition, leading to high PHA yields and predictable kinetics. In this study, a novel isolate of S. stutzeri demonstrated the potential to produce PHA under submerged culture conditions, utilizing cheese whey (CW) hydrolysate as the carbon source. The cell biomass increased to 11.31 g/L when using cheese whey (CW) hydrolysate, surpassing the 9.89 g/L obtained with glucose as the control carbon source. This resulted in a PHA yield of up to 7.47 g/L, with PHA content ranging from 64.1–66.5% of the cell dry weight. The presence of growth-promoting factors in CW positively influenced both biomass accumulation and PHA synthesis. The higher cell biomass and increased PHA productivity indicate that this PHA-producing strain of S. stutzeri efficiently assimilated nitrogen from the whey. This efficiency may be attributed to a higher uptake rate across the cell membrane, potentially facilitated by the nitrogen being in a non-ionic form with reduced pH dependency during membrane transport. 3.3 Statistical significance and influence of parameters on PHA production To enhance PHA production by S. stutzeri utilizing CW hydrolysate, key physiological process parameters—such as carbon-to-phosphate (C/P) ratio (25–200 (w/w)), fermentation time (12–168 h), inoculum to substrate ratio (0.5–14.5 (v/v) %), carbon-to-nitrogen (C/N) ratio (10–65 (w/w)) —were systematically evaluated and optimized. The highest yield of 65.2% was achieved at a C/P ratio of 150, fermentation time of 120 h, inoculum ratio of 10%, and a C/N ratio of 50, indicating that high carbon availability and prolonged fermentation favoured PHA accumulation. In contrast, the lowest yield of 22.1% was observed under short fermentation time (24 h) and low inoculum (1%), despite a high C/P ratio, suggesting the importance of both cell density and time. C/P ratio and fermentation time showed a strong positive correlation with yield, and inoculum levels above 5.5%, particularly at 14.5%, significantly enhanced PHA production, reaching up to 61.1%. A C/N ratio in the range of 35–50 appeared optimal for balancing nutrient supply. Moderate conditions, such as a C/P ratio of 100, inoculum of 5.5%, and C/N ratio of 35, consistently yielded around 45–47%. Conversely, lower inoculum levels (≤ 1%) and low C/N ratios (≤ 20) generally resulted in poor yields. The data suggest that a synergistic effect of sufficient carbon, optimal nitrogen limitation, and higher biomass levels over time is key to maximizing PHA biosynthesis. Table 1 Using 4 factor Central Composite Design for high PHA production with actual and residual responses for all experimental set Run Factor 1 Factor 2 Factor 3 Factor 4 Response 1 A:C/P ratio (w/w) B: Fermentation time hrs C: Inoculum to substrate ratio (v/v) % D: C/N ratio (w/w) PHA production mg/100ml Actual Value Predicted Value Residual Value 1 150 120 10 50 65.2 65.2 65.47 -0.2705 2 150 24 10 20 28.4 28.4 29.01 -0.6077 3 50 120 10 50 55.45 55.45 56.12 -0.6732 4 150 120 1 20 34.3 34.3 33.5 0.7987 5 100 72 5.5 50 47.2 47.2 46.42 0.7827 6 75 72 5.5 35 43.5 43.50 44.62 -1.12 7 100 168 5.5 35 41.5 41.5 41.34 0.1562 8 150 24 1 20 22.1 22.1 21.48 0.6211 9 50 24 10 50 41.4 41.4 42.19 -0.7884 10 150 24 1 50 31.4 31.4 32.01 -0.6069 11 150 24 10 50 42.2 42.2 41.67 0.5268 12 100 72 5.5 35 47.1 47.1 45.33 1.77 13 100 72 0.5 35 34.6 34.6 35.75 -1.15 14 50 120 1 20 25.4 25.4 25.98 -0.5790 15 100 72 5.5 10 28.6 28.6 29.27 -0.6704 16 100 72 14.5 35 61.1 61.1 60.71 0.3911 17 200 72 5.5 35 51.6 51.6 51.49 0.1105 18 100 72 6.5 35 47.5 47.5 47.16 0.3399 19 50 24 1 20 24.1 24.1 23.82 0.2808 20 100 72 5.5 35 45.1 45.1 45.33 -0.2322 21 50 24 1 50 29.4 29.4 29.03 0.3654 22 100 72 5.5 35 45.1 45.1 45.33 -0.2322 23 150 120 10 20 53.1 53.1 53.52 -0.4175 24 150 120 1 50 42.8 42.8 43.32 -0.5167 25 25 72 5.5 35 44.2 44.2 44.2 0.0026 26 50 120 10 20 50.1 50.1 49.48 0.6172 27 100 12 5.5 35 30.3 30.3 30.59 -0.2929 28 50 120 1 50 31.1 31.1 30.48 0.6181 29 100 72 5.5 65 41.4 41.4 41.09 0.3117 30 50 24 10 20 35.1 35.1 34.84 0.4645 The relationship between the predicted and experimental values was statistically evaluated using Analysis of Variance (ANOVA) to assess the reliability of the quadratic polynomial model in predicting PHA Mass fraction yield (%). The ANOVA results for the model, highlights the statistical significance of the regression terms. The processed data obtained from the experimental runs facilitated the estimation of regression coefficients, which were subsequently used to construct the predictive polynomial equation for PHA yield. This model enables a quantitative understanding of the individual and interactive effects of the input variables on PHA production. Second order polynomial equation is as follows: PHA Mass fraction yield (%) = + 45.33 + 1.75 A + 6.49 B + 8.29 C + 4.29 D + 2.47 AB -0.8719 AC + 1.33 AD + 3.12 BC -0.1781 BD + 0.5344 CD + 0.6635 A² -4.24 B² -0.3021 C² -3.21 D² where (A) carbon-to-phosphorus (C/P) ratio, (B) fermentation time, (C) inoculum-to-substrate ratio, and (D) carbon-to-nitrogen (C/N) ratio of the culture medium Table 2 ANOVA table depicting the model validation Source Sum of Squares df Mean Square F-value p-value Model 3454.07 14 246.72 295.86 < 0.0001 significant A-C/P ratio 65.94 1 65.94 79.07 < 0.0001 B-Fermentation time 837.27 1 837.27 1004.04 < 0.0001 C-Inoculum to substrate ratio 1324.11 1 1324.11 1587.84 < 0.0001 D-C/N ratio 425.83 1 425.83 510.64 < 0.0001 AB 97.27 1 97.27 116.64 < 0.0001 AC 12.16 1 12.16 14.59 0.0017 AD 28.22 1 28.22 33.84 < 0.0001 BC 155.94 1 155.94 187.00 < 0.0001 BD 0.5077 1 0.5077 0.6088 0.4474 CD 4.57 1 4.57 5.48 0.0335 A² 8.41 1 8.41 10.09 0.0063 B² 312.59 1 312.59 374.85 < 0.0001 C² 1.52 1 1.52 1.83 0.1965 D² 217.47 1 217.47 260.78 < 0.0001 Residual 12.51 15 0.8339 Lack of Fit 9.84 13 0.7571 0.5678 0.7895 not significant Pure Error 2.67 2 1.33 Cor Total 3466.58 29 Factors affecting the % biomass PHA content can be observed through the 3D contour plots. Post-analysis of the design was performed to determine point prediction and confirm the predicted PHA mass fraction yield %, which was estimated at 56.47% using the following factors: C/P ratio 147.654(w/w), fermentation time 69.543 hours, inoculum to substrate ratio 8.83 (v/v) %, and C/N ratio 45.879 (w/w). After conducting triplicate experimental trials, a mean PHA production value of 66.5 ± 0.68% was observed higher than the estimated one by the design expert software. Further the cell kinetics studies were performed and are presented in Table 3 . Table 3 Cell kinetics studies for PHB production using S. stutzeri BPSNITW100893 DCW (g/L) PHA (g/L) PHA mass fraction yield% Biomass productivity (g/L/h) PHA productivity (g/L/h) 12.9 9.39 72.24% 1.806 1.305 13.11 10.18 78.30% 1.807 1.414 11.45 8.91 68.56% 1.806 1.239 12.49 ± 0.89 9.49 ± 0.64 73.03 ± 4.91% 1.806 ± 0.001 1.319 ± 0.089 3.4 Characterization of PHA produced by S. stutzeri 3.4.1 Nuclear magnetic resonance (NMR) The purified polyhydroxybutyrate (PHA) was characterized using proton nuclear magnetic resonance (1H NMR) spectroscopy as shown in Fig. 5. The analysis was performed by dissolving the PHA sample in deuterated chloroform (CDCl 3 ) at a concentration of 1 mg/ml and acquiring a spectrum on a Bruker Avance II 400 MHz spectrometer available in Central Research Instrumentation Facility, NIT Warangal. This method is well established for the structural elucidation of PHA, allowing the identification of characteristic signals corresponding to the polymer backbone and side chain methyl groups. The use of CDCl 3 as a solvent ensures good stability of PHA and minimal interference in the spectrum, facilitating the detection of chemical shifts and coupling patterns associated with the polymer. High resolution NMR spectroscopy, particularly at 400 MHz, enhances signal dispersion and provides detailed insights into the purity and composition of the sample, as demonstrated in the similar studies of PHA. In Fig. 5, peak at 5.28 ppm (A, quartet) corresponds to the methine proton (-CH-) adjacent to the ester oxygen. The splitting into a quartet suggests coupling with three equivalent protons (likely a methyl group). Peaks at 2.63 ppm (B, doublet of doublets) and 2.49 ppm (C, doublet of doublets) represents the methylene protons (-CH2-) in the polymer backbone. The splitting pattern indicates coupling with both the methine proton (A) and possibly the methyl protons (D or E). Peaks at 1.28 ppm (D, singlet) corresponds to one of the methine groups (-CH3) at the end of the polymer chain or branch. 0.86 ppm (E, singlet), this peak likely represents another methyl group (-CH3), slightly shifted downfield due to its specific chemical environment. Observations of peak at 5.28(A), 2.63(B), 2.49(C) and 1.28(D) corresponding to respective groups methyl, methylene and methine protons confirms of PHA monomer(Narayanan et al. 2021 ) . 3.4.2 Fourier Transform Infrared analysis FT-IR Analysis of the functional groups of purified PHA produced by Stutzerimonas stutzeri BPSNITW100893 was investigated by Bruker Alpha II ATR-FT-IR spectrophotometer that was equipped with opacus (analysis software) for Windows v.10 available in Central Research Instrumentation Facility, NIT Warangal. The following conditions were used: Spectral range, 4000–400 cm − 1; window material with 16 scans and resolution was adjusted to 4 cm − 1 (Kołodyńska et al. 2020 ). Figure 6 demonstrates the FT-IR peaks of the PHA compound, The strong and sharp peak at 1723.75 cm⁻¹ is a key indicator of PHA, representing the ester carbonyl (C = O) stretching vibration. This is the most prominent and diagnostic band for polyester-based biopolymers such as PHA. The C–H stretching vibrations are evident from the peaks at 2978.69 cm⁻¹ and 2934.57 cm⁻¹, which are typical for the methyl (–CH₃) and methylene (–CH₂) groups in the aliphatic backbone of PHA. Further confirming its structure, C–O–C and C–O stretching vibrations appear at 1281.23 cm⁻¹, 1222.45 cm⁻¹, 1183.55 cm⁻¹, and 1057.33 cm⁻¹, consistent with the ester functional groups present in the PHA polymer chain. The bending vibrations associated with the methyl and methylene groups are seen around 1381.06 cm⁻¹, while smaller peaks around 980.11 cm⁻¹ and lower suggest skeletal vibrations of the polymer backbone. The broad and less intense absorption band at 3436.61 cm⁻¹ could indicate O–H stretching, possibly from absorbed moisture or slight hydroxyl group contamination, which is common in polymer samples but does not alter the identification (Koller and Rodríguez-Contreras 2015 ; Nwinyi and Owolabi 2019 ; Narayanan et al. 2021 ; Christensen et al. 2023 ). 