Production of polyhydroxybutyrate (PHB) in Scenedesmus acutus using a low-cost substrate

Production of polyhydroxybutyrate (PHB) in Scenedesmus acutus using a low-cost substrate | 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 Production of polyhydroxybutyrate (PHB) in Scenedesmus acutus using a low-cost substrate Alejandro Valdez-Calderon, Saúl López-Gómez, Arian Espinosa-Roa, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8399498/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 12 You are reading this latest preprint version Abstract The importance of conventional plastics is undeniable; however, their non-biodegradability makes them one of the biggest environmental problems. Among the alternatives to mitigate environmental damages, the production of polyhydroxyalkanoates (PHB) provides a biodegradable solution, additional to their mechanical and thermal properties, making them accessible to a wide variety of applications. In general, biopolymers accumulate as energy and carbon storage material in microorganisms such as bacteria and microalgae. In the present study, the impact of different sources like carbon, glucose, nitrogen and sodium was tested on the production of PHB in the cultures of the microalgae S. acutus . To evaluate the effect of the variables, a fractional Taguchi experimental design was devised and executed, thus, 16 experimental runs and 3 replicas in each treatment were considered. Results showed calculated concentrations of the biopolymer in a range from 7.5–34.7% w/w dry weight. Additionally, the PHB was identified by spectroscopic and thermogravimetric analysis. Statistical analysis was performed using Minitab 16, where differences in biomass production, PHB concentration in g/L and the percentage of PHB were analyzed. Likewise, a Pareto diagram was used to consider the biomass production results, with glucose, biomass-glucose, and biomass-sodium as determining factors in the PHB production. The present research provides significant data on critical factors related with PHA production, for the first time to our knowledge, thus showing Scenedesmus acutus as a PHB producer through low-cost medium providing a promising candidate for industrial scale-up. Polyhydroxybutyrate (PHB) Scenedesmus acutus biopolymers microalgae-based production Figures Figure 1 Figure 2 Figure 3 1. Introduction The global demand for polymerical materials continues to increase driven by the growth in the consumption of conventional plastics, however, the production of petroleum derivatives has been compromised due their high price, caused by the amount of energy consumed during manufacturing. Conventional plastics have a significant negative impact on the environment; they are primarily derived from crude oil, a non-renewable natural resource; exhibit poor biodegradability, leading to their accumulation in landfills and marine environments for centuries; moreover, their incineration releases harmful chemical substances; they enter the food chain in the form of microplastics; and their widespread use places increasing pressure on existing waste management infrastructure. Due to the previous reasons, alternatives have been sought to replace them, as bioplastics [ 1 – 3 ]. Bioplastics are materials that, given appropriate factors of humidity, soil composition and temperature, present a short degradation period compared to conventional ones [ 4 – 5 ]. Other advantages are improvement on soil fertility, diversified feedstock, reduced carbon footprint, managed end of life, reduced greenhouse gas emission, reduced reliance on fossil full, sustainable and ecofriendly [ 6 – 9 ]. Bioplastics can be divided into 3 categories such as biobased non degradable plastic, biobased biodegradable plastics and fossil based biodegradable plastics [ 10 ]. Biodegradable biobased plastics such as polylactic acid (PLA), polyhydroxyalkanoates (PHAs), starch, and cellulose are widely used. Their demand has increased due to their origin from renewable sources, as well as their ability to undergo biodegradation. Among these materials, starch has been the most extensively utilized owing to its abundance in natural environments [ 11 – 12 ]; however, its production has declined, possibly because starch constitutes a fundamental resource for the food industry [ 13 ]. A strong alternative is PHAs, an emerging family of aliphatic polyesters whose global market was expected to reach an annual value of USD 93 million by the end of 2023, and to nearly double by 2028 with USD 195 million [ 14 – 16 ]. These bioplastics are naturally synthesized by different types of bacteria and microalgae, accumulating in the form of insoluble particles in their cytoplasm [ 17 ]. For 2019, it represented 1.2% of the production volumes of plastics of biological origin and increased up to 1.7% by the end of 2020. Although there are reports of up to 150 different types of PHAs, the monomers of 4 to 6 carbon atoms are the most commonly studied, as in the case of poly-3-hydroxybutyrate (PHB) [ 18 – 19 ]. For PHB, it is known that there are more than 300 bacteria used for production, including genera such as, Alcaligenes , Azotobacter , Bacillus , Pseudomonas , Rhizobium , Rhodospirillum and Cupriavidus , the latter being C. necator the largest producer of PHB in yield with high levels (90%) [ 20 – 22 ]. For microalgae, genera such as Chloroella , Desmodesmus , Ralstonia and Spirulina have been previously reported, producing this bioplastic with maximum production levels of up to 45% (in relation to their biomass) [ 1 , 23 – 25 ]. Although, the values are lower than in the case of bacteria, there are several advantages inherent to microalgae cultivation, such as, easy to monitor, high production of biomass, cost effective, ecofriendly and harnessed to produce various bioactive compounds for commercial use, producing an added value [ 26 – 27 ]. The variation in PHB production in microalgae has been studied and associated with different nutrients and changes in their growth conditions. Previous studies have reported the influence of macronutrients such as glucose and micronutrients such as nitrogen, phosphorus, sodium and iron [ 28 – 29 ], and the use of low-cost sources such as supplemental organic carbon [ 30 ] and vegetable waste such as cassava peel [ 31 ]. In this work it is reported the use of a low-cost medium for the growth of S. acutus in 16 different nutrient variants to obtain PHB, and evaluated the results. 2. Materials and Methods 2.1 Microalgae Strain and Culture Medium All experiments were carried out with biomass from Scenedesmus acutus obtained from the repository of the Technological University of the Metropolitan Area of the Valley of Mexico “UTVAM” (Tizayuca, México; 19°51′59″N 98°59′04″W). This species was cultured in laboratory conditions at room temperature with purified water, receiving constant aeration under a white LED light (10–40 W/m; 900–3300 lumens/m), using commercial bicarbonate as a carbon source and fertilizer (Ferti Plus™) as a source of nitrogen and micronutrients. For the different tests, biomass recovered from this medium, known as “traditional medium”, was used. Sixteen different media were proposed to promote PHB production, with variations in nitrogen and carbon sources, while maintaining the base culture growth conditions. 2.2 Experimental design Scenedesmus acutus was grown in sixteen different modified culture media that were prepared to have diverse nutrient conditions. The culture media were prepared by selecting factors found in previous research. Four relevant factors in the generation of PHB during the cultivation of microalgae were considered: carbon, glucose, nitrogen and sodium. To determine the relevant factors in Scenedesmus acutus , an experimental Taguchi matrix was designed. The Taguchi method uses orthogonal arrays, which stipulate the way of conducting the minimal number of experiments that will give the information of all the factors that affect the performance parameter [ 22 ]. A binary factorial experimental design was formulated with the help of Minitab software where the different interactions of these factors were explored, taking into account their absence and excess, resulting in a total of 16 formulated (Table 1 ). Table 1 Factor levels of Taguchi design. Variable (source) Name Levels Low High N (Fertilizer) A 0 mL/L 2 mL/L C (Bicarbonate) B 2.5 g/L 5 g/L Na (sodium nitrate) C 0 g/L 2 g/L Glucose D 0 g/L 3 g/L 2.3 Culture Conditions Cells of microalgae of S. acutus kept in 1 L of protein medium were used as inoculum (5 mL) for liquid cultures. The experiment was carried out for 7 days in 600 mL plastic Erlenmeyer containing 1 mL per liter of Bold basal medium in 500-mL of purified water at 25ºC, under continuous illumination with cold white light fluorescent lamps (100 µmol m 2 /sec) and atmospheric CO 2 (200 mL/sec). At the end of cultivation, the bioplastic production was analyzed. 