Production of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) by Haloferax mediterranei Using Candy Industry Waste as Raw Materials

Citation: Simó-Cabrera, L.; García-Chumillas, S.; Benitez-Benitez, S.J.; Cánovas, V.; Monzó, F.; Pire, C.; Martínez-Espinosa, R.M. Production of Poly(3-hydroxybutyrate-co-3hydroxyvalerate) (PHBV) by Haloferax mediterranei Using Candy Industry Waste as Raw Materials. Bioengineering 2024,11, 870. https://doi.org/10.3390/ bioengineering11090870 Academic Editors: Dirk Holtmann and Martin Koller Received: 29 June 2024 Revised: 9 August 2024 Accepted: 26 August 2024 Published: 27 August 2024 Copyright: © 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). bioengineering Article Production of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) by Haloferax mediterranei Using Candy Industry Waste as Raw Materials Lorena Simó-Cabrera 1,2 , Salvador García-Chumillas 3,4 , Sergio J. Benitez-Benitez 3, Verónica Cánovas 4, Fuensanta Monzó3, Carmen Pire 1,2 and Rosa María Martínez-Espinosa 1,2,* 1Biochemistry, Molecular Biology, Edaphology and Agricultural Chemistry Department, Faculty of Science, University of Alicante, Carretera San Vicente del Raspeig s/n-03690 San Vicente del Raspeig, E-03690 Alicante, Spain; [email protected] (L.S.-C.); carmen.pir[email protected] (C.P.) 2Multidisciplinary Institute for Environmental Studies “Ramón Margalef”, University of Alicante, Ap. 99, E-03080 Alicante, Spain 3Technological Centre of Footwear and Plastic of the Region of Murcia (CETEC) Avda, Europa 4-5, E-30840 Alhama de Murcia, Spain; [email protected]g (S.G.-C.); [email protected]g (S.J.B.-B.); [email protected] (F.M.) 4Cetec Biotechnology, Avda, Europa 4-5, E-30840 Alhama de Murcia, Spain; v[email protected] *Correspondence: [email protected]; Tel.: +34-965903400 (ext. 1258 or 8841) Abstract: The haloarchaeon Haloferax mediterranei synthesizes poly(3-hydroxybutyrate-co-3hydroxyvalerate) (PHBV) under unfavorable nutritional conditions without the addition of any precursor to the culture, which is an advantage compared to other microbial counterparts able to synthesize polyhydroxyalkanoates (PHA). PHBV is a biodegradable polymer showing physiochemical properties of biotechnological and biomedical interest and can be used as an alternative to plastics made from chemical synthesis (which are not environmentally friendly). The versatile metabolism of H. mediterranei makes the use of waste as a carbon source for cellular growth and PHA synthesis possible. In this work, cellular growth and the production and characterization of PHBV using two different types of confectionery waste were analyzed and compared with cellular growth and PHBV synthesis in a standard culture media with glucose of analytical grade as a carbon source. The PHBV granules produced were analyzed by TEM and the biopolymer was isolated and characterized by GC-MS, FTIR NMR, and DSC. The results reveal that H. mediterranei can use these two residues (R1 and R2) for pure PHBV production, achieving 0.256 and 0.983 g PHBV/L, respectively, which are among the highest yields so far described using for the first-time waste from the candy industry. Thus, a circular economy-based process has been designed to optimize the upscaling of PHBV production by using haloarchaea as cell factories and valorizing confectionery waste. Keywords: haloarchaea; polyhydroxyalkanoates (PHA); poly(3-hydroxybutyrate) (PHB); poly(3hydroxyvalerate) (PHV); poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV); candy waste; bioplastics 1. Introduction In the last decade, there has been a massive increase in waste generation due to processes of anthropogenic origin in general, particularly industrial activities [ 1 ]. Worldwide municipal solid waste generation is expected to increase to 2.59 billion tons annually by 2030 and to 3.40 billion tons by 2050 [ 2 ]. As an example, over 30% of food is lost or wasted annually, an estimated 1.32 billion tons end up in landfills where food waste goes through a series of bioconversions, which are usually harmful to the environment [ 3 ]. Regarding the global concerns facing pollution, current plastic pollution is one of the biggest global concerns, affecting marine and terrestrial ecosystems, animal populations, and human health [ 4 ]. It is estimated that around 140 million tons of plastics are produced annually Bioengineering 2024,11, 870. https://doi.org/10.3390/bioengineering11090870 https://www.mdpi.com/journal/bioengineering Bioengineering 2024,11, 