4. Conclusion The present study demonstrates the effective utilization of cheese whey as a carbon source for PHB production by a novel isolated strain of S. stutzeri . The strain exhibited high cell biomass and PHB accumulation under optimized conditions, confirming its robust metabolic potential for biopolymer synthesis. To the best of our knowledge, this is the first report directly employing S. stutzeri with cheese whey as the primary carbon source for PHA production, as no prior literature has documented this specific combination. The findings highlight both the valorization of dairy industry waste and the promising capabilities of S. stutzeri for sustainable bioplastic production. This study thus opens new avenues for cost-effective and environmentally friendly biopolymer manufacturing using agro-industrial residues. Declarations Author Contribution Bhanu Pratap Singh:Conceptualization, Investigation, Literature review, Resources, Writing original draftSatish Babu Rajulapati: Conceptualization, Supervision, Data curation, Writing – review & editing.Sridhar Pilli:Supervision, Writing - review & editing; Resources.R. D Tyagi:Review & Writing-editing, Resources References Acharjee SA, Bharali P, Gogoi B et al (2023) PHA-Based Bioplastic: a Potential Alternative to Address Microplastic Pollution. Water Air Soil Pollut 234 Amelia TSM, Govindasamy S, Tamothran AM et al (2019) Applications of PHA in agriculture. Biotechnological Applications of Polyhydroxyalkanoates. Springer Singapore, pp 347–361 Asiri F, Chu KH (2022) Valorization of agro-industrial wastes into polyhydroxyalkanoates-rich single-cell proteins to enable a circular waste-to-feed economy. Chemosphere 309. https://doi.org/10.1016/j.chemosphere.2022.136660 ASTM Committee (2021) Standard Specification for Labeling of Plastics Designed to be Aerobically Composted in Municipal or Industrial Facilities Bose SA, Rajulapati SB, Velmurugan S et al (2023) Process intensification of biopolymer polyhydroxybutyrate production by pseudomonas putida SS9: A statistical approach. Chemosphere 313. https://doi.org/10.1016/j.chemosphere.2022.137350 Brennan B, Lawler J, Regan F (2021) Recovery of viable ammonia-nitrogen products from agricultural slaughterhouse wastewater by membrane contactors: A review. Environ Sci (Camb) 7:259–273 Carvalheira M, Hilliou L, Oliveira CSS et al (2022) Polyhydroxyalkanoates from industrial cheese whey: Production and characterization of polymers with differing hydroxyvalerate content. Curr Res Biotechnol 4:211–220. https://doi.org/10.1016/j.crbiot.2022.03.004 Christensen M, Chiciudean I, Jablonski P et al (2023) Towards high-throughput screening (HTS) of polyhydroxyalkanoate (PHA) production via Fourier transform infrared (FTIR) spectroscopy of Halomonas sp. R5-57 and Pseudomonas sp. MR4-99. PLoS ONE 18. https://doi.org/10.1371/journal.pone.0282623 Farid NFSM, Ariffin H, Mamat MRZ et al (2015) Non-solvent-based pretreatment of poly(3-hydroxybutyrate) for improved bio-based crotonic acid production. RSC Adv 5:33546–33553. https://doi.org/10.1039/c5ra03017j Food and Agriculture Organization of the United States (2022) Crops and livestock products census of India Gonzfilez Siso MI (1996) THE BIOTECHNOLOGICAL UTILIZATION OF CHEESE WHEY. A REVIEW Gutt B, Kehl K, Ren Q, Boesel LF (2016) Using ANOVA Models to Compare and Optimize Extraction Protocols of P3HBHV from Cupriavidus necator. Ind Eng Chem Res 55:10355–10365. https://doi.org/10.1021/acs.iecr.6b02694 Hahn S, Hennecke D (2022) Final Report WP4-Comparison between natural and synthetic polymers Sponsor. European Chemicals Industry Council (Cefic Koller M, Rodríguez-Contreras A (2015) Techniques for tracing PHA-producing organisms and for qualitative and quantitative analysis of intra- and extracellular PHA. Eng Life Sci 15:558–581 Kołodyńska D, Fila D, Hubicki Z (2020) Recovery of lanthanum(III) and nickel(II) ions from acidic solutions by the highly effective ion exchanger. Molecules 25. https://doi.org/10.3390/molecules25163718 Mamat MRZ, Ariffin H, Hassan MA, Mohd Zahari MAK (2014) Bio-based production of crotonic acid by pyrolysis of poly(3-hydroxybutyrate) inclusions. J Clean Prod 83:463–472. https://doi.org/10.1016/j.jclepro.2014.07.064 Naik P, Ranade V (2020) Mathematical Modelling as a Tool to Optimize PHA Production by Massilia spp. J Adv Biol Biotechnol 19–29. https://doi.org/10.9734/jabb/2020/v23i230140 Narayanan M, Kandasamy G, Murali P et al (2021) Optimization and production of polyhydroxybutyrate from sludge by Bacillus cereus categorized through FT-IR and NMR analyses. J Environ Chem Eng 9. https://doi.org/10.1016/j.jece.2020.104908 Nwinyi OC, Owolabi TA (2019) Scanning electron microscopy and Fourier transmission analysis of polyhydroxyalkanoates isolated from bacteria species from abattoir in Ota, Nigeria. J King Saud Univ Sci 31:285–298. https://doi.org/10.1016/j.jksus.2017.08.003 Parodi A, Jorea A, Fagnoni M et al (2021) Bio-based crotonic acid from polyhydroxybutyrate: synthesis and photocatalyzed hydroacylation. Green Chem 23:3420–3427. https://doi.org/10.1039/d1gc00421b Pati S, Maity S, Dash A et al (2020) Biocompatible PHB Production from Bacillus Species Under Submerged and Solid-State Fermentation and Extraction Through Different Downstream Processing. Curr Microbiol 77:1203–1209. https://doi.org/10.1007/s00284-020-01922-7 Pérez-Padilla V, Molina-Henares MA, Udaondo Z et al (2025) Genetic basis of biofilm formation and salt adaptation in the plant-beneficial strain Stutzerimonas stutzeri MJL19. Appl Microbiol Biotechnol 109. https://doi.org/10.1007/s00253-025-13523-0 Pires AF, Marnotes NG, Rubio OD et al (2021) Dairy by-products: A review on the valorization of whey and second cheese whey. Foods 10 Prazeres AR, Carvalho F, Rivas J (2012) Cheese whey management: A review. J Environ Manage 110:48–68 Ratnaningrum D, Endah ES, Saraswaty V et al (2020) The effect of sodium hypochlorite concentration on extraction of poly-β-hidroxy-butyrate (PHB) produced from soil bacteria Burkholderia sp B37. In: IOP Conference Series: Earth and Environmental Science. Institute of Physics Publishing Reddy MV, Nandan Reddy VU, Chang YC (2022) Integration of anaerobic digestion and chain elongation technologies for biogas and carboxylic acids production from cheese whey. J Clean Prod 364. https://doi.org/10.1016/j.jclepro.2022.132670 Salvà-Serra F, Pérez-Pantoja D, Donoso RA et al (2023) Comparative genomics of Stutzerimonas balearica (Pseudomonas balearica): diversity, habitats, and biodegradation of aromatic compounds. Front Microbiol 14. https://doi.org/10.3389/fmicb.2023.1159176 Udourioh1 GA, Solomon2 MM, Okolie3 JA TITLE PAGE-Food Science of Animal Resources-Article Title A Review of the Valorization of Dairy Industry Wastes through Thermochemical, Biological, and Integrated Processes for Value-Added Products Running Title (within 10 words) Valorization of Dairy Industry Wastes for Value-Added Products: A Review Valentino F, Karabegovic L, Majone M et al (2015) Polyhydroxyalkanoate (PHA) storage within a mixed-culture biomass with simultaneous growth as a function of accumulation substrate nitrogen and phosphorus levels. Water Res 77:49–63. https://doi.org/10.1016/j.watres.2015.03.016 Zhang B, Zhou J, Wu J et al (2025) Unlocking N2O respiratory pathways in Stutzerimonas stutzeri PRE-2: Implications for reducing N2O emissions from estuaries. Mar Environ Res 206. https://doi.org/10.1016/j.marenvres.2025.107044 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-7168043\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":509041490,\"identity\":\"af627cb7-7e8d-45d5-9563-bbc370cb41ce\",\"order_by\":0,\"name\":\"Bhanu Pratap Singh\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"National Institute of Technology - Warangal\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Bhanu\",\"middleName\":\"Pratap\",\"lastName\":\"Singh\",\"suffix\":\"\"},{\"id\":509041491,\"identity\":\"72858eb4-8ee7-468f-b6d3-3661ba233572\",\"order_by\":1,\"name\":\"Satish Babu Rajulapati\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5klEQVRIie3RvYrCQBSG4S8IbpNgGxtzC2PjNoq3ckLASsFLsBobwdbLsFq1m3CKbQLbDmixS2B6EcRUu/6gxSIT7SzmLQcezgcDuFwvmvJGCgIVBbq+2EHlSqoEogcJLsQXuJ2xFY2/0rRYbqL3sL///jkwamPl8dBCRJaAg8w0V7PBQhAxwozAMxvBkXiSvbkefIREa0AD7NuGTXOkheTuXPfNmURlBDqBCiTHR1I9E1FGhM4FB9Ikq4lphdT79ZtZPCoZFufbQm46i7fE1A/tXqPxybyzDvuff/qmZ4DL5XK57vQH5VFYbwS0i18AAAAASUVORK5CYII=\",\"orcid\":\"\",\"institution\":\"National Institute of Technology - Warangal\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Satish\",\"middleName\":\"Babu\",\"lastName\":\"Rajulapati\",\"suffix\":\"\"},{\"id\":509041495,\"identity\":\"d2f99bf1-d3c5-4beb-b1c6-764d212c5580\",\"order_by\":2,\"name\":\"Sridhar Pilli\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"National Institute of Technology - Warangal\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Sridhar\",\"middleName\":\"\",\"lastName\":\"Pilli\",\"suffix\":\"\"},{\"id\":509041497,\"identity\":\"f43ddb51-deab-4a3f-8234-b14de8ab7402\",\"order_by\":3,\"name\":\"R. D. Tyagi\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Dongguan University of Technology\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"R.\",\"middleName\":\"D.\",\"lastName\":\"Tyagi\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2025-07-20 07:08:19\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-7168043/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-7168043/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":90688066,\"identity\":\"59885d44-006d-4407-89d4-50d9a7edd4dd\",\"added_by\":\"auto\",\"created_at\":\"2025-09-05 17:30:32\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":493034,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eMolecular identification and screening of \\u003cem\\u003eS. stutzeri\\u003c/em\\u003e BPSNITW100893\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7168043/v1/03a1f5e3de39847c77fcdbac.png\"},{\"id\":90688064,\"identity\":\"b99b1815-d33b-41ae-806a-14834b244fbd\",\"added_by\":\"auto\",\"created_at\":\"2025-09-05 17:30:32\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":503429,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e(a, b, c, d, e, f): 3-D contour plots illustrating the interactive effects of the pairs of independent variables for optimized PHA mole fraction yield (%).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7168043/v1/df0dbdaa93b55160989618b7.png\"},{\"id\":90688067,\"identity\":\"9915224d-8be3-4e0e-9eb4-222e8737cbfc\",\"added_by\":\"auto\",\"created_at\":\"2025-09-05 17:30:32\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":73328,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003csup\\u003e1\\u003c/sup\\u003eH NMR of PHA produced from\\u003cem\\u003e S. stutzeri \\u003c/em\\u003eBPSNITW100893 using cheese whey as substrate\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7168043/v1/0f2ce26b8cea61fa6efd3425.png\"},{\"id\":90688862,\"identity\":\"a93ef10a-36d4-4a66-8d54-eab2d87b8746\",\"added_by\":\"auto\",\"created_at\":\"2025-09-05 17:38:32\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":87809,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eFT-IR analysis of PH\\u003cem\\u003eA extracted from S. stutzeri \\u003c/em\\u003eBPSNITW100893 using cheese whey\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7168043/v1/c752a961699db326a6e98599.png\"},{\"id\":92389213,\"identity\":\"f085c6c2-6983-440f-98e2-2a1dca723c63\",\"added_by\":\"auto\",\"created_at\":\"2025-09-29 08:16:56\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":2361526,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7168043/v1/44412d8d-9e77-46bd-b7bb-be247d3670c6.pdf\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Bioconversion of whey to Polyhydroxyalkanoate (PHA): Process Optimization and Yield Enhancement\",\"fulltext\":[{\"header\":\"1. Introduction\",\"content\":\"\\u003cp\\u003eBiodegradable polymers are defined by various standards, with institutions such as ASTM, ISO, and DIN offering slightly different criteria based on microbial action and degradation outcomes (ASTM Committee \\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e; Hahn and Hennecke \\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e). Polyhydroxybutyrate (PHA), a type of biodegradable thermoplastic, has garnered interest for its biocompatibility and environmental benefits (Amelia et al. \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e; Acharjee et al. \\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e). However, high production cost, especially due to the use of expensive carbon sources and complex processing, limit its industrial viability. In contrast, dairy industry waste streams, such as cheese whey and associated wastewater, present a promising, nutrient-rich but underutilized resource for sustainable PHA production (Pires et al. \\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e). Cheese whey (CW), a by-product of cheese production, is generated in massive quantities worldwide. Producing 1 kg of cheese requires approximately 10 kg of milk, resulting in about 9 kg of by-products, primarily whey (Prazeres et al. \\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e2012\\u003c/span\\u003e). As of the FAO 2022 report, India\\u0026rsquo;s cheese production reached approximately 23.1\\u0026nbsp;million tonnes, generating an estimated 208\\u0026nbsp;million tonnes of cheese whey annually (Food and Agriculture Organization of the United Nations, 2022). The environmental implications of such vast whey volumes are significant due to CW\\u0026rsquo;s high biochemical oxygen demand (BOD), which ranges from 15,000 to 35,000 mg/L. This high organic load contributes to CO₂ emissions and presents serious risks to aquatic ecosystems (Gonzfilez Siso \\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e1996\\u003c/span\\u003e; Carvalheira et al. \\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e). Notably, the valorization of 10,000 litres of whey can offset about 0.10 to 0.24 tonnes of CO₂, demonstrating its potential environmental benefit when diverted from waste streams to useful applications. CW is a nutrient-rich substrate, typically containing around 44 g/L of lactose, 6 g/L of proteins, and 4 g/L of lipids, along with small amounts of poorly biodegradable proteins (Reddy et al. \\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e). These characteristics make whey a promising feedstock for microbial fermentation processes, particularly in the production of biodegradable plastics such as polyhydroxybutyrate (PHA). However, its inherent limitations\\u0026mdash;like a low carbon-to-nitrogen (C/N) ratio and complex, variable composition\\u0026mdash;can complicate direct microbial utilization. Nonetheless, research has shown that certain microbial strains, particularly those from the Pseudomonas genus, and native consortia isolated from dairy effluents, are capable of efficiently converting CW into PHA. This not only addresses the waste management challenge but also aligns with the principles of a circular economy, turning dairy industry by-products into eco-friendly, high-value materials.