2.4 Dry cell weight determination (DCW) To determine dry cell weight (DCW), culture samples (10 mL) were centrifuged (15,000 rpm, 15 min, 4°C) and cell pellets were washed with deionized water after removing supernatant. The harvested cell pellets were then vacuum-dried until reaching a constant weight and DCW was then measured [ 32 ]. 2.5 PHB extraction, quantification and characterization The PHB was extracted based on the methodology of [ 28 ], the microalgae were separated from the culture medium by centrifuging at 4000 rpm and the biomass pellets obtained were dried by leaving them at 90°C for 40 h. After that, the samples were resuspended in distilled water at a ratio of 1:10 (m/v) and 1 mL of 2 N HCl was added at a temperature of 40–50°C for 2 hours in a water bath. After this process, it was centrifuged (4000 rpm) for 20 minutes, the pellets were recovered and 5 mL of chloroform was added, allowing it to rest for 24 hours. All supply materials were purchased from Meyer and Sigma-Aldrich, and were used without further purification. The solvents were purified by distillation over appropriate drying agents. Photophysical properties were measured by electronic absorption spectra obtained by a Perkin Elmer LAMBDA 2S UV-Vis Spectrophotometer in a 100–800 nm range. Afterwards, the chemical properties of PHAs were recorded and scanned by an Agilent Technologies Cary 600 Fourier Transform Infrared Spectroscope (FTIR), in a range of 600 to 4000 wave number (cm − 1 ). After PHAs dissolution in CDCl 3 , a Varian Inova 400 Nuclear Magnetic Resonance Spectrometer (RMN) was used to record Proton Nuclear Magnetic Resonance ( 1 H-NMR) spectra at 20ºC, and their chemical shifts (δ) were reported in ppm. Additionally, the Differential Scanning Calorimetry (DSC) curve was measured on a Differential Scanning Calorimeter (DSC Discovey 2500, TA Instruments) under a nitrogen flow of 50 mL/min, in the temperature range of 25–300°C with heating and cooling rates of 10°C/min on samples in 40 µL aluminum crucibles sealed with pierced lids. 3. Results The biopolymers obtained were characterized by UV spectroscopy, FTIR spectroscopy and DSC. In all the experiments, the typical signals of the biopolymer were identified, so for practical purposes only the results are shown for A2 (Fig. 2 ), which was the experimental one with the highest percentage of PHB calculated. For the optical characterization of PHB using UV-visible spectroscopy, its maximum absorption was identified at 217 nm (Fig. 2 a), making evident a low absorption capacity where the observable band corresponds to its S 0 -S 1 electronic transition (π → π*) of the carbonyl group. In the case of FTIR spectroscopy (Fig. 2 b), firstly two bands were observed at 2990 cm − 1 and 2940 cm − 1 , corresponding to carbons C–H with sp 2 and sp 3 geometries, continuing with a band around 1722 cm − 1 , referring to the carbonyl group of the ester bond, followed by the band at 1276 cm − 1 of the –CH group, all of them coincide with the bands of biopolymer. The band observed at 1455 cm − 1 refers to the asymmetric deformation of the CH 3 bond, while the band at 1377 cm − 1 is assigned to the asymmetric movement of the methyl groups. In addition, the bands observed at 1331 cm − 1 are characteristic of the asymmetric and symmetric vibration of the C–O–C group. Each crop component was considered as a variable that could potentially affect PHB production. Glucose, bicarbonate, fertilizer and sodium nitrate levels were considered, in principle, influential. A two-level full factorial design (2 4 ) would imply a total of 16 experiments, plus the replications necessary for the evaluation of the degree of coincidence between the results. Therefore, a two-level Taguchi design involving 16 random runs was selected. Table 2 shows the design matrix for the experiment with the quantities contributed by each of the variables and the total yield of PHB production in percentage and m/v concentration. Table 2 Design matrix response values for Taguchi design with PHB values. Run Glucose (gL − 1 ) Carbon (gL − 1 ) Nitrogen (gL − 1 ) Sodium (gL − 1 ) Biomass (gL − 1 ) PHB (gL − 1 ) PHB (% w/w) 1 0 2.5 0 0 0.085 ± 0.003 0.018 ± 0.002 21.56 ± 1.4 2 0 5 0 0 0.057 ± 0.013 0.014 ± 0.002 24.70 ± 0.9 3 0 2.5 2 0 0.085 ± 0.007 0.010 ± 0.002 12.05 ± 2.1 4 0 5 2 0 0.091 ± 0.003 0.014 ± 0.001 16.13 ± 1.5 5 0 2.5 0 2 0.079 ± 0.003 0.012 ± 0.002 15.13 ± 1.8 6 0 5 0 2 0.103 ± 0.005 0.015 ± 0.002 15.88 ± 1.2 7 0 2.5 2 2 0.091 ± 0.007 0.008 ± 0.002 9.08 ± 2.2 8 0 5 2 2 0.119 ± 0.003 0.026 ± 0.001 22.15 ± 1.7 9 3 2.5 0 0 0.084 ± 0.010 0.012 ± 0.001 14.39 ± 1.9 10 3 5 0 0 0.082 ± 0.007 0.009 ± 0.001 11.68 ± 1.5 11 3 2.5 2 0 0.391 ± 0.009 0.029 ± 0.002 7.50 ± 0.8 12 3 5 2 0 0.259 ± 0.007 0.028 ± 0.002 10.80 ± 0.5 13 3 2.5 0 2 0.184 ± 0.014 0.015 ± 0.003 8.27 ± 1.2 14 3 5 0 2 0.211 ± 0.013 0.022 ± 0.002 10.23 ± 0.8 15 3 2.5 2 2 0.188 ± 0.008 0.019 ± 0.004 10.04 ± 1.9 16 3 5 2 2 0.170 ± 0.009 0.022 ± 0.001 13.35 ± 1.4 The biomass growth in the 16 different variants, after the established days, is shown in Table 2 and its statistical analysis is visualized in Fig. 2 a. The range of values ​​was determined between 0.57 − 0.391 gL − 1 , with the maximum being condition run 11, where sodium was omitted, and the lowest being condition run 2, which contained sodium as the only nutrient in the medium. From its statistical analysis, significant differences were observed between medium conditions 11, 12, 14 and 13 (in decreasing order of biomass quantity) and all the other runs, and it is important to highlight that they had the highest biomass generation performance. The remaining runs were very similar in their biomass values ​​as observed in runs 1, 3–7, 9 and 10 with no significant differences. Now moving on to the analysis of PHB production (Fig. 2 b, Table 2 ) the highest values ​​in a range of 0.025–0.030 gL − 1 were presented in runs 11, 12 and 8 (in decreasing order). In 8 it was obtained in the absence of glucose and for 11 and 12 sodium was omitted. There was no significant difference between the three conditions with the highest concentrations, but it is important to emphasize that there was a significant difference in the remaining runs. The remaining runs had similar ranges of PHB production, so it was observed that runs 1, 2, 4–6, 9, 13, 7, and 16 were very similar. It is important to mention that the percentage of PHB obtained reflects the improvements in the methodology and media conditions, since this relationship involves the concentration of bioplastics relative to the dry biomass generated in each particular case. According to the statistical analysis of % PHB shown in Fig. 2 c, the maximum value for condition 2 was 24.7%, followed by 8 and 1 with values ​​of 22.15% and 21.56%, respectively; the three maximum values ​​did not show significant differences. For cases 3–7, 9, and 10, no differences were found, and their range was 12–16% PHB. The run with the lowest percentage was condition 11 with 7.50%. The analysis of results using Minitab 16 lets discard the less significant factors and interactions. For this experiment, the factors were considered significant at p < 0.05. In the first, the four factors referring to nutrients were analyzed and in a second study, the values of biomass produced were added. With the above, two Pareto diagrams were created, shown in Fig. 3 . In the second Pareto, glucose can be seen as the determining factor individually, followed by a combination of biomass generated with glucose and biomass with sodium respectively. 