870 2 of 16 from different sources [ 5 ]. If the current production and waste management situation continues, by 2050, around 9000 million tons of plastic waste will be recycled, 12,000 million tons will be incinerated, and 12,000 million tons will have accumulated in the natural environment [6]. Among other processes, food production, processing, packaging, and plastic production and usage are integrated into many processes that finally contribute to global waste production. Production waste is a current concern for the candy industry due to the strict regulations regarding food safety: contamination risks cannot be assumed and companies see their food waste increased to ensure the quality of the final product [ 7 ]. The efficiency of resource utilization and waste materials recovery can be enhanced through a circular economy, thus decreasing the emission of fossil carbon during extraction and manufacturing processes. In this context, biotechnological-based procedures can enhance the bioeconomy by using waste generated from the food industry for various applications [3]. The growing concern about the effects of plastic pollution on ecosystems and organisms has drawn attention to sustainable materials as an alternative to fossil-fuel plastics. From this point of view, bioplastics are potential substitutes for conventional plastics due to their inherent biodegradability, biocompatibility, and wide range of applications, as well as a production process more friendly to the environment [ 8 ]. Polyhydroxyalkanoates (PHAs) are widely recognized as biopolymers due to their applications in the pharmaceutical, medical, and food sectors [ 9 – 11 ]. These biological polymers are mainly produced by microbial fermentation processes and are accumulated within microbial cells as granules that serve as carbon and energy storage. The synthesis of these granules takes place when an excess of carbon source is available under nutrient-limiting conditions such as in phosphorus, oxygen, or nitrogen or in a fluctuating pH of the media [ 12 ]. Then, the stored PHA granules could be used by microorganisms as a carbon source when the nutrients are limited [13]. Several microbial members of the Bacteria and Archaea domains have been tested during the last two decades for PHA production. Considering the high efficiency of some species able to produce PHAs, those microorganisms are thus revealed as good candidates to be used as cell factories to produce PHA such as poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxyvalerate) (PHV) [ 14 – 18 ]. For example, among halophilic microorganisms, the haloarchaeon Haloferax mediterranei and the Halomonas species from the bacterial domain have been extensively studied for efficient PHA production. Up to now, H. mediterranei has been reported to synthesize PHBV and poly(3-hydroxybutyrate-co-3-hydroxyvalerateco-4-hydroxybutyrate) (PHBV4HB), whilst Halomonas species can accumulate PHB, PHBV, and poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [ 18 , 19 ]. These two genera have been used as model representatives of each domain to study PHA biosynthesis. In both cases, it has been reported that C/N balance, P limitation, overexpression of the genes coding for PHA synthases, and fine-tuning regulation of the TCA cycle are essential to increase the production of PHA [ 16 , 19 – 22 ]. In the case of H. mediterranei, cells can synthesize PHBV, a more versatile and economically favorable biopolymer than PHB, without the addition of any precursor, which makes this species the most interesting in terms of biotechnological PHBV production compared to its bacterial counterparts [ 18 ]. This species can probably use the largest range of single carbon sources (glycerol, glucose, starch, etc.) as well as industrial waste as part of the culture formulation and presents a high growth rate and metabolic versatility [ 16 , 23 , 24 ]. An important drawback in industrial PHA production by microorganisms is the cost of raw materials, which involves 40–48% of the final costs [25], hence the need to find alternative carbon sources for the growth media promoting the synthesis of PHBV. Only after achieving this aim would the production of PHBV be scalable, economically competitive, and low in time consumption compared to the production of other plastics. The use of waste in the production of PHAs remains poorly explored; considering the lack of homogeneity in the composition of waste, one of the main current concerns is to optimize circular economy-based processes in which the final PHA produced using waste shows the desirable chemical composition and physicochemical properties for targeted industrial and biotechnological applications [10,14,16]. Bioengineering 2024,11, 870 3 of 16 This work aims to study H. mediterranei growth as well as PHBV production using waste from the candy industry to optimize a process that is cost-effective, low in time consumption, and environmentally friendly. To the best of our knowledge, this is the first time that confectionary residues are used for this purpose, thus providing a scalable process based on a circular economy. 