\\u003c/p\\u003e\\u003cp\\u003eTo enhance PHA yields from CW, careful optimization of bioconversion conditions is critical. Important factors include maintaining ideal C/N and C/P ratios (Valentino et al. \\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e), regulating pH, temperature, and aeration, and selecting effective cultivation strategies. Two-stage fermentation\\u0026mdash;where microbial growth precedes a nutrient-limitation phase\\u0026mdash;has proven effective in boosting PHA accumulation. Additionally, pre-treatment techniques improve substrate quality and fermentation efficiency. Fed-batch and semi-continuous fermentation methods help mitigate substrate inhibition, while recent advances in genetic engineering and omics technologies are enabling the development of highly efficient microbial strains. Through such integrated biotechnological innovations, cheese whey is increasingly being transformed from a significant pollutant into a valuable resource for sustainable bioplastic production.\\u003c/p\\u003e\\u003cp\\u003e\\u003cem\\u003eStutzerimonas stutzeri\\u003c/em\\u003e is a bacterial domain, Proteobacteria phylum, Gammaproteobacteria class, a metabolically versatile, Gram-negative bacterium known for its ecological adaptability and broad biotechnological potential that was reclassified in 2021 from the genus Pseudomonas based on phylogenomics. \\u003cem\\u003eS. stutzeri\\u003c/em\\u003e is widely distributed in soil, marine sediments, wastewater, and other contaminated environments. It exhibits notable capabilities in nitrogen cycling, particularly denitrification and, in some strains, nitrogen fixation. Owing to its ability to thrive under nutrient-limited and stress conditions, \\u003cem\\u003eS. stutzeri\\u003c/em\\u003e has been employed in the bioremediation of hydrocarbons, heavy metals, and other industrial pollutants (Salv\\u0026agrave;-Serra et al. \\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e; Zhang et al. \\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e2025\\u003c/span\\u003e; P\\u0026eacute;rez-Padilla et al. \\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e2025\\u003c/span\\u003e). Importantly, certain strains of the same genus are capable of accumulating polyhydroxybutyrate (PHA), a biodegradable polymer, under optimized carbon-rich and nitrogen-limited conditions. This makes it a promising strain for sustainable bioplastic production using low-cost substrates such as dairy wastewater or cheese whey. High genetic diversity, salt tolerance, and substrate flexibility of this novel strain further support its role in environmental biotechnology and circular waste valorization.\\u003c/p\\u003e\\u003cp\\u003eAs dairy wastewater volumes continue to rise alongside global dairy demand, efficient valorisation through optimized microbial fermentation and process innovation will play an increasingly important role in fostering a circular bioeconomy (Asiri and Chu \\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e). This study aims to investigate the efficiency and underlying mechanisms of PHA production from dairy wastewater using a novel \\u003cem\\u003eS. stutzeri\\u003c/em\\u003e strain, with the dual aim of mitigating environmental pollution and lowering the production cost of biodegradable plastics with high purity.\\u003c/p\\u003e\"},{\"header\":\"2. Materials and Methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e\\n \\u003ch2\\u003e2.1 Isolation, Screening, and culture maintenance of Lipolytic microbes\\u003c/h2\\u003e\\n \\u003cp\\u003eFood waste for microbial strain isolation used in this study was collected from the Institute Food Court - C at the National Institute of Technology (NIT), Warangal, India. The waste was composted aerobically through pit composting by burying it 25 cm below ground behind the mess facility for 45 days. Microbial isolation was carried out using serial dilution and spread plate techniques, followed by screening with Sudan Black B staining, Nile Blue A, and Nile Red assays to identify colonies capable of metabolizing complex organic substrates into polyhydroxyalkanoates (PHAs) (Godbole et al. 2016). Seven isolates were identified as responsible for producing PHA. Morphological characterization and biochemical assays were performed. Further identification of the isolate was confirmed through 16S rRNA gene sequencing. The selected strains were cultivated in nitrogen-balanced Mineral Salt Medium (NB-MSM) supplemented with a 1 molar glucose solution (10 mL/L) as the primary carbon source, using a shake flask with an incubation speed of 150 rpm and a temperature of 30℃. NB-MSM composed of (per litre): 10 g C\\u003csub\\u003e6\\u003c/sub\\u003eH\\u003csub\\u003e12\\u003c/sub\\u003eO\\u003csub\\u003e6\\u003c/sub\\u003e, 2 g (NH\\u003csub\\u003e4\\u003c/sub\\u003e)\\u003csub\\u003e2\\u003c/sub\\u003eSO\\u003csub\\u003e4\\u003c/sub\\u003e, 6.8 g Na\\u003csub\\u003e2\\u003c/sub\\u003eHPO\\u003csub\\u003e4\\u003c/sub\\u003e.7H\\u003csub\\u003e2\\u003c/sub\\u003eO, 1.5 g KH\\u003csub\\u003e2\\u003c/sub\\u003ePO\\u003csub\\u003e4\\u003c/sub\\u003e, 100 mg CaCl\\u003csub\\u003e2\\u003c/sub\\u003e.2H\\u003csub\\u003e2\\u003c/sub\\u003eO, 60 mg NH\\u003csub\\u003e4\\u003c/sub\\u003e-Fe-(III) citrate, 200 mg MgSO\\u003csub\\u003e4\\u003c/sub\\u003e.7H\\u003csub\\u003e2\\u003c/sub\\u003eO and 1 ml trace elements solution. The composition of trace elements in 1 L of solution is as follows: 100 mg ZnSO\\u003csub\\u003e4\\u003c/sub\\u003e\\u0026middot;7H\\u003csub\\u003e2\\u003c/sub\\u003eO, 30 mg MnCl\\u003csub\\u003e2\\u003c/sub\\u003e\\u0026middot;4H\\u003csub\\u003e2\\u003c/sub\\u003eO, 300 mg H\\u003csub\\u003e3\\u003c/sub\\u003eBO\\u003csub\\u003e3\\u003c/sub\\u003e, 200 mg CoCl\\u003csub\\u003e2\\u003c/sub\\u003e.6H\\u003csub\\u003e2\\u003c/sub\\u003eO, 20 mg CuSO\\u003csub\\u003e4\\u003c/sub\\u003e\\u0026middot;5H\\u003csub\\u003e2\\u003c/sub\\u003eO, 20 mg NiCl\\u003csub\\u003e2\\u003c/sub\\u003e.6H\\u003csub\\u003e2\\u003c/sub\\u003eO, and 30 mg NaMoO\\u003csub\\u003e4\\u003c/sub\\u003e (Bose et al. \\u003cspan class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e). One high-yielding novel strain, \\u003cem\\u003eStutzerimonas stutzeri\\u003c/em\\u003e BPSNITW100893, was selected for better results in terms of a maximum 6.41 g/L PHA biomass produced (approximately 61.32% PHA biomass yield). The isolated strain of \\u003cem\\u003eS. stutzeri\\u003c/em\\u003e was maintained on nutrient agar plates at 4\\u0026deg;C for short-term storage and further experimental use, and subculturing was performed once every thirty days to keep them alive.\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv id=\\\"Sec4\\\" class=\\\"Section2\\\"\\u003e\\n \\u003ch2\\u003e2.2 Pre-treatment of cheese whey as a carbon source\\u003c/h2\\u003e\\n \\u003cp\\u003eCheese whey was collected in a 5 L sterilized container from a domestic dairy near Warangal, India. In this study, the thermo-chemical method is used for pre-treatment to remove excess proteins before using whey lactose as a carbon source. CW is subjected to thermal pretreatment by boiling for 15 minutes at 100\\u0026deg;C, followed by cooling to room temperature. It was then filtered, and the whey supernatant pH 7.3\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.15 was adjusted to neutral pH 7.0\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.1 using 1N (NaOH/ H₂SO₄) and used as a feed for microbial growth and PHA production.\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv id=\\\"Sec5\\\" class=\\\"Section2\\\"\\u003e\\n \\u003ch2\\u003e2.3 PHA accumulation using submerged culture\\u003c/h2\\u003e\\n \\u003cp\\u003eThe organism was cultivated using a nitrogen-balanced (NB-MSM), to enhance the intracellular accumulation of PHA, bacterial cultures were grown in a nitrogen-deficient medium (ND-MSM), as previously described by (Brennan et al. \\u003cspan class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e). PHA production using a two-stage batch cultivation strategy by \\u003cem\\u003eS. stutzeri\\u003c/em\\u003e was studied with 250 mL Erlenmeyer flasks with 100 mL of media containing deproteinized cheese whey, which was replaced with glucose (10 g/L) used for control growth media. In the first stage, bacterial cells were cultivated in NB-MSM at 30\\u0026deg;C with agitation at 150 rpm until they reached the exponential growth phase. For enhanced PHA accumulation, the culture was then transitioned to a nitrogen-limited (ND-MSM) medium in the second stage. The initial nitrogen concentration in the medium was maintained at 2.0 g/L using ammonium sulphate (NH\\u003csub\\u003e4\\u003c/sub\\u003e)\\u003csub\\u003e2\\u003c/sub\\u003eSO\\u003csub\\u003e4\\u003c/sub\\u003e as the nitrogen source. To assess the influence of nitrogen availability on PHA accumulation, bacterial growth and PHA content were monitored across varying concentrations of the nitrogen source (0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0 g/L).\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv id=\\\"Sec6\\\" class=\\\"Section2\\\"\\u003e\\n \\u003ch2\\u003e2.4 Extraction and Recovery of PHA\\u003c/h2\\u003e\\n \\u003cp\\u003ePHA extraction was carried out following the dispersion method using sodium hypochlorite and chloroform (Pati et al. \\u003cspan class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e; Bose et al. \\u003cspan class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e). Cells were harvested by centrifugation at 10,000 rpm for 20 minutes at 4℃. The collected pellets were washed sequentially with deionized water and phosphate-buffered saline (PBS). After washing, the pellets were air-dried for 20 minutes, and the dry cell weight (DCW) was recorded using Eq.\\u0026nbsp;1.\\u003c/p\\u003e\\n \\u003cdiv id=\\\"Equa\\\" class=\\\"Equation\\\"\\u003e\\n \\u003cdiv class=\\\"mathdisplay\\\" id=\\\"FileID_Equa\\\" name=\\\"EquationSource\\\"\\u003e$$\\\\:DCW(g/L)\\\\:=\\\\:(Final\\\\:dry\\\\:weight\\\\:-\\\\:Initial\\\\:weight)/Volume\\\\:of\\\\:culture\\\\:\\\\left(L\\\\right)$$\\u003c/div\\u003e\\n \\u003c/div\\u003e\\n \\u003cp\\u003eThe extraction process involved adding chloroform and sodium hypochlorite (6%w/v) solution to washed cell pellets in a 1:1 volume ratio (12.5 \\u0026micro;L (v/v) each) adjusted proportionally to the dry cell weight to ensure complete lysis and efficient PHA extraction (Ratnaningrum et al. \\u003cspan class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e). The mixture was then incubated at 30\\u0026deg;C for 12 hours in borosilicate glass reagent bottles with leak proof blue screw cap. Following incubation, the solution was centrifuged at 10,000 rpm for 10 minutes at room temperature, leading to the formation of three distinct layers. The bottom chloroform layer, containing the dissolved PHA, was carefully separated and transferred to a clean ampoule for volume measurement. To precipitate the PHA, a methanol and water mixture (7:3 v/v) was added at five times the volume of the chloroform extracted. Further centrifugation at 10,000 rpm for 20 minutes at 4℃ enhanced the precipitation of PHA and calculated using Eq.\\u0026nbsp;2 denoting the PHA content in biomass (PHA mass fraction or yield %).