4. Discussion The results obtained in this study demonstrate that the culture conditions evaluated, particularly light intensity, nitrogen and sodium availability, glucose and bicarbonate concentration, the commercial fertilizer used as the base of the medium, and the culture growth time, exert a determining influence on polyhydroxybutyrate (PHB) accumulation and biomass generation in Scenedesmus acutus . Modulation of the carbon-nitrogen ratio emerged as an essential element for inducing the intracellular storage of this biopolymer, consistent with reports for other green microalgae species [ 33 ]. The biomass obtained under the different experimental combinations showed variations attributable to the interaction between light intensity, nutrient availability, and the photosynthetic capacity of the culture. High light intensities favored higher growth rates, possibly due to greater photosynthetic activity, while the glucose supply acted as an external carbon source capable of sustaining metabolism even under nitrogen deficiency [ 34 ]. This pattern has been widely documented in Scenedesmus spp. [ 35 ]. However, excessive light levels could generate photo-oxidative stress, which would explain some of the occasional decreases in biomass observed [ 36 ]. Comparison with previous studies shows that the biomass obtained (0.39–0.57 g/L) is lower than that reported in conventional media such as BG-11 or agro-industrial wastewater (2–3 g/L), but it is within similar or higher ranges than those obtained with low-cost media [ 37 – 38 ]. This confirms that the use of a commercial fertilizer as the base of the medium represents a viable economic alternative for large-scale intensive crops [ 39 ]. Statistical analysis using a two-level Taguchi design allowed for the identification of conditions with significant differences. Runs 11, 12, and 14 generated the greatest biomass, particularly when sodium was omitted, suggesting that this nutrient could limit the growth of S. acutus at certain concentration ranges. Regarding PHB accumulation, the results show that nutritional stress, especially nitrogen or sodium restriction, induces a metabolic imbalance that favors the diversion of carbon toward the synthesis of polyhydroxyalkanoates. Runs 11, 12, and 8 recorded the highest absolute concentrations (0.025–0.030 g/L), while the relative percentages of PHB reached values ​​up to 24.7%. These figures are comparable to values ​​reported in Scenedesmus obliquus and Chlorella vulgaris [ 25 ] and, in some cases, higher than recent studies of Scenedesmus spp. [ 38 ]. Comparatively, the PHB percentages obtained are lower than those reported for cyanobacteria such as Spirulina sp. or Nostoc muscorum (30–31%) under extreme nutrient limitation conditions, but higher than those of strains such as Synechocystis sp. and Botryococcus braunii , which typically range from 10–17% [ 40 – 41 ]. These results show that S. acutus has an intermediate-to-high performance within the microalgal spectrum for PHB production, and that its accumulation depends strongly on the availability of external carbon and the type of nutrient limitation imposed. Characterization of the biopolymer using UV-visible spectroscopy, FTIR, and DSC thermal analysis confirmed the nature of PHB in all treatments, revealing maximum UV absorption at 217 nm, corresponding to the π→π* electronic transition of the carbonyl group. Characteristic FTIR bands of PHB: 2990 and 2940 cm⁻¹ (C–H sp2/sp3), 1722 cm⁻¹ (carbonyl of the ester), 1455 and 1377 cm⁻¹ (vibrations of CH₃), 1331–1276 cm⁻¹ (C–O–C vibration of the ester), the slight variations observed in relative intensities could be attributed to the presence of impurities or cellular remnants, suggesting the need to optimize extraction and purification processes using combined methods [ 42 ]. Analysis with Minitab identified glucose as the most influential factor on PHB production, followed by glucose-biomass and sodium-biomass interactions. The agreement between experimental values ​​and estimated main effects suggests that the predictive model accurately describes the culture dynamics. However, the nature of the Taguchi design limits the detection of complex interactions; therefore, future studies could consider full factorial designs or response surface models [ 43 ]. These results suggest that Scenedesmus acutus has significant potential as a PHB producer, especially under nutritional stress and in the presence of external carbon sources. The combination of cost-effectiveness, experimental optimization strategies, and reliable structural characterization of the biopolymer suggests the feasibility of integrating this microalga into industrial bioplastics production schemes [ 44 ]. However, significant challenges remain: improving extraction yields, reducing energy costs, and evaluating production performance in open and semi-closed systems. Addressing these issues will pave the way for more efficient and sustainable processes that can be economically competitive for PHB production from microalgae. 5. Conclusions The genus Scenedesmus has been scarcely explored in the field of biopolymer production. This work, specifically for the species Scenedesmus acutus , reports the production of PHB for the first time. It was carried out using different variables, starting with low-cost sources. It was possible to identify that the limiting factor of glucose and its combination with nitrogen had significant results derived from its statistical analysis in a balance of biomass generation and PHB production. This, along with the addition of the biomass experiment, was corroborated in a Pareto-type multivariate study, where glucose was identified as the main factor, followed by glucose and biomass generation. The range of % PHB in the results we obtained (7.50–24.70%) reflects the importance of the variation in nutrients associated with the production of bioplastic, so we contribute to the optimization of resources with low-cost reagents and leaves open the possibility of continuing to optimize the obtaining of PHB in Scenedesmus , a bioplastic that increases its use every year from a microalga that expands its applications. Declarations Author Contribution AVC contributed to Conceptualization, Methodology, Formal Analysis, Investigation, and Writing – Original Draft Preparation. SLG participated in Methodology, Data Curation, Formal Analysis, and Writing – Review & Editing. ARE contributed to Software development, Validation, Visualization, and Data Curation. BKGP was involved in Investigation, Resources management, and Writing , Review & Editing. JRGR contributed to Conceptualization, Supervision. MAGM participated in Methodology, Validation, Visualization, and Writing – Review & Editing. All authors reviewed and approved the final version of the manuscript and agree to be accountable for all aspects of the work. Acknowledgement The Acknowledgments section should include information on the source of any financial support received for the work being published, before the References. The authors acknowledge the financial support provided by the Secretaría de Ciencia, Humanidades, Tecnología e Innovación (SECIHTI), Mexico, which made this research possible. The authors also express their sincere gratitude to Engineer Angélica María Soto Herrera for her valuable technical support as the laboratory technician in charge of the Process Laboratory at the Universidad Tecnológica de la Zona Metropolitana del Valle de México (UTVAM). Data Availability The data underlying this article are available in the article and its online supplementary material. 