2. Materials and Methods 2.1. Candy Waste Used as a Carbon Source Two different types of solid waste (Table 1) from the candy industry were kindly supplied by the company Vidal Golosinas SA (Murcia, Spain). Both types of waste (Residue 1: R1 and Residue 2: R2) were obtained during the manufacturing of gummies. To use R1 and R2 as carbon sources for microbial growth and PHBV production, R1 was added to the medium before autoclaving. R2 was diluted to 20% in distilled water using a blender and subsequently sterilized using a Steritop/Receiver Flask, Merck KGaA, Darmstadt, Germany filter with a vacuum pump. Then, the solution was added to the autoclaved medium at room temperature. The waste was sterilized differently because R2 cannot be autoclaved, as it becomes caramelized during the high-temperature process, resulting in it no longer being bioavailable. The major polysaccharide identified in both residues was starch (98% starch-rich). Both residues were added to the medium at 1% (w/v; 1 g/100 mL) as a final concentration. Table 1. Nutritional composition of waste (R1 and R2). The nutritional quantification was provided by the company “Vidal Golosinas” S.A. (Spain). Average Values Residue 1 Residue 2 Total fat (g/100 g) 0 0 Saturated fat (g/100 g) 0 0 Total carbohydrate (g/100 g) 91 83 Sugars (g/100 g) 90 73 Protein (g/100 g) 0.3 4.4 Salt (g/100 g) 0.01 0.16 Energy/100 g 1558 kJ 374 kcal 1505 kJ 354 kcal Pictures of each waste Bioengineering 2024, 11, x FOR PEER REVIEW 3 of 16 in which the final PHA produced using waste shows the desirable chemical composition and physicochemical properties for targeted industrial and biotechnological applications [10,14,16]. This work aims to study H. mediterranei growth as well as PHBV production using waste from the candy industry to optimize a process that is cost-effective, low in time consumption, and environmentally friendly. To the best of our knowledge, this is the first time that confectionary residues are used for this purpose, thus providing a scalable process based on a circular economy. 2. Materials and Methods 2.1. Candy Waste Used as a Carbon Source Two different types of solid waste (Table 1) from the candy industry were kindly supplied by the company Vidal Golosinas SA (Murcia, Spain). Both types of waste (Residue 1: R1 and Residue 2: R2) were obtained during the manufacturing of gummies. To use R1 and R2 as carbon sources for microbial growth and PHBV production, R1 was added to the medium before autoclaving. R2 was diluted to 20% in distilled water using a blender and subsequently sterilized using a Steritop/Receiver Flask, Merck KGaA, Darmstadt, Germany filter with a vacuum pump. Then, the solution was added to the autoclaved medium at room temperature. The waste was sterilized differently because R2 cannot be autoclaved, as it becomes caramelized during the high-temperature process, resulting in it no longer being bioavailable. The major polysaccharide identified in both residues was starch (98% starch-rich). Both residues were added to the medium at 1% (w/v; 1 g/100 mL) as a final concentration. Table 1. Nutritional composition of waste (R1 and R2). The nutritional quantification was provided by the company “Vidal Golosinas” S.A. (Spain). Average Values Residue 1 Residue 2 Total fat (g/100 g) 0 0 Saturated fat (g/100 g) 0 0 Total carbohydrate (g/100 g) 91 83 Sugars (g/100 g) 90 73 Protein (g/100 g) 0.3 4.4 Salt (g/100 g) 0.01 0.16 Energy/100 g 1558 kJ 374 kcal 1505 kJ 354 kcal Pictures of each waste 2.2. Microorganism Used and Growth Conditions The haloarchaeon Haloferax mediterranei R-4 (ATCC 33500) has been used as a model organism in this work. Cells were grown in a mineral minimal medium (MM) with potassium nitrate as the sole source of nitrogen. In this study, anhydrous glucose (99%), was used as a carbon source in control cultures according to previous recipes optimized for the growth