\\u003c/p\\u003e\\n \\u003cdiv id=\\\"Equb\\\" class=\\\"Equation\\\"\\u003e\\n \\u003cdiv class=\\\"mathdisplay\\\" id=\\\"FileID_Equb\\\" name=\\\"EquationSource\\\"\\u003e$$\\\\:PHB\\\\:content\\\\:in\\\\:biomass=\\\\frac{Weight\\\\:of\\\\:PHB\\\\:\\\\left(powder\\\\right)\\\\:obtained\\\\left(in\\\\:grams\\\\right)}{Dry\\\\:cell\\\\:weight\\\\:\\\\left(in\\\\:grams\\\\right)}*100$$\\u003c/div\\u003e\\n \\u003c/div\\u003e\\n \\u003cp\\u003e\\u003cbr\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003eThe resulting pellets were separated, and 0.1 mL of concentrated sulfuric acid (H₂SO₄) was added to the pellets. The mixture was then boiled at 100\\u0026deg;C for 25 minutes and allowed to cool. A standard calibration curve for crotonic acid was prepared using known concentrations, and the PHA content was quantified by referencing a standard calibration curve with the standard graph and using the purity formula as in Eq.\\u0026nbsp;3 (Mamat et al. \\u003cspan class=\\\"CitationRef\\\"\\u003e2014\\u003c/span\\u003e; Farid et al. \\u003cspan class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e; Parodi et al. \\u003cspan class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e; Bose et al. \\u003cspan class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e).\\u003c/p\\u003e\\n \\u003cdiv id=\\\"Equc\\\" class=\\\"Equation\\\"\\u003e\\n \\u003cdiv class=\\\"mathdisplay\\\" id=\\\"FileID_Equc\\\" name=\\\"EquationSource\\\"\\u003e$$\\\\:PHB\\\\:Purity\\\\left(\\\\%\\\\right)=\\\\frac{Expected\\\\:Crotonic\\\\:Acid\\\\:\\\\left(\\\\mu\\\\:g/mL\\\\right)from\\\\:pure\\\\:PHB}{Measured\\\\:Crotonic\\\\:Acid\\\\:(\\\\mu\\\\:g/mL))}\\\\times\\\\:100$$\\u003c/div\\u003e\\n \\u003c/div\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv id=\\\"Sec7\\\" class=\\\"Section2\\\"\\u003e\\n \\u003ch2\\u003e2.5 Statistical optimization of culture media\\u003c/h2\\u003e\\n \\u003cp\\u003eIn this study, a rotatable Central Composite Design (rCCD) was employed to optimize the production of high cell density biomass using deproteinized cheese whey as a carbon source. The optimization focused on four key factors: carbon to nitrogen ratio (C/N), carbon to phosphorus ratio (C/P), fermentation time, and inoculum to substrate ratio. The C/N and C/P ratios were varied by adjusting the concentrations of ammonium sulphate and potassium dihydrogen phosphate, while keeping the carbon input (from cheese whey) constant. To fix the desired carbon-to-nitrogen (C/N) and carbon-to-phosphorus (C/P) ratios in the cheese whey-based medium, the total carbon content was first estimated based on the lactose concentration in whey, assuming that lactose comprises approximately 40% carbon by weight. For instance, with 20 g/L lactose, the carbon content was calculated as 16 g/L. Based on the selected C/N and C/P ratios in the experimental design, the required nitrogen and phosphorus concentrations were determined by using the formulas:\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:\\\\:\\\\text{R}\\\\text{e}\\\\text{q}\\\\text{u}\\\\text{i}\\\\text{r}\\\\text{e}\\\\text{d}\\\\:\\\\text{N}=\\\\text{C}\\\\text{a}\\\\text{r}\\\\text{b}\\\\text{o}\\\\text{n}\\\\:(\\\\text{g}/\\\\text{L})/(\\\\text{C}/\\\\text{N}\\\\:\\\\text{r}\\\\text{a}\\\\text{t}\\\\text{i}\\\\text{o})\\\\:\\\\)\\u003c/span\\u003e\\u003c/span\\u003eand \\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:\\\\text{R}\\\\text{e}\\\\text{q}\\\\text{u}\\\\text{i}\\\\text{r}\\\\text{e}\\\\text{d}\\\\:\\\\text{P}=\\\\:\\\\text{C}\\\\text{a}\\\\text{r}\\\\text{b}\\\\text{o}\\\\text{n}\\\\:(\\\\text{g}/\\\\text{L})/(\\\\text{C}/\\\\text{P}\\\\:\\\\text{r}\\\\text{a}\\\\text{t}\\\\text{i}\\\\text{o})\\\\)\\u003c/span\\u003e\\u003c/span\\u003e. External sources of nitrogen and phosphorus\\u0026mdash;ammonium sulphate and potassium dihydrogen phosphate, respectively\\u0026mdash;were added to the medium in amounts calculated to meet these ratios. The required mass of each compound was calculated using their respective nitrogen and phosphorus content by weight (approximately 21.2% for (NH\\u003csub\\u003e4\\u003c/sub\\u003e)\\u003csub\\u003e2\\u003c/sub\\u003eSO\\u003csub\\u003e4\\u003c/sub\\u003e and 22.8% for KH₂PO₄), ensuring precise control over nutrient levels in the medium while maintaining a constant carbon input from whey. This approach allowed systematic evaluation of the effects of nutrient balance on biomass accumulation. Four independent variables were selected based on their critical role in microbial growth and fermentation efficiency and were designated as: X\\u003csub\\u003e1\\u003c/sub\\u003e, X\\u003csub\\u003e2\\u003c/sub\\u003e, X\\u003csub\\u003e3\\u003c/sub\\u003e and X\\u003csub\\u003e4\\u003c/sub\\u003e. The CCD generated a total of 30 experimental runs, which included factorial points, axial points, and 6 center points to ensure robustness and to detect any curvature in the response surface.\\u003c/p\\u003e\\n \\u003cp\\u003eThe results from experimental designs using a shake flask were analyzed and interpreted using Design Expert software V 13.0.5.0. Based on the cell density response and the interaction effects among the studied variables, multiple regression analysis was performed to model the relationship between the independent variables and the response. A second-order polynomial equation was fitted to the experimental data obtained from the Central Composite Design (CCD). This model allowed for the evaluation of both linear and quadratic effects, as well as two-way interactions between the variables. The general form of the second-order polynomial model is given as:\\u003c/p\\u003e\\n \\u003cp\\u003eY\\u0026thinsp;=\\u0026thinsp;\\u0026beta;₀ + \\u0026Sigma;\\u0026beta;\\u003csub\\u003ei\\u003c/sub\\u003eX\\u003csub\\u003ei\\u003c/sub\\u003e + \\u0026Sigma;\\u0026beta;\\u003csub\\u003ei\\u003c/sub\\u003e\\u003csub\\u003ei\\u003c/sub\\u003eX\\u003csub\\u003ei\\u003c/sub\\u003e\\u0026sup2; + \\u0026Sigma;\\u0026Sigma;\\u0026beta;\\u003csub\\u003ei\\u003c/sub\\u003eⱼX\\u003csub\\u003ei\\u003c/sub\\u003eXⱼ + \\u0026epsilon;\\u003c/p\\u003e\\n \\u003cp\\u003ewhere Y is the predicted response, X\\u003csub\\u003ei\\u003c/sub\\u003e and Xⱼ represent the coded levels of the independent variables (C/N ratio, C/P ratio, fermentation time, and inoculum to substrate ratio), \\u0026beta;₀ is the intercept, \\u0026beta;\\u003csub\\u003ei\\u003c/sub\\u003e are the linear coefficients, \\u0026beta;\\u003csub\\u003ei\\u003c/sub\\u003e\\u003csub\\u003ei\\u003c/sub\\u003e are the quadratic coefficients, \\u0026beta;\\u003csub\\u003ei\\u003c/sub\\u003eⱼ are the interaction coefficients for i\\u0026thinsp;\\u0026ne;\\u0026thinsp;j, \\u0026epsilon; is the random error. This model was used to analyze the influence of each factor and their interactions, and to predict optimal conditions for maximum biomass production, omitting statistically not significant parameters (where p\\u0026thinsp;\\u0026gt;\\u0026thinsp;0.05).\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e\\n \\u003ch2\\u003e2.6 Data analysis and Model validation of CCD\\u003c/h2\\u003e\\n \\u003cp\\u003eThe regression model is used to predict optimal conditions and evaluate the importance of various factors. ANOVA (Analysis of Variance) helps determine the statistical relevance of the regression model and its components by breaking down total data variation into contributions from the model (regression), lack of fit, and pure error. The F-test compares the mean square of the regression with that of the residual error to assess model significance. A high F-value and a low p-value (usually below 0.05) indicate a statistically significant model (Gutt et al. \\u003cspan class=\\\"CitationRef\\\"\\u003e2016\\u003c/span\\u003e)\\u003c/p\\u003e\\n \\u003cp\\u003eResidual analysis is conducted to check the model\\u0026apos;s suitability by examining the differences between observed and predicted values. Common diagnostic tools include the normal probability plot, which confirms if residuals follow a normal distribution; the residuals vs. fitted values plot, which identifies non-random patterns that may signal issues with the model; and Cook\\u0026rsquo;s distance and leverage plots, which detect influential points that may unduly affect model outcomes. The Lack-of-Fit test distinguishes variation due to model inadequacy from variation due to pure error (repeated measurements). A significant result (p-value\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05) indicates that the model may not fit the data well and might need to be improved, such as by including interaction terms or transforming variables. After validating the model, visual tools like 3D response surface plots and 2D contour plots are employed to explore factor interactions and determine optimal response conditions. These visualizations help clarify how input variables influence outcomes, aiding in the identification of ideal settings and further aiding validation feasibility (Naik and Ranade \\u003cspan class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e)\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv id=\\\"Sec9\\\" class=\\\"Section2\\\"\\u003e\\n \\u003ch2\\u003e2.7 PHA characterization\\u003c/h2\\u003e\\n \\u003cp\\u003eCharacterization of the biopolymer sample is essential to evaluate its structural, thermal, morphological, and mechanical properties, ensuring its suitability for intended applications. Structural analysis is typically performed using Fourier-transform infrared spectroscopy (FTIR) to identify functional groups and confirm the presence of characteristic PHA peaks, such as the carbonyl (C\\u0026thinsp;=\\u0026thinsp;O) stretch around 1725 cm⁻\\u0026sup1;. Nuclear magnetic resonance (NMR) spectroscopy, particularly \\u0026sup1;H and \\u0026sup1;\\u0026sup3;C NMR, further confirms the molecular structure and monomer composition.\\u003c/p\\u003e\\n\\u003c/div\\u003e\"},{\"header\":\"3. Results and Discussions\",\"content\":\"\\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e\\n \\u003ch2\\u003e3.1 Molecular Identification of the novel isolated strain\\u003c/h2\\u003e\\n \\u003cp\\u003eThe strain isolated was examined for the sequence similarities of the 16S rRNA gene sequence using the BLASTN 2.3.1\\u0026thinsp;+\\u0026thinsp;programme and NCBI sequence viewer 3.51.1. The database alignments of BLAST analysis showed 99.87% similarity with the 16S rRNA gene sequence of Pseudomonas stutzeri A1501(GenBank: Accession no. CP000304.1). Figure 1 represents the isolated strain cultures on an agar plate, a dendrogram illustrating the relationship among eleven isolates belonging to the genus \\u003cem\\u003eStutzerimonas\\u003c/em\\u003e, bar 0.003 represents substitution.\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e\\n \\u003ch2\\u003e3.2 PHA production using cheese whey hydrolysate\\u003c/h2\\u003e\\n \\u003cp\\u003eWhey composition analysis reveals that it contains approximately 7.3% of total sugars, 0.65% proteins, 0.41% fats, including 0.57% minerals before pre-treatment. After pre-treatment, total sugars (7.1%) and minerals (0.54%) are mostly retained while proteins (\\u0026lt;\\u0026thinsp;0.1%) are precipitated and removed, and fats (\\u0026lt;\\u0026thinsp;0.1%) are reduced. Pure sugars such as glucose, fructose, sucrose, xylose, and mannose are frequently used as model substrates in research and scale-up studies because they are easily metabolizable by many PHA-producing bacteria and allow controlled fermentation conditions due to their consistent composition, leading to high PHA yields and predictable kinetics. In this study, a novel isolate of \\u003cem\\u003eS. stutzeri\\u003c/em\\u003e demonstrated the potential to produce PHA under submerged culture conditions, utilizing cheese whey (CW) hydrolysate as the carbon source. The cell biomass increased to 11.31 g/L when using cheese whey (CW) hydrolysate, surpassing the 9.89 g/L obtained with glucose as the control carbon source. This resulted in a PHA yield of up to 7.47 g/L, with PHA content ranging from 64.1\\u0026ndash;66.5% of the cell dry weight. The presence of growth-promoting factors in CW positively influenced both biomass accumulation and PHA synthesis. The higher cell biomass and increased PHA productivity indicate that this PHA-producing strain of \\u003cem\\u003eS. stutzeri\\u003c/em\\u003e efficiently assimilated nitrogen from the whey. This efficiency may be attributed to a higher uptake rate across the cell membrane, potentially facilitated by the nitrogen being in a non-ionic form with reduced pH dependency during membrane transport.