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Biomolecules 10(12):1628. https://doi.org/10.3390/biom10121628 Shayesteh H, Laird DW, Hughes LJ, Nematollahi MA, Kakhki AM, Moheimani NR (2023) Co-producing phycocyanin and bioplastic in Arthrospira platensis using carbon-rich wastewater. BioTech 12(3):49. https://doi.org/10.3390/biotech12030049 Moraes-Mourão MM, Xavier LP, Urbatzka R, Figueiroa LB, Costa CEFd, Dias CGBT, Schneider MPC, Vasconcelos V, Santos AV (2021) Characterization and biotechnological potential of intracellular polyhydroxybutyrate by Stigeoclonium sp. B23 using cassava peel as carbon source. Polymers 13(5):687. https://doi.org/10.3390/polym13050687 Khomlaem C, Aloui H, Deshmukh AR, Yun JH, Kim HS, Napathorn SC, Kim BS (2020) Defatted Chlorella biomass as a renewable carbon source for polyhydroxyalkanoates and carotenoids co-production. Algal Res 51:102068. https://doi.org/10.1016/j.algal.2020.102068 Agarwal P, Soni R, Kaur P, Madan A, Mishra R, Pandey J et al (2022) Cyanobacteria as a promising alternative for sustainable environment: synthesis of biofuel and biodegradable plastics. Front Microbiol 13:939347. https://doi.org/10.3389/fmicb.2022.939347 Morales-Sánchez D, Martinez-Rodriguez OA, Martinez A (2017) Heterotrophic cultivation of microalgae: production of metabolites of commercial interest. J Chem Technol Biotechnol 92(5):925–936 Ziganshina EE, Bulynina SS, Yureva KA, Ziganshin AM (2023) Optimization of photoautotrophic growth regimens of Scenedesmaceae alga: the influence of light conditions and carbon dioxide concentrations. Appl Sci 13(23):12753 Cheloni G, Slaveykova V (2018) Photo-oxidative stress in green algae and cyanobacteria. React Oxyg Species 5(14):126–133 Reyna-Martinez R, Gomez-Flores R, López-Chuken U, Quintanilla-Licea R, Caballero-Hernandez D, Rodríguez-Padilla C, Beltrán-Rocha JC, Tamez-Guerra P (2018) Antitumor activity of Chlorella sorokiniana and Scenedesmus sp. microalgae native of Nuevo León State, México. PeerJ 6:e4358. https://doi.org/10.7717/peerj.4358 Sánchez-Pineda PA, López-Pacheco IY, Villalba-Rodríguez AM, Godínez-Alemán JA, González-González RB, Parra-Saldívar R, Iqbal HMN (2024) Enhancing the production of PHA in Scenedesmus sp. by the addition of green synthesized nitrogen, phosphorus, and nitrogen–phosphorus-doped carbon dots. Biotechnol Biofuels 17:77. https://doi.org/10.1186/s13068-024-02522-4 Palanisami K, Nagothu US (2024) Expanding hill water management. India’s Water Future in a Changing Climate. Springer Nature, Singapore, pp 203–227 Kavitha G, Kurinjimalar C, Sivakumar K, Kaarthik M, Aravind R, Palani P, Rengasamy R (2016) Optimization of polyhydroxybutyrate production utilizing wastewater as nutrient source by Botryococcus braunii Kütz using response surface methodology. Int J Biol Macromol 93:534–542. https://doi.org/10.1016/j.ijbiomac.2016.09.019 Ansari S, Fatma T (2016) Cyanobacterial polyhydroxybutyrate (PHB): screening, optimization and characterization. PLoS ONE 11(6):e0158168. https://doi.org/10.1371/journal.pone.0158168 Kumari P, Kiran BR, Mohan SV (2022) Polyhydroxybutyrate production by Chlorella sorokiniana SVMIICT8 under nutrient-deprived mixotrophy. Bioresour Technol 354:127135. https://doi.org/10.1016/j.biortech.2022.127135 Okolie JA, Epelle EI, Nanda S, Castello D, Dalai AK, Kozinski JA (2021) Modeling and process optimization of hydrothermal gasification for hydrogen production: a comprehensive review. J Supercrit Fluids 173:105199 Nandal M, Khyalia P, Ghalawat A, Jugiani H, Kaur M, Laura JS (2022) Review on the use of microalgae biomass for bioplastics synthesis: a sustainable and green approach to control plastic pollution. Pollution 8(3):844–859 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 19 Feb, 2026 Reviews received at journal 19 Feb, 2026 Reviews received at journal 18 Feb, 2026 Reviewers agreed at journal 08 Feb, 2026 Reviewers agreed at journal 08 Feb, 2026 Reviews received at journal 06 Jan, 2026 Reviewers agreed at journal 29 Dec, 2025 Reviewers agreed at journal 29 Dec, 2025 Reviewers invited by journal 26 Dec, 2025 Editor assigned by journal 23 Dec, 2025 Submission checks completed at journal 23 Dec, 2025 First submitted to journal 18 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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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-8399498","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":567066535,"identity":"2bfa7dcd-5748-406e-8504-dc6dfbed99ee","order_by":0,"name":"Alejandro Valdez-Calderon","email":"","orcid":"","institution":"Universidad Tecnológica de la Zona Metropolitana del Valle de México,","correspondingAuthor":false,"prefix":"","firstName":"Alejandro","middleName":"","lastName":"Valdez-Calderon","suffix":""},{"id":567066536,"identity":"fa05254e-b651-4c4d-a073-bd4f6098fdc3","order_by":1,"name":"Saúl López-Gómez","email":"","orcid":"","institution":"Universidad Tecnológica de la Zona Metropolitana del Valle de México,","correspondingAuthor":false,"prefix":"","firstName":"Saúl","middleName":"","lastName":"López-Gómez","suffix":""},{"id":567066537,"identity":"f4ccd394-d2b0-4e41-9206-075a1568a992","order_by":2,"name":"Arian Espinosa-Roa","email":"","orcid":"","institution":"Centro de Investigación en Química Aplícada","correspondingAuthor":false,"prefix":"","firstName":"Arian","middleName":"","lastName":"Espinosa-Roa","suffix":""},{"id":567066538,"identity":"76e85051-28f3-443c-86b7-ab143ae0c0cb","order_by":3,"name":"Brenda Karen González-Pérez","email":"","orcid":"","institution":"Universidad Tecnológica de la Zona Metropolitana del Valle de México,","correspondingAuthor":false,"prefix":"","firstName":"Brenda","middleName":"Karen","lastName":"González-Pérez","suffix":""},{"id":567066539,"identity":"7665830e-6949-4180-bc80-7df6bad6f5bf","order_by":4,"name":"José Roberto 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16:30:52","extension":"html","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":137109,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8399498/v1/b5ac537f9379e760cffa030a.html"},{"id":99234451,"identity":"b4fd813d-bd1f-4a0e-9a9d-a715355fdcb4","added_by":"auto","created_at":"2025-12-30 12:57:50","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":52161,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of PHB obtained where a) UV-vis spectrum; b) IR spectrum and; c) DSC thermogram.\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8399498/v1/2a9c5fbf17d6fe1830fdc3b0.png"},{"id":99234450,"identity":"127a3c4e-7b2c-4b35-85f4-fbc1ee85c36c","added_by":"auto","created_at":"2025-12-30 12:57:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":115604,"visible":true,"origin":"","legend":"\u003cp\u003eStatical analysis of runs 1-16 for a) biomass generation of \u003cem\u003eS. acutus\u003c/em\u003e b) PHB concentration and c) PHB yield.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8399498/v1/b32a607c1b5e088f9daac47b.png"},{"id":99234452,"identity":"76445dc6-d6fb-46eb-9800-ded52b7de501","added_by":"auto","created_at":"2025-12-30 12:57:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":64370,"visible":true,"origin":"","legend":"\u003cp\u003ePareto diagram showing the relevant nutrient values for each case.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8399498/v1/39d9b1066fe4640af2b37a7e.png"},{"id":99323813,"identity":"4b913c31-8432-4c97-97d1-6161f9c2753d","added_by":"auto","created_at":"2025-12-31 16:46:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":910139,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8399498/v1/1d6e9bac-bd0e-4c7d-9358-74d2e33b5d27.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Production of polyhydroxybutyrate (PHB) in Scenedesmus acutus using a low-cost substrate ","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe global demand for polymerical materials continues to increase driven by the growth in the consumption of conventional plastics, however, the production of petroleum derivatives has been compromised due their high price, caused by the amount of energy consumed during manufacturing. Conventional plastics have a significant negative impact on the environment; they are primarily derived from crude oil, a non-renewable natural resource; exhibit poor biodegradability, leading to their accumulation in landfills and marine environments for centuries; moreover, their incineration releases harmful chemical substances; they enter the food chain in the form of microplastics; and their widespread use places increasing pressure on existing waste management infrastructure. Due to the previous reasons, alternatives have been sought to replace them, as bioplastics [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBioplastics are materials that, given appropriate factors of humidity, soil composition and temperature, present a short degradation period compared to conventional ones [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Other advantages are improvement on soil fertility, diversified feedstock, reduced carbon footprint, managed end of life, reduced greenhouse gas emission, reduced reliance on fossil full, sustainable and ecofriendly [\u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Bioplastics can be divided into 3 categories such as biobased non degradable plastic, biobased biodegradable plastics and fossil based biodegradable plastics [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBiodegradable biobased plastics such as polylactic acid (PLA), polyhydroxyalkanoates (PHAs), starch, and cellulose are widely used. Their demand has increased due to their origin from renewable sources, as well as their ability to undergo biodegradation. Among these materials, starch has been the most extensively utilized owing to its abundance in natural environments [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]; however, its production has declined, possibly because starch constitutes a fundamental resource for the food industry [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA strong alternative is PHAs, an emerging family of aliphatic polyesters whose global market was expected to reach an annual value of USD 93\u0026nbsp;million by the end of 2023, and to nearly double by 2028 with USD 195\u0026nbsp;million [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. These bioplastics are naturally synthesized by different types of bacteria and microalgae, accumulating in the form of insoluble particles in their cytoplasm [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. For 2019, it represented 1.2% of the production volumes of plastics of biological origin and increased up to 1.7% by the end of 2020. Although there are reports of up to 150 different types of PHAs, the monomers of 4 to 6 carbon atoms are the most commonly studied, as in the case of poly-3-hydroxybutyrate (PHB) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFor PHB, it is known that there are more than 300 bacteria used for production, including genera such as, \u003cem\u003eAlcaligenes\u003c/em\u003e, \u003cem\u003eAzotobacter\u003c/em\u003e, \u003cem\u003eBacillus\u003c/em\u003e, \u003cem\u003ePseudomonas\u003c/em\u003e, \u003cem\u003eRhizobium\u003c/em\u003e, \u003cem\u003eRhodospirillum\u003c/em\u003e and \u003cem\u003eCupriavidus\u003c/em\u003e, the latter being \u003cem\u003eC. necator\u003c/em\u003e the largest producer of PHB in yield with high levels (90%) [\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. For microalgae, genera such as \u003cem\u003eChloroella\u003c/em\u003e, \u003cem\u003eDesmodesmus\u003c/em\u003e, \u003cem\u003eRalstonia\u003c/em\u003e and \u003cem\u003eSpirulina\u003c/em\u003e have been previously reported, producing this bioplastic with maximum production levels of up to 45% (in relation to their biomass) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Although, the values are lower than in the case of bacteria, there are several advantages inherent to microalgae cultivation, such as, easy to monitor, high production of biomass, cost effective, ecofriendly and harnessed to produce various bioactive compounds for commercial use, producing an added value [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe variation in PHB production in microalgae has been studied and associated with different nutrients and changes in their growth conditions. Previous studies have reported the influence of macronutrients such as glucose and micronutrients such as nitrogen, phosphorus, sodium and iron [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], and the use of low-cost sources such as supplemental organic carbon [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] and vegetable waste such as cassava peel [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this work it is reported the use of a low-cost medium for the growth of \u003cem\u003eS. acutus\u003c/em\u003e in 16 different nutrient variants to obtain PHB, and evaluated the results.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Microalgae Strain and Culture Medium\u003c/h2\u003e \u003cp\u003eAll experiments were carried out with biomass from \u003cem\u003eScenedesmus acutus\u003c/em\u003e obtained from the repository of the Technological University of the Metropolitan Area of the Valley of Mexico \u0026ldquo;UTVAM\u0026rdquo; (Tizayuca, M\u0026eacute;xico; 19\u0026deg;51\u0026prime;59\u0026Prime;N 98\u0026deg;59\u0026prime;04\u0026Prime;W). This species was cultured in laboratory conditions at room temperature with purified water, receiving constant aeration under a white LED light (10\u0026ndash;40 W/m; 900\u0026ndash;3300 lumens/m), using commercial bicarbonate as a carbon source and fertilizer (Ferti Plus\u0026trade;) as a source of nitrogen and micronutrients.\u003c/p\u003e \u003cp\u003eFor the different tests, biomass recovered from this medium, known as \u0026ldquo;traditional medium\u0026rdquo;, was used. Sixteen different media were proposed to promote PHB production, with variations in nitrogen and carbon sources, while maintaining the base culture growth conditions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Experimental design\u003c/h2\u003e \u003cp\u003e \u003cem\u003eScenedesmus acutus\u003c/em\u003e was grown in sixteen different modified culture media that were prepared to have diverse nutrient conditions. The culture media were prepared by selecting factors found in previous research. Four relevant factors in the generation of PHB during the cultivation of microalgae were considered: carbon, glucose, nitrogen and sodium. To determine the relevant factors in \u003cem\u003eScenedesmus acutus\u003c/em\u003e, an experimental Taguchi matrix was designed. The Taguchi method uses orthogonal arrays, which stipulate the way of conducting the minimal number of experiments that will give the information of all the factors that affect the performance parameter [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. A binary factorial experimental design was formulated with the help of Minitab software where the different interactions of these factors were explored, taking into account their absence and excess, resulting in a total of 16 formulated (Table\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFactor levels of Taguchi design.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eVariable (source)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eName\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eLevels\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLow\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHigh\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN (Fertilizer)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 mL/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2 mL/L\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC (Bicarbonate)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.5 g/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5 g/L\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNa (sodium nitrate)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 g/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2 g/L\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlucose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 g/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3 g/L\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Culture Conditions\u003c/h2\u003e \u003cp\u003eCells of microalgae of \u003cem\u003eS. acutus\u003c/em\u003e kept in 1 L of protein medium were used as inoculum (5 mL) for liquid cultures. The experiment was carried out for 7 days in 600 mL plastic Erlenmeyer containing 1 mL per liter of Bold basal medium in 500-mL of purified water at 25\u0026ordm;C, under continuous illumination with cold white light fluorescent lamps (100 \u0026micro;mol m\u003csup\u003e2\u003c/sup\u003e/sec) and atmospheric CO\u003csub\u003e2\u003c/sub\u003e (200 mL/sec). At the end of cultivation, the bioplastic production was analyzed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Dry cell weight determination (DCW)\u003c/h2\u003e \u003cp\u003eTo determine dry cell weight (DCW), culture samples (10 mL) were centrifuged (15,000 rpm, 15 min, 4\u0026deg;C) and cell pellets were washed with deionized water after removing supernatant. The harvested cell pellets were then vacuum-dried until reaching a constant weight and DCW was then measured [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 PHB extraction, quantification and characterization\u003c/h2\u003e \u003cp\u003eThe PHB was extracted based on the methodology of [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], the microalgae were separated from the culture medium by centrifuging at 4000 rpm and the biomass pellets obtained were dried by leaving them at 90\u0026deg;C for 40 h. After that, the samples were resuspended in distilled water at a ratio of 1:10 (m/v) and 1 mL of 2 N HCl was added at a temperature of 40\u0026ndash;50\u0026deg;C for 2 hours in a water bath. After this process, it was centrifuged (4000 rpm) for 20 minutes, the pellets were recovered and 5 mL of chloroform was added, allowing it to rest for 24 hours.