of this strain [26], while it was replaced by industrial waste from the candy industry to analyze cell growth and PHBV production. The composition of the MM was as follows: 0.7 g NaBr, 0.2 g NaHCO3, 6 g KCl, 41.5 g MgCl2·6H2O, 59.3 g MgSO4·7 H2O, and 234 g NaCl. All the compounds were dissolved in 25% distilled water (25% w/v salted water (SW)). After autoclaving, 0.005 g/L FeCl3, 1% KNO3, and 0.001 M Na2HPO4·12 H2O/NaH2PO4·2 H2O were added. The pH value of the culture media was set to 7.3 using Bioengineering 2024, 11, x FOR PEER REVIEW 3 of 16 in which the final PHA produced using waste shows the desirable chemical composition and physicochemical properties for targeted industrial and biotechnological applications [10,14,16]. This work aims to study H. mediterranei growth as well as PHBV production using waste from the candy industry to optimize a process that is cost-effective, low in time consumption, and environmentally friendly. To the best of our knowledge, this is the first time that confectionary residues are used for this purpose, thus providing a scalable process based on a circular economy. 2. Materials and Methods 2.1. Candy Waste Used as a Carbon Source Two different types of solid waste (Table 1) from the candy industry were kindly supplied by the company Vidal Golosinas SA (Murcia, Spain). Both types of waste (Residue 1: R1 and Residue 2: R2) were obtained during the manufacturing of gummies. To use R1 and R2 as carbon sources for microbial growth and PHBV production, R1 was added to the medium before autoclaving. R2 was diluted to 20% in distilled water using a blender and subsequently sterilized using a Steritop/Receiver Flask, Merck KGaA, Darmstadt, Germany filter with a vacuum pump. Then, the solution was added to the autoclaved medium at room temperature. The waste was sterilized differently because R2 cannot be autoclaved, as it becomes caramelized during the high-temperature process, resulting in it no longer being bioavailable. The major polysaccharide identified in both residues was starch (98% starch-rich). Both residues were added to the medium at 1% (w/v; 1 g/100 mL) as a final concentration. Table 1. Nutritional composition of waste (R1 and R2). The nutritional quantification was provided by the company “Vidal Golosinas” S.A. (Spain). Average Values Residue 1 Residue 2 Total fat (g/100 g) 0 0 Saturated fat (g/100 g) 0 0 Total carbohydrate (g/100 g) 91 83 Sugars (g/100 g) 90 73 Protein (g/100 g) 0.3 4.4 Salt (g/100 g) 0.01 0.16 Energy/100 g 1558 kJ 374 kcal 1505 kJ 354 kcal Pictures of each waste 2.2. Microorganism Used and Growth Conditions The haloarchaeon Haloferax mediterranei R-4 (ATCC 33500) has been used as a model organism in this work. Cells were grown in a mineral minimal medium (MM) with potassium nitrate as the sole source of nitrogen. In this study, anhydrous glucose (99%), was used as a carbon source in control cultures according to previous recipes optimized for the growth of this strain [26], while it was replaced by industrial waste from the candy industry to analyze cell growth and PHBV production. The composition of the MM was as follows: 0.7 g NaBr, 0.2 g NaHCO3, 6 g KCl, 41.5 g MgCl2·6H2O, 59.3 g MgSO4·7 H2O, and 234 g NaCl. All the compounds were dissolved in 25% distilled water (25% w/v salted water (SW)). After autoclaving, 0.005 g/L FeCl3, 1% KNO3, and 0.001 M Na2HPO4·12 H2O/NaH2PO4·2 H2O were added. The pH value of the culture media was set to 7.3 using 2.2. Microorganism Used and Growth Conditions The haloarchaeon Haloferax mediterranei R-4 (ATCC 33500) has been used as a model organism in this work. Cells were grown in a mineral minimal medium (MM) with potassium nitrate as the sole source of nitrogen. In this study, anhydrous glucose (99%), was used as a carbon source in control cultures according to previous recipes optimized for the growth of this strain [ 26 ], while it was replaced by industrial waste from the candy industry to analyze cell growth and PHBV production. The composition of the MM was as follows: 0.7 g NaBr, 0.2 g NaHCO 3 , 6 g KCl, 41.5 g MgCl 2· 6H 2 O, 59.3 g MgSO 4· 7H 2 O, and 234 g NaCl. All the compounds were dissolved in 25% distilled water (25% w/vsalted water (SW)). After autoclaving, 0.005 g/L FeCl 3 , 1% KNO 3 , and 0.001 M Na 2 HPO 4· 12H 2 O/NaH 2 PO 4· 2H 2 O were added. The pH value of the culture media was set to 7.3 using HCl or NaOH. For each condition tested (control and the two candy waste), 1 L cultures were incubated in 3 L Erlenmeyer because of proper