\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e\\n \\u003ch2\\u003e3.3 Statistical significance and influence of parameters on PHA production\\u003c/h2\\u003e\\n \\u003cp\\u003eTo enhance PHA production by \\u003cem\\u003eS. stutzeri\\u003c/em\\u003e utilizing CW hydrolysate, key physiological process parameters\\u0026mdash;such as carbon-to-phosphate (C/P) ratio (25\\u0026ndash;200 (w/w)), fermentation time (12\\u0026ndash;168 h), inoculum to substrate ratio (0.5\\u0026ndash;14.5 (v/v) %), carbon-to-nitrogen (C/N) ratio (10\\u0026ndash;65 (w/w)) \\u0026mdash;were systematically evaluated and optimized. The highest yield of 65.2% was achieved at a C/P ratio of 150, fermentation time of 120 h, inoculum ratio of 10%, and a C/N ratio of 50, indicating that high carbon availability and prolonged fermentation favoured PHA accumulation. In contrast, the lowest yield of 22.1% was observed under short fermentation time (24 h) and low inoculum (1%), despite a high C/P ratio, suggesting the importance of both cell density and time. C/P ratio and fermentation time showed a strong positive correlation with yield, and inoculum levels above 5.5%, particularly at 14.5%, significantly enhanced PHA production, reaching up to 61.1%. A C/N ratio in the range of 35\\u0026ndash;50 appeared optimal for balancing nutrient supply. Moderate conditions, such as a C/P ratio of 100, inoculum of 5.5%, and C/N ratio of 35, consistently yielded around 45\\u0026ndash;47%. Conversely, lower inoculum levels (\\u0026le;\\u0026thinsp;1%) and low C/N ratios (\\u0026le;\\u0026thinsp;20) generally resulted in poor yields. The data suggest that a synergistic effect of sufficient carbon, optimal nitrogen limitation, and higher biomass levels over time is key to maximizing PHA biosynthesis.\\u003c/p\\u003e\\n \\u003cdiv class=\\\"gridtable\\\"\\u003e\\n \\u003ctable id=\\\"Tab1\\\" border=\\\"1\\\"\\u003e\\n \\u003ccaption language=\\\"En\\\"\\u003e\\n \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 1\\u003c/div\\u003e\\n \\u003cdiv class=\\\"CaptionContent\\\"\\u003e\\n \\u003cp\\u003eUsing 4 factor Central Composite Design for high PHA production with actual and residual responses for all experimental set\\u003c/p\\u003e\\n \\u003c/div\\u003e\\n \\u003c/caption\\u003e\\n \\u003cthead\\u003e\\n \\u003ctr\\u003e\\n \\u003cth align=\\\"left\\\" rowspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003eRun\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eFactor 1\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eFactor 2\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eFactor 3\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eFactor 4\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eResponse 1\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/th\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eA:C/P ratio\\u003c/p\\u003e\\n \\u003cp\\u003e(w/w)\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eB: Fermentation time\\u003c/p\\u003e\\n \\u003cp\\u003ehrs\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eC: Inoculum to substrate ratio\\u003c/p\\u003e\\n \\u003cp\\u003e(v/v) %\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eD: C/N ratio\\u003c/p\\u003e\\n \\u003cp\\u003e(w/w)\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003ePHA production\\u003c/p\\u003e\\n \\u003cp\\u003emg/100ml\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eActual Value\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003ePredicted Value\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eResidual\\u003c/p\\u003e\\n \\u003cp\\u003eValue\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/thead\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e150\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e120\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e10\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e50\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e65.2\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e65.2\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e65.47\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e-0.2705\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e2\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e150\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e24\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e10\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e20\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e28.4\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e28.4\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e29.01\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e-0.6077\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e3\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e50\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e120\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e10\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e50\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e55.45\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e55.45\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e56.12\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e-0.6732\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e4\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e150\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e120\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e20\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e34.3\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e34.3\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e33.5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e0.7987\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e100\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e72\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e5.5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e50\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e47.2\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e47.2\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e46.42\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e0.7827\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e6\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e75\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e72\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e5.5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e35\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e43.5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e43.50\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e44.62\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e-1.12\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e7\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e100\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e168\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e5.5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e35\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e41.5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e41.5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e41.34\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e0.1562\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e8\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e150\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e24\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e20\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e22.1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e22.1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e21.48\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e0.6211\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e9\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e50\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e24\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e10\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e50\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e41.4\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e41.4\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e42.19\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e-0.7884\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e10\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e150\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e24\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e50\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e31.4\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e31.4\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e32.01\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e-0.6069\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e11\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e150\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e24\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e10\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e50\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e42.2\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e42.2\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e41.67\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e0.5268\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e12\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e100\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e72\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e5.5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e35\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e47.1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e47.1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e45.33\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e1.77\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e13\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e100\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e72\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e0.5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e35\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e34.6\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e34.6\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e35.75\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e-1.15\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e14\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e50\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e120\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e20\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e25.4\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e25.4\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e25.98\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e-0.5790\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e15\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e100\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e72\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e5.5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e10\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e28.6\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e28.6\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e29.27\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e-0.6704\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e16\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e100\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e72\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e14.5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e35\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e61.1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e61.1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e60.71\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e0.3911\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e17\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e200\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e72\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e5.5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e35\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e51.6\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e51.6\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e51.49\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e0.1105\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e18\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e100\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e72\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e6.5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e35\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e47.5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e47.5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e47.16\