\u003c/p\u003e \u003cp\u003eAll supply materials were purchased from Meyer and Sigma-Aldrich, and were used without further purification. The solvents were purified by distillation over appropriate drying agents. Photophysical properties were measured by electronic absorption spectra obtained by a Perkin Elmer LAMBDA 2S UV-Vis Spectrophotometer in a 100\u0026ndash;800 nm range. Afterwards, the chemical properties of PHAs were recorded and scanned by an Agilent Technologies Cary 600 Fourier Transform Infrared Spectroscope (FTIR), in a range of 600 to 4000 wave number (cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). After PHAs dissolution in CDCl\u003csub\u003e3\u003c/sub\u003e, a Varian Inova 400 Nuclear Magnetic Resonance Spectrometer (RMN) was used to record Proton Nuclear Magnetic Resonance (\u003csup\u003e1\u003c/sup\u003eH-NMR) spectra at 20\u0026ordm;C, and their chemical shifts (δ) were reported in ppm. Additionally, the Differential Scanning Calorimetry (DSC) curve was measured on a Differential Scanning Calorimeter (DSC Discovey 2500, TA Instruments) under a nitrogen flow of 50 mL/min, in the temperature range of 25\u0026ndash;300\u0026deg;C with heating and cooling rates of 10\u0026deg;C/min on samples in 40 \u0026micro;L aluminum crucibles sealed with pierced lids.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003eThe biopolymers obtained were characterized by UV spectroscopy, FTIR spectroscopy and DSC. In all the experiments, the typical signals of the biopolymer were identified, so for practical purposes only the results are shown for A2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), which was the experimental one with the highest percentage of PHB calculated.\u003c/p\u003e \u003cp\u003eFor the optical characterization of PHB using UV-visible spectroscopy, its maximum absorption was identified at 217 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), making evident a low absorption capacity where the observable band corresponds to its S\u003csub\u003e0\u003c/sub\u003e-S\u003csub\u003e1\u003c/sub\u003e electronic transition (π \u0026rarr; π*) of the carbonyl group. In the case of FTIR spectroscopy (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), firstly two bands were observed at 2990 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 2940 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, corresponding to carbons C\u0026ndash;H with sp\u003csup\u003e2\u003c/sup\u003e and sp\u003csup\u003e3\u003c/sup\u003e geometries, continuing with a band around 1722 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, referring to the carbonyl group of the ester bond, followed by the band at 1276 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of the \u0026ndash;CH group, all of them coincide with the bands of biopolymer. The band observed at 1455 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e refers to the asymmetric deformation of the CH\u003csub\u003e3\u003c/sub\u003e bond, while the band at 1377 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is assigned to the asymmetric movement of the methyl groups. In addition, the bands observed at 1331 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e are characteristic of the asymmetric and symmetric vibration of the C\u0026ndash;O\u0026ndash;C group.\u003c/p\u003e \u003cp\u003eEach crop component was considered as a variable that could potentially affect PHB production. Glucose, bicarbonate, fertilizer and sodium nitrate levels were considered, in principle, influential. A two-level full factorial design (2\u003csup\u003e4\u003c/sup\u003e) would imply a total of 16 experiments, plus the replications necessary for the evaluation of the degree of coincidence between the results. Therefore, a two-level Taguchi design involving 16 random runs was selected. Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the design matrix for the experiment with the quantities contributed by each of the variables and the total yield of PHB production in percentage and m/v concentration.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDesign matrix response values for Taguchi design with PHB values.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRun\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGlucose\u003c/p\u003e \u003cp\u003e(gL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCarbon\u003c/p\u003e \u003cp\u003e(gL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNitrogen\u003c/p\u003e \u003cp\u003e(gL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSodium\u003c/p\u003e \u003cp\u003e(gL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eBiomass\u003c/p\u003e \u003cp\u003e(gL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePHB\u003c/p\u003e \u003cp\u003e(gL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003ePHB\u003c/p\u003e \u003cp\u003e(% w/w)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.085\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e0.018\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e21.56\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.057\u0026thinsp;\u0026plusmn;\u0026thinsp;0.013\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e0.014\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e24.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.085\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e0.010\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e12.05\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.091\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e0.014\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e16.13\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.079\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e0.012\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e15.13\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.103\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e0.015\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e15.88\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.091\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e0.008\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e9.08\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.119\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e0.026\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e22.15\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.084\u0026thinsp;\u0026plusmn;\u0026thinsp;0.010\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e0.012\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e14.39\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.082\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e0.009\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e11.68\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.391\u0026thinsp;\u0026plusmn;\u0026thinsp;0.009\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e0.029\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e7.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.259\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e0.028\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e10.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.184\u0026thinsp;\u0026plusmn;\u0026thinsp;0.014\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e0.015\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e8.27\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.211\u0026thinsp;\u0026plusmn;\u0026thinsp;0.013\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e0.022\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e10.