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e0.3399\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e19\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e50\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e24\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e20\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e24.1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e24.1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e23.82\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e0.2808\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e20\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e100\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e72\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e5.5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e35\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e45.1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e45.1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e45.33\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e-0.2322\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e21\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e50\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e24\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e50\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e29.4\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e29.4\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e29.03\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e0.3654\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e22\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e100\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e72\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e5.5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e35\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e45.1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e45.1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e45.33\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e-0.2322\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e23\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e150\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e120\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e10\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e20\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e53.1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e53.1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e53.52\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e-0.4175\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e24\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e150\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e120\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e50\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e42.8\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e42.8\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e43.32\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e-0.5167\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e25\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e25\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e72\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e5.5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e35\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e44.2\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e44.2\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e44.2\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e0.0026\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e26\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e50\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e120\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e10\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e20\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e50.1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e50.1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e49.48\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e0.6172\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e27\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e100\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e12\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e5.5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e35\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e30.3\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e30.3\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e30.59\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e-0.2929\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e28\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e50\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e120\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e50\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e31.1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e31.1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e30.48\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e0.6181\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e29\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e100\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e72\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e5.5\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e65\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e41.4\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e41.4\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e41.09\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e0.3117\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e30\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e50\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e24\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e10\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e20\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e35.1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e35.1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e34.84\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e0.4645\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n \\u003c/table\\u003e\\n \\u003c/div\\u003e\\n \\u003cp\\u003eThe relationship between the predicted and experimental values was statistically evaluated using Analysis of Variance (ANOVA) to assess the reliability of the quadratic polynomial model in predicting PHA Mass fraction yield (%). The ANOVA results for the model, highlights the statistical significance of the regression terms. The processed data obtained from the experimental runs facilitated the estimation of regression coefficients, which were subsequently used to construct the predictive polynomial equation for PHA yield. This model enables a quantitative understanding of the individual and interactive effects of the input variables on PHA production. Second order polynomial equation is as follows:\\u003c/p\\u003e\\n \\u003cp\\u003ePHA Mass fraction yield (%)\\u0026thinsp;=\\u0026thinsp;+\\u0026thinsp;45.33\\u0026thinsp;+\\u0026thinsp;1.75 A\\u0026thinsp;+\\u0026thinsp;6.49 B\\u0026thinsp;+\\u0026thinsp;8.29 C\\u0026thinsp;+\\u0026thinsp;4.29 D\\u0026thinsp;+\\u0026thinsp;2.47 AB -0.8719 AC\\u0026thinsp;+\\u0026thinsp;1.33 AD\\u0026thinsp;+\\u0026thinsp;3.12 BC -0.1781 BD\\u0026thinsp;+\\u0026thinsp;0.5344 CD\\u0026thinsp;+\\u0026thinsp;0.6635 A\\u0026sup2; -4.24 B\\u0026sup2; -0.3021 C\\u0026sup2; -3.21 D\\u0026sup2;\\u003c/p\\u003e\\n \\u003cp\\u003ewhere (A) carbon-to-phosphorus (C/P) ratio, (B) fermentation time, (C) inoculum-to-substrate ratio, and (D) carbon-to-nitrogen (C/N) ratio of the culture medium\\u003c/p\\u003e\\n \\u003cdiv class=\\\"gridtable\\\"\\u003e\\n \\u003ctable id=\\\"Tab2\\\" border=\\\"1\\\"\\u003e\\n \\u003ccaption language=\\\"En\\\"\\u003e\\n \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 2\\u003c/div\\u003e\\n \\u003cdiv class=\\\"CaptionContent\\\"\\u003e\\n \\u003cp\\u003eANOVA table depicting the model validation\\u003c/p\\u003e\\n \\u003c/div\\u003e\\n \\u003c/caption\\u003e\\n \\u003cthead\\u003e\\n \\u003ctr\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eSource\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eSum of Squares\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003edf\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eMean Square\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eF-value\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003ep-value\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/th\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/thead\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eModel\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e3454.07\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e14\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e246.72\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e295.86\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e\\u0026lt;\\u0026thinsp;0.0001\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003esignificant\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eA-C/P ratio\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e65.94\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e65.94\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e79.07\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e\\u0026lt;\\u0026thinsp;0.0001\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eB-Fermentation time\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e837.27\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e837.27\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e1004.04\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e\\u0026lt;\\u0026thinsp;0.0001\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eC-Inoculum to substrate ratio\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e1324.11\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e1324.11\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e1587.84\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e\\u0026lt;\\u0026thinsp;0.0001\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eD-C/N ratio\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e425.83\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e425.83\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e510.64\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e\\u0026lt;\\u0026thinsp;0.0001\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eAB\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e97.27\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e97.27\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e116.64\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e\\u0026lt;\\u0026thinsp;0.0001\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eAC\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e12.16\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e12.16\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e14.59\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e0.0017\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eAD\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e28.22\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e28.22\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e33.84\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e\\u0026lt;\\u0026thinsp;0.0001\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eBC\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e155.94\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e155.94\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e187.00\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e\\u0026lt;\\u0026thinsp;0.0001\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eBD\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e0.5077\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e0.5077\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e0.6088\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e0.4474\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eCD\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e4.57\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e4.57\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e5.48\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e0.0335\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eA\\u0026sup2;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e8.41\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e8.41\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e10.09\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e0.0063\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eB\\u0026sup2;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e312.59\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e312.59\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e374.85\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e\\u0026lt;\\u0026thinsp;0.0001\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eC\\u0026sup2;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e1.52\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e1.52\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e1.83\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e0.1965\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eD\\u0026sup2;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e217.47\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e217.47\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e260.78\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e\\u0026lt;\\u0026thinsp;0.0001\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eResidual\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e12.51\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e15\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e0.8339\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eLack