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.188\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e0.019\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e10.04\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.170\u0026thinsp;\u0026plusmn;\u0026thinsp;0.009\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e0.022\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e13.35\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe biomass growth in the 16 different variants, after the established days, is shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and its statistical analysis is visualized in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea. The range of values ​​was determined between 0.57\u0026thinsp;\u0026minus;\u0026thinsp;0.391 gL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, with the maximum being condition run 11, where sodium was omitted, and the lowest being condition run 2, which contained sodium as the only nutrient in the medium. From its statistical analysis, significant differences were observed between medium conditions 11, 12, 14 and 13 (in decreasing order of biomass quantity) and all the other runs, and it is important to highlight that they had the highest biomass generation performance. The remaining runs were very similar in their biomass values ​​as observed in runs 1, 3\u0026ndash;7, 9 and 10 with no significant differences.\u003c/p\u003e \u003cp\u003eNow moving on to the analysis of PHB production (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) the highest values ​​in a range of 0.025\u0026ndash;0.030 gL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were presented in runs 11, 12 and 8 (in decreasing order). In 8 it was obtained in the absence of glucose and for 11 and 12 sodium was omitted. There was no significant difference between the three conditions with the highest concentrations, but it is important to emphasize that there was a significant difference in the remaining runs. The remaining runs had similar ranges of PHB production, so it was observed that runs 1, 2, 4\u0026ndash;6, 9, 13, 7, and 16 were very similar. It is important to mention that the percentage of PHB obtained reflects the improvements in the methodology and media conditions, since this relationship involves the concentration of bioplastics relative to the dry biomass generated in each particular case.\u003c/p\u003e \u003cp\u003eAccording to the statistical analysis of % PHB shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, the maximum value for condition 2 was 24.7%, followed by 8 and 1 with values ​​of 22.15% and 21.56%, respectively; the three maximum values ​​did not show significant differences. For cases 3\u0026ndash;7, 9, and 10, no differences were found, and their range was 12\u0026ndash;16% PHB. The run with the lowest percentage was condition 11 with 7.50%.\u003c/p\u003e \u003cp\u003eThe analysis of results using Minitab 16 lets discard the less significant factors and interactions. For this experiment, the factors were considered significant at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. In the first, the four factors referring to nutrients were analyzed and in a second study, the values of biomass produced were added. With the above, two Pareto diagrams were created, shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. In the second Pareto, glucose can be seen as the determining factor individually, followed by a combination of biomass generated with glucose and biomass with sodium respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe results obtained in this study demonstrate that the culture conditions evaluated, particularly light intensity, nitrogen and sodium availability, glucose and bicarbonate concentration, the commercial fertilizer used as the base of the medium, and the culture growth time, exert a determining influence on polyhydroxybutyrate (PHB) accumulation and biomass generation in \u003cem\u003eScenedesmus acutus\u003c/em\u003e. Modulation of the carbon-nitrogen ratio emerged as an essential element for inducing the intracellular storage of this biopolymer, consistent with reports for other green microalgae species [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe biomass obtained under the different experimental combinations showed variations attributable to the interaction between light intensity, nutrient availability, and the photosynthetic capacity of the culture. High light intensities favored higher growth rates, possibly due to greater photosynthetic activity, while the glucose supply acted as an external carbon source capable of sustaining metabolism even under nitrogen deficiency [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. This pattern has been widely documented in \u003cem\u003eScenedesmus\u003c/em\u003e spp. [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. However, excessive light levels could generate photo-oxidative stress, which would explain some of the occasional decreases in biomass observed [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eComparison with previous studies shows that the biomass obtained (0.39\u0026ndash;0.57 g/L) is lower than that reported in conventional media such as BG-11 or agro-industrial wastewater (2\u0026ndash;3 g/L), but it is within similar or higher ranges than those obtained with low-cost media [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. This confirms that the use of a commercial fertilizer as the base of the medium represents a viable economic alternative for large-scale intensive crops [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eStatistical analysis using a two-level Taguchi design allowed for the identification of conditions with significant differences. Runs 11, 12, and 14 generated the greatest biomass, particularly when sodium was omitted, suggesting that this nutrient could limit the growth of S. acutus at certain concentration ranges.\u003c/p\u003e \u003cp\u003eRegarding PHB accumulation, the results show that nutritional stress, especially nitrogen or sodium restriction, induces a metabolic imbalance that favors the diversion of carbon toward the synthesis of polyhydroxyalkanoates. Runs 11, 12, and 8 recorded the highest absolute concentrations (0.025\u0026ndash;0.030 g/L), while the relative percentages of PHB reached values ​​up to 24.7%. These figures are comparable to values ​​reported in \u003cem\u003eScenedesmus obliquus\u003c/em\u003e and \u003cem\u003eChlorella vulgaris\u003c/em\u003e [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] and, in some cases, higher than recent studies of Scenedesmus spp. [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eComparatively, the PHB percentages obtained are lower than those reported for cyanobacteria such as \u003cem\u003eSpirulina\u003c/em\u003e sp. or \u003cem\u003eNostoc muscorum\u003c/em\u003e (30\u0026ndash;31%) under extreme nutrient limitation conditions, but higher than those of strains such as \u003cem\u003eSynechocystis\u003c/em\u003e sp. and \u003cem\u003eBotryococcus braunii\u003c/em\u003e, which typically range from 10\u0026ndash;17% [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThese results show that \u003cem\u003eS. acutus\u003c/em\u003e has an intermediate-to-high performance within the microalgal spectrum for PHB production, and that its accumulation depends strongly on the availability of external carbon and the type of nutrient limitation imposed.\u003c/p\u003e \u003cp\u003eCharacterization of the biopolymer using UV-visible spectroscopy, FTIR, and DSC thermal analysis confirmed the nature of PHB in all treatments, revealing maximum UV absorption at 217 nm, corresponding to the π\u0026rarr;π* electronic transition of the carbonyl group. Characteristic FTIR bands of PHB: 2990 and 2940 cm⁻\u0026sup1; (C\u0026ndash;H sp2/sp3), 1722 cm⁻\u0026sup1; (carbonyl of the ester), 1455 and 1377 cm⁻\u0026sup1; (vibrations of CH₃), 1331\u0026ndash;1276 cm⁻\u0026sup1; (C\u0026ndash;O\u0026ndash;C vibration of the ester), the slight variations observed in relative intensities could be attributed to the presence of impurities or cellular remnants, suggesting the need to optimize extraction and purification processes using combined methods [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAnalysis with Minitab identified glucose as the most influential factor on PHB production, followed by glucose-biomass and sodium-biomass interactions. The agreement between experimental values ​​and estimated main effects suggests that the predictive model accurately describes the culture dynamics. However, the nature of the Taguchi design limits the detection of complex interactions; therefore, future studies could consider full factorial designs or response surface models [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThese results suggest that \u003cem\u003eScenedesmus acutus\u003c/em\u003e has significant potential as a PHB producer, especially under nutritional stress and in the presence of external carbon sources. The combination of cost-effectiveness, experimental optimization strategies, and reliable structural characterization of the biopolymer suggests the feasibility of integrating this microalga into industrial bioplastics production schemes [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHowever, significant challenges remain: improving extraction yields, reducing energy costs, and evaluating production performance in open and semi-closed systems. Addressing these issues will pave the way for more efficient and sustainable processes that can be economically competitive for PHB production from microalgae.