of Fit\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e9.84\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e13\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e0.7571\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e0.5678\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e0.7895\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003enot significant\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003ePure Error\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e2.67\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e2\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e1.33\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eCor Total\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e3466.58\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e29\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n \\u003c/table\\u003e\\n \\u003c/div\\u003e\\n \\u003cp\\u003eFactors affecting the % biomass PHA content can be observed through the 3D contour plots.\\u003c/p\\u003e\\n \\u003cp\\u003ePost-analysis of the design was performed to determine point prediction and confirm the predicted PHA mass fraction yield %, which was estimated at 56.47% using the following factors: C/P ratio 147.654(w/w), fermentation time 69.543 hours, inoculum to substrate ratio 8.83 (v/v) %, and C/N ratio 45.879 (w/w). After conducting triplicate experimental trials, a mean PHA production value of 66.5\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.68% was observed higher than the estimated one by the design expert software. Further the cell kinetics studies were performed and are presented in Table \\u003cspan class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e.\\u003c/p\\u003e\\n \\u003cdiv class=\\\"gridtable\\\"\\u003e\\n \\u003ctable id=\\\"Tab3\\\" border=\\\"1\\\"\\u003e\\n \\u003ccaption language=\\\"En\\\"\\u003e\\n \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 3\\u003c/div\\u003e\\n \\u003cdiv class=\\\"CaptionContent\\\"\\u003e\\n \\u003cp\\u003eCell kinetics studies for PHB production using S. stutzeri BPSNITW100893\\u003c/p\\u003e\\n \\u003c/div\\u003e\\n \\u003c/caption\\u003e\\n \\u003cthead\\u003e\\n \\u003ctr\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eDCW\\u003c/p\\u003e\\n \\u003cp\\u003e(g/L)\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003ePHA\\u003c/p\\u003e\\n \\u003cp\\u003e(g/L)\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003ePHA mass fraction yield%\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eBiomass productivity\\u003c/p\\u003e\\n \\u003cp\\u003e(g/L/h)\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003ePHA productivity\\u003c/p\\u003e\\n \\u003cp\\u003e(g/L/h)\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/thead\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e12.9\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e9.39\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e72.24%\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e1.806\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e1.305\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e13.11\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e10.18\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e78.30%\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e1.807\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e1.414\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e11.45\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e8.91\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e68.56%\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e1.806\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e1.239\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003e12.49\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.89\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e9.49\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.64\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e73.03\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;4.91%\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e1.806\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.001\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e1.319\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.089\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n \\u003c/table\\u003e\\n \\u003c/div\\u003e\\n \\u003cp\\u003e\\u003cbr\\u003e\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv id=\\\"Sec14\\\" class=\\\"Section2\\\"\\u003e\\n \\u003ch2\\u003e3.4 Characterization of PHA produced by \\u003cem\\u003eS. stutzeri\\u003c/em\\u003e\\u003c/h2\\u003e\\n \\u003cdiv id=\\\"Sec15\\\" class=\\\"Section3\\\"\\u003e\\n \\u003ch2\\u003e3.4.1 Nuclear magnetic resonance (NMR)\\u003c/h2\\u003e\\n \\u003cp\\u003eThe purified polyhydroxybutyrate (PHA) was characterized using proton nuclear magnetic resonance (1H NMR) spectroscopy as shown in Fig.\\u0026nbsp;5. The analysis was performed by dissolving the PHA sample in deuterated chloroform (CDCl\\u003csub\\u003e3\\u003c/sub\\u003e) at a concentration of 1 mg/ml and acquiring a spectrum on a Bruker Avance II 400 MHz spectrometer available in Central Research Instrumentation Facility, NIT Warangal. This method is well established for the structural elucidation of PHA, allowing the identification of characteristic signals corresponding to the polymer backbone and side chain methyl groups. The use of CDCl\\u003csub\\u003e3\\u003c/sub\\u003e as a solvent ensures good stability of PHA and minimal interference in the spectrum, facilitating the detection of chemical shifts and coupling patterns associated with the polymer. High resolution NMR spectroscopy, particularly at 400 MHz, enhances signal dispersion and provides detailed insights into the purity and composition of the sample, as demonstrated in the similar studies of PHA. In Fig. 5, peak at 5.28 ppm (A, quartet) corresponds to the methine proton (-CH-) adjacent to the ester oxygen. The splitting into a quartet suggests coupling with three equivalent protons (likely a methyl group). Peaks at 2.63 ppm (B, doublet of doublets) and 2.49 ppm (C, doublet of doublets) represents the methylene protons (-CH2-) in the polymer backbone. The splitting pattern indicates coupling with both the methine proton (A) and possibly the methyl protons (D or E). Peaks at 1.28 ppm (D, singlet) corresponds to one of the methine groups (-CH3) at the end of the polymer chain or branch. 0.86 ppm (E, singlet), this peak likely represents another methyl group (-CH3), slightly shifted downfield due to its specific chemical environment. Observations of peak at 5.28(A), 2.63(B), 2.49(C) and 1.28(D) corresponding to respective groups methyl, methylene and methine protons confirms of PHA monomer(Narayanan et al. \\u003cspan class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e) .\\u003c/p\\u003e\\n \\u003c/div\\u003e\\n \\u003cdiv id=\\\"Sec16\\\" class=\\\"Section3\\\"\\u003e\\n \\u003ch2\\u003e3.4.2 Fourier Transform Infrared analysis\\u003c/h2\\u003e\\n \\u003cp\\u003eFT-IR Analysis of the functional groups of purified PHA produced by \\u003cem\\u003eStutzerimonas stutzeri\\u003c/em\\u003e BPSNITW100893 was investigated by Bruker Alpha II ATR-FT-IR spectrophotometer that was equipped with opacus (analysis software) for Windows v.10 available in Central Research Instrumentation Facility, NIT Warangal. The following conditions were used: Spectral range, 4000\\u0026ndash;400 cm\\u0026thinsp;\\u0026minus;\\u0026thinsp;1; window material with 16 scans and resolution was adjusted to 4 cm\\u0026thinsp;\\u0026minus;\\u0026thinsp;1 (Kołodyńska et al. \\u003cspan class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e). Figure 6 demonstrates the FT-IR peaks of the PHA compound, The strong and sharp peak at 1723.75 cm⁻\\u0026sup1; is a key indicator of PHA, representing the ester carbonyl (C\\u0026thinsp;=\\u0026thinsp;O) stretching vibration. This is the most prominent and diagnostic band for polyester-based biopolymers such as PHA. The C\\u0026ndash;H stretching vibrations are evident from the peaks at 2978.69 cm⁻\\u0026sup1; and 2934.57 cm⁻\\u0026sup1;, which are typical for the methyl (\\u0026ndash;CH₃) and methylene (\\u0026ndash;CH₂) groups in the aliphatic backbone of PHA. Further confirming its structure, C\\u0026ndash;O\\u0026ndash;C and C\\u0026ndash;O stretching vibrations appear at 1281.23 cm⁻\\u0026sup1;, 1222.45 cm⁻\\u0026sup1;, 1183.55 cm⁻\\u0026sup1;, and 1057.33 cm⁻\\u0026sup1;, consistent with the ester functional groups present in the PHA polymer chain. The bending vibrations associated with the methyl and methylene groups are seen around 1381.06 cm⁻\\u0026sup1;, while smaller peaks around 980.11 cm⁻\\u0026sup1; and lower suggest skeletal vibrations of the polymer backbone. The broad and less intense absorption band at 3436.61 cm⁻\\u0026sup1; could indicate O\\u0026ndash;H stretching, possibly from absorbed moisture or slight hydroxyl group contamination, which is common in polymer samples but does not alter the identification (Koller and Rodr\\u0026iacute;guez-Contreras \\u003cspan class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e; Nwinyi and Owolabi \\u003cspan class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e; Narayanan et al. \\u003cspan class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e; Christensen et al. \\u003cspan class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e).\\u003c/p\\u003e\\n \\u003c/div\\u003e\\n\\u003c/div\\u003e\"},{\"header\":\"4. Conclusion\",\"content\":\"\\u003cp\\u003eThe present study demonstrates the effective utilization of cheese whey as a carbon source for PHB production by a novel isolated strain of \\u003cem\\u003eS. stutzeri\\u003c/em\\u003e. The strain exhibited high cell biomass and PHB accumulation under optimized conditions, confirming its robust metabolic potential for biopolymer synthesis. To the best of our knowledge, this is the first report directly employing \\u003cem\\u003eS. stutzeri\\u003c/em\\u003e with cheese whey as the primary carbon source for PHA production, as no prior literature has documented this specific combination. The findings highlight both the valorization of dairy industry waste and the promising capabilities of \\u003cem\\u003eS. stutzeri\\u003c/em\\u003e for sustainable bioplastic production. This study thus opens new avenues for cost-effective and environmentally friendly biopolymer manufacturing using agro-industrial residues.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003ch2\\u003eAuthor Contribution\\u003c/h2\\u003e\\u003cp\\u003eBhanu Pratap Singh:Conceptualization, Investigation, Literature review, Resources, Writing original draftSatish Babu Rajulapati: Conceptualization, Supervision, Data curation, Writing \\u0026ndash; review \\u0026amp; editing.Sridhar Pilli:Supervision, Writing - review \\u0026amp; editing; Resources.R. D Tyagi:Review \\u0026amp; Writing-editing, Resources\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eAcharjee SA, Bharali P, Gogoi B et al (2023) PHA-Based Bioplastic: a Potential Alternative to Address Microplastic Pollution. Water Air Soil Pollut 234\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eAmelia TSM, Govindasamy S, Tamothran AM et al (2019) Applications of PHA in agriculture. Biotechnological Applications of Polyhydroxyalkanoates. Springer Singapore, pp 347\\u0026ndash;361\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eAsiri F, Chu KH (2022) Valorization of agro-industrial wastes into polyhydroxyalkanoates-rich single-cell proteins to enable a circular waste-to-feed economy. Chemosphere 309. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1016/j.chemosphere.2022.136660\\u003c/span\\u003e\\u003cspan address=\\\"10.1016/j.chemosphere.2022.136660\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eASTM Committee (2021) Standard Specification for Labeling of Plastics Designed to be Aerobically Composted in Municipal or Industrial Facilities\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eBose SA, Rajulapati SB, Velmurugan S et al (2023) Process intensification of biopolymer polyhydroxybutyrate production by pseudomonas putida SS9: A statistical approach. Chemosphere 313. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1016/j.chemosphere.2022.137350\\u003c/span\\u003e\\u003cspan address=\\\"10.1016/j.chemosphere.2022.137350\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eBrennan B, Lawler J, Regan F (2021) Recovery of viable ammonia-nitrogen products from agricultural slaughterhouse wastewater by membrane contactors: A review. Environ Sci (Camb) 7:259\\u0026ndash;273\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eCarvalheira M, Hilliou L, Oliveira CSS et al (2022) Polyhydroxyalkanoates from industrial cheese whey: Production and characterization of polymers with differing hydroxyvalerate content. Curr Res Biotechnol 4:211\\u0026ndash;220. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1016/j.crbiot.2022.03.004\\u003c/span\\u003e\\u003cspan address=\\\"10.1016/j.crbiot.2022.03.004\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eChristensen M, Chiciudean I, Jablonski P et al (2023) Towards high-throughput screening (HTS) of polyhydroxyalkanoate (PHA) production via Fourier transform infrared (FTIR) spectroscopy of Halomonas sp. R5-57 and Pseudomonas sp. MR4-99. PLoS ONE 18. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1371/journal.pone.0282623\\u003c/span\\u003e\\u003cspan address=\\\"10.1371/journal.pone.0282623\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eFarid NFSM, Ariffin H, Mamat MRZ et al (2015) Non-solvent-based pretreatment of poly(3-hydroxybutyrate) for improved bio-based crotonic acid production. RSC Adv 5:33546\\u0026ndash;33553. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1039/c5ra03017j\\u003c/span\\u003e\\u003cspan address=\\\"10.1039/c5ra03017j\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eFood and Agriculture Organization of the United States (2022) Crops and livestock products census of India\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eGonzfilez Siso MI (1996) THE BIOTECHNOLOGICAL UTILIZATION OF CHEESE WHEY. A REVIEW\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eGutt B, Kehl K, Ren Q, Boesel LF (2016) Using ANOVA Models to Compare and Optimize Extraction Protocols of P3HBHV from Cupriavidus necator. Ind Eng Chem Res 55:10355\\u0026ndash;10365. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1021/acs.iecr.6b02694\\u003c/span\\u003e\\u003cspan address=\\\"10.1021/acs.iecr.6b02694\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eHahn S, Hennecke D (2022) Final Report WP4-Comparison between natural and synthetic polymers Sponsor. European Chemicals Industry Council (Cefic\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eKoller M, Rodr\\u0026iacute;guez-Contreras A (2015) Techniques for tracing PHA-producing organisms and for qualitative and quantitative analysis of intra- and extracellular PHA. Eng Life Sci 15:558\\u0026ndash;581\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eKołodyńska D, Fila D, Hubicki Z (2020) Recovery of lanthanum(III) and nickel(II) ions from acidic solutions by the highly effective ion exchanger. Molecules 25. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.3390/molecules25163718\\u003c/span\\u003e\\u003cspan address=\\\"10.3390/molecules25163718\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eMamat MRZ, Ariffin H, Hassan MA, Mohd Zahari MAK (2014) Bio-based production of crotonic acid by pyrolysis of poly(3-hydroxybutyrate) inclusions. J Clean Prod 83:463\\u0026ndash;472. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1016/j.jclepro.2014.07.064\\u003c/span\\u003e\\u003cspan address=\\\"10.1016/j.jclepro.2014.07.064\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eNaik P, Ranade V (2020) Mathematical Modelling as a Tool to Optimize PHA Production by Massilia spp. J Adv Biol Biotechnol 19\\u0026ndash;29. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.9734/jabb/2020/v23i230140\\u003c/span\\u003e\\u003cspan address=\\\"10.9734/jabb/2020/v23i230140\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eNarayanan M, Kandasamy G, Murali P et al (2021) Optimization and production of polyhydroxybutyrate from sludge by Bacillus cereus categorized through FT-IR and NMR analyses. J Environ Chem Eng 9. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1016/j.jece.2020.104908\\u003c/span\\u003e\\u003cspan address=\\\"10.1016/j.jece.2020.104908\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eNwinyi OC, Owolabi TA (2019) Scanning electron microscopy and Fourier transmission analysis of polyhydroxyalkanoates isolated from bacteria species from abattoir in Ota, Nigeria. J King Saud Univ Sci 31:285\\u0026ndash;298. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1016/j.jksus.2017.08.003\\u003c/span\\u003e\\u003cspan address=\\\"10.1016/j.jksus.2017.08.003\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eParodi A, Jorea A, Fagnoni M et al (2021) Bio-based crotonic acid from polyhydroxybutyrate: synthesis and photocatalyzed hydroacylation. Green Chem 23:3420\\u0026ndash;3427. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1039/d1gc00421b\\u003c/span\\u003e\\u003cspan address=\\\"10.1039/d1gc00421b\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003ePati S, Maity S, Dash A et al (2020) Biocompatible PHB Production from Bacillus Species Under Submerged and Solid-State Fermentation and Extraction Through Different Downstream Processing. Curr Microbiol 77:1203\\u0026ndash;1209. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1007/s00284-020-01922-7\\u003c/span\\u003e\\u003cspan address=\\\"10.1007/s00284-020-01922-7\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eP\\u0026eacute;rez-Padilla V, Molina-Henares MA, Udaondo Z et al (2025) Genetic basis of biofilm formation and salt adaptation in the plant-beneficial strain Stutzerimonas stutzeri MJL19. Appl Microbiol Biotechnol 109. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1007/s00253-025-13523-0\\u003c/span\\u003e\\u003cspan address=\\\"10.1007/s00253-025-13523-0\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003ePires AF, Marnotes NG, Rubio OD et al (2021) Dairy by-products: A review on the valorization of whey and second cheese whey. Foods 10\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003ePrazeres AR, Carvalho F, Rivas J (2012) Cheese whey management: A review. J Environ Manage 110:48\\u0026ndash;68\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eRatnaningrum D, Endah ES, Saraswaty V et al (2020) The effect of sodium hypochlorite concentration on extraction of poly-β-hidroxy-butyrate (PHB) produced from soil bacteria Burkholderia sp B37. In: IOP Conference Series: Earth and Environmental Science. Institute of Physics Publishing\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eReddy MV, Nandan Reddy VU, Chang YC (2022) Integration of anaerobic digestion and chain elongation technologies for biogas and carboxylic acids production from cheese whey. J Clean Prod 364. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1016/j.jclepro.2022.132670\\u003c/span\\u003e\\u003cspan address=\\\"10.1016/j.jclepro.2022.132670\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eSalv\\u0026agrave;-Serra F, P\\u0026eacute;rez-Pantoja D, Donoso RA et al (2023) Comparative genomics of Stutzerimonas balearica (Pseudomonas balearica): diversity, habitats, and biodegradation of aromatic compounds. Front Microbiol 14. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.3389/fmicb.2023.1159176\\u003c/span\\u003e\\u003cspan address=\\\"10.3389/fmicb.2023.1159176\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eUdourioh1 GA, Solomon2 MM, Okolie3 JA TITLE PAGE-Food Science of Animal Resources-Article Title A Review of the Valorization of Dairy Industry Wastes through Thermochemical, Biological, and Integrated Processes for Value-Added Products Running Title (within 10 words) Valorization of Dairy Industry Wastes for Value-Added Products: A Review\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eValentino F, Karabegovic L, Majone M et al (2015) Polyhydroxyalkanoate (PHA) storage within a mixed-culture biomass with simultaneous growth as a function of accumulation substrate nitrogen and phosphorus levels. Water Res 77:49\\u0026ndash;63. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1016/j.watres.2015.03.016\\u003c/span\\u003e\\u003cspan address=\\\"10.1016/j.watres.2015.03.016\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eZhang B, Zhou J, Wu J et al (2025) Unlocking N2O respiratory pathways in Stutzerimonas stutzeri PRE-2: Implications for reducing N2O emissions from estuaries. Mar Environ Res 206. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1016/j.marenvres.2025.107044\\u003c/span\\u003e\\u003cspan address=\\\"10.1016/j.marenvres.2025.107044\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Polyhydroxyalkanoate, cheese whey hydrolysate, polymers, waste valorization\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-7168043/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-7168043/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003ePolyhydroxyalkanoate (PHA) is a biodegradable biopolymer with significant potential as an eco-friendly substitute for conventional plastics. This study investigates microbial bio-transformation and enhanced biosynthesis of polyhydroxyalkanoate (PHA) using cheese whey (a dairy industry processing waste) as a substrate. The process employs \\u003cem\\u003eStutzerimonas stutzeri\\u003c/em\\u003e BPSNITW100893, a novel strain isolated from food waste generated at the Institute Food Court-C of NIT Warangal, which produces higher PHA, compared to six isolated strains from initial screening using mineral salt media (MSM) and characterized using FTIR and NMR. A rotatable Central Composite Design (rCCD) based optimization using four key factors remarkably enhanced production under optimal physiological conditions, i.e., C/P ratio (147.6 w/w), fermentation time (69.6 hours), inoculum to substrate ratio 8.83 (v/v) %, C/N ratio 45.9 (w/w). A high PHA mass fraction yield % of 66.51% was observed as compared to the predicted yield of 56.48% from cheese whey hydrolysate as feed. Scale-up studies were successfully conducted up to 3 L with optimized parameters confirmed by cell proliferation studies. These studies demonstrated high productivity with a maximum PHA mole fraction yield of 73.03\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;4.92% and a productivity rate of 1.319\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.089 g/L/h, highlighting the potential of dairy processing waste as a substrate for sustainable biopolymer production and waste valorization.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Bioconversion of whey to Polyhydroxyalkanoate (PHA): Process Optimization and Yield Enhancement\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-09-05 17:30:27\",\"doi\":\"10.21203/rs.3.rs-7168043/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"4a7cca17-c53a-4e76-a479-3fe06520466f\",\"owner\":[],\"postedDate\":\"September 5th, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2025-09-29T08:08:46+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2025-09-05 17:30:27\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-7168043\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-7168043\",\"identity\":\"rs-7168043\",\"version\":[\"v1\"]},\"buildId\":\"8U1c8b4HqxoKbykW_rLl7\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}