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eThe genus \u003cem\u003eScenedesmus\u003c/em\u003e has been scarcely explored in the field of biopolymer production. This work, specifically for the species \u003cem\u003eScenedesmus acutus\u003c/em\u003e, reports the production of PHB for the first time. It was carried out using different variables, starting with low-cost sources. It was possible to identify that the limiting factor of glucose and its combination with nitrogen had significant results derived from its statistical analysis in a balance of biomass generation and PHB production. This, along with the addition of the biomass experiment, was corroborated in a Pareto-type multivariate study, where glucose was identified as the main factor, followed by glucose and biomass generation.\u003c/p\u003e \u003cp\u003eThe range of % PHB in the results we obtained (7.50\u0026ndash;24.70%) reflects the importance of the variation in nutrients associated with the production of bioplastic, so we contribute to the optimization of resources with low-cost reagents and leaves open the possibility of continuing to optimize the obtaining of PHB in \u003cem\u003eScenedesmus\u003c/em\u003e, a bioplastic that increases its use every year from a microalga that expands its applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAVC contributed to Conceptualization, Methodology, Formal Analysis, Investigation, and Writing \u0026ndash; Original Draft Preparation. SLG participated in Methodology, Data Curation, Formal Analysis, and Writing \u0026ndash; Review \u0026amp; Editing. ARE contributed to Software development, Validation, Visualization, and Data Curation. BKGP was involved in Investigation, Resources management, and Writing , Review \u0026amp; Editing. JRGR contributed to Conceptualization, Supervision. MAGM participated in Methodology, Validation, Visualization, and Writing \u0026ndash; Review \u0026amp; Editing. All authors reviewed and approved the final version of the manuscript and agree to be accountable for all aspects of the work.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe Acknowledgments section should include information on the source of any financial support received for the work being published, before the References. The authors acknowledge the financial support provided by the Secretar\u0026iacute;a de Ciencia, Humanidades, Tecnolog\u0026iacute;a e Innovaci\u0026oacute;n (SECIHTI), Mexico, which made this research possible. The authors also express their sincere gratitude to Engineer Ang\u0026eacute;lica Mar\u0026iacute;a Soto Herrera for her valuable technical support as the laboratory technician in charge of the Process Laboratory at the Universidad Tecnol\u0026oacute;gica de la Zona Metropolitana del Valle de M\u0026eacute;xico (UTVAM).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data underlying this article are available in the article and its online supplementary material.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbdo SM, Ali GH (2019) Analysis of polyhydroxybutyrate and bioplastic production from microalgae. 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Pollution 8(3):844\u0026ndash;859\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"world-journal-of-microbiology-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wibi","sideBox":"Learn more about [World Journal of Microbiology and Biotechnology](https://www.springer.com/journal/11274)","snPcode":"11274","submissionUrl":"https://submission.nature.com/new-submission/11274/3","title":"World Journal of Microbiology and Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Polyhydroxybutyrate (PHB), Scenedesmus acutus, biopolymers, microalgae-based production","lastPublishedDoi":"10.21203/rs.3.rs-8399498/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8399498/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe importance of conventional plastics is undeniable; however, their non-biodegradability makes them one of the biggest environmental problems. Among the alternatives to mitigate environmental damages, the production of polyhydroxyalkanoates (PHB) provides a biodegradable solution, additional to their mechanical and thermal properties, making them accessible to a wide variety of applications. In general, biopolymers accumulate as energy and carbon storage material in microorganisms such as bacteria and microalgae. In the present study, the impact of different sources like carbon, glucose, nitrogen and sodium was tested on the production of PHB in the cultures of the microalgae \u003cem\u003eS. acutus\u003c/em\u003e. To evaluate the effect of the variables, a fractional Taguchi experimental design was devised and executed, thus, 16 experimental runs and 3 replicas in each treatment were considered. Results showed calculated concentrations of the biopolymer in a range from 7.5\u0026ndash;34.7% w/w dry weight. Additionally, the PHB was identified by spectroscopic and thermogravimetric analysis. Statistical analysis was performed using Minitab 16, where differences in biomass production, PHB concentration in g/L and the percentage of PHB were analyzed. Likewise, a Pareto diagram was used to consider the biomass production results, with glucose, biomass-glucose, and biomass-sodium as determining factors in the PHB production. The present research provides significant data on critical factors related with PHA production, for the first time to our knowledge, thus showing \u003cem\u003eScenedesmus acutus\u003c/em\u003e as a PHB producer through low-cost medium providing a promising candidate for industrial scale-up.\u003c/p\u003e","manuscriptTitle":"Production of polyhydroxybutyrate (PHB) in Scenedesmus acutus using a low-cost substrate","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-30 12:57:45","doi":"10.21203/rs.3.rs-8399498/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-19T11:04:47+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-19T08:31:19+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-18T13:55:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"72193933541046100570871772803492799803","date":"2026-02-09T02:04:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"257839771576684736936790754010092055250","date":"2026-02-08T13:06:31+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-06T21:11:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"291455191542924264396495908697522277536","date":"2025-12-30T01:15:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"26693321622857277179648792628881605960","date":"2025-12-29T20:02:43+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-26T09:53:57+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-23T07:25:44+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-23T05:57:39+00:00","index":"","fulltext":""},{"type":"submitted","content":"World Journal of Microbiology and Biotechnology","date":"2025-12-19T01:01:48+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"world-journal-of-microbiology-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wibi","sideBox":"Learn more about [World Journal of Microbiology and Biotechnology](https://www.springer.com/journal/11274)","snPcode":"11274","submissionUrl":"https://submission.nature.com/new-submission/11274/3","title":"World Journal of Microbiology and Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"1bb98bfc-fdeb-4e07-ae1a-938bf8389008","owner":[],"postedDate":"December 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-21T09:53:30+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-30 12:57:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8399498","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8399498","identity":"rs-8399498","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}