aeration (quintuplicates were prepared for all growth conditions). Cultures were inoculated with 10 mL of preadapted H. mediterranei R-4 (ATCC 33500) cells (preadapted cells are cells previously grown under Bioengineering 2024,11, 870 4 of 16 the conditions described) grown until the mid-stationary phase of growth (OD 600 = 1.2). Cultures were incubated in an orbital shaking incubator at 170 rpm and 37 ◦ C until the stationary phase was reached. The growth of the cells was monitored by measuring the OD at 600 nm. The correlation between optical density at a wavelength of 600 nm (OD 600 ) and cell number for H. mediterranei (Cells/mL = OD 600 × 9.69 × 10 8 ) was estimated using a cell staining protocol with SYBR green (N ′ ,N ′ -dimethyl-N-[4-[(E)-(3-methyl-1,3-benzothiazol-2ylidene)methyl]-1-phenylquinolin-1-ium-2-yl]-N-propylpropane-1,3-diamine) [27,28]. 2.3. Analysis of the PHA Granules within the Cells by Transmission Electron Microscopy (TEM) In total, 20 mL of the cell culture in the stationary phase (OD 600nm = 1.95) were harvested by centrifugation at 13,000 × gRCF for 30 min for TEM analysis. Cells were washed twice with a saline buffer (10% NaCl, 0.1 M sodium phosphate buffer, pH 7.2) and resuspended in a 2.5% (v/v) glutaraldehyde solution in a saline buffer for primary fixation overnight at 4 ◦ C. Following the primary fixation, the cells were pelleted and washed three times with saline buffer. The cell pellets were then fixed with 1.0% osmium tetroxide in saline buffer (1% v/v) for 2 h at 4 ◦ C and subsequently washed three times with a saline buffer. A third fixative solution was added to the cells; 0.5% (w/v) uranyl acetate solution (in Veronal-acetate buffer) and cells were incubated overnight in the dark at 4 ◦ C. The ensuing steps were performed according to the procedure described by Tian and coworkers [ 29 ]. Photomicrographs were taken with a Philips Tecnai 12 Electron Microscope (Carl Zeiss, Jena, Germany) capable of imaging negatively stained samples between 80 kV and 120 kV and equipped with a megaview III digital camera. 2.4. PHA Extraction Method Biomass was harvested at the stationary phase by centrifugation at 25,861.9 × gRCF for 30 min. Then, the cells were washed with 10% NaCl and allowed to shake with 0.1% SDS (w/v) until solubilized. Subsequently, the pellet was separated and frozen at − 80 ◦ C to proceed with lyophilization. The lyophilized pellet was treated with hot chloroform at 62 ◦ C for 6 h, followed by precipitation with 10 volumes of pre-chilled methanol. To collect PHBV, the precipitate was centrifuged at 4000 × gRCF for 30 min and PHA was dried at 40 ◦ C to remove all residual solvent and obtain a constant weight. The pellet, corresponding to raw PHA, was recovered and weighed. The polymer yield was determined by dividing the weight of PHA obtained after extraction by the cell dry weight (CDW), gPHA/gCDW. 2.5. Characterization by Attenuated Total Reflect and Fourier-Transform Infrared Spectroscopy (ATR–FTIR) The spectrum was recorded using a Spectrometer (Bruker Vertex 70, Bruker, Billerica, MA, USA) with an ATR accessory from 400 to 4000 cm −1 , with a resolution of 4 cm −1 and averaged over 32 scans. The extracted polymer has been compared with a commercial PHBV (PHBV Y1000P from Tianam Biopolymer, Ningbo, China). 2.6. Nuclear Magnetic Resonance (NMR) NMR spectra were recorded using a Bruker 400 MHz. All NMR spectra were recorded at 25 ◦ C unless otherwise stated. Chemical shifts ( δ ) are reported in parts per million (ppm) and referenced to CDCl 3 ( 1 H: 7.26 ppm). The content in the monomer 3-hydroxyvalerate (3HV) was achieved from the integration of the resonance peaks assigned to the methyl protons from the monomeric units 3HV and 3HB in 1H NMR, using the equation 3HV(%)=I3HV I3HV +I3HB ·100 where I 3HV and I 3HB refer to the integration of the methyl protons of the two monomers (3HV and 3HB, respectively). Bioengineering 2024,11, 870 5 of 16 2.7. Methanolysis and PHA Quantification by Gas Chromatography (GC) The methanolysis protocol was carried out as described by Kumar et al. in 2017 [ 30 ]. In total, 50 mg of lyophilized cells (frozen and dried) were weighed and transferred to a PYREX tube with a plastic screw cap with PTFE-lined rubber. To extract the polymer, the intracellular PHBV underwent a methanolysis reaction that gave rise to the methyl esters of the β -hydroxycarboxylic acids. Then, 2 mL of chloroform, 0.3 mL of 98% sulfuric acid, and 1.7 mL of methanol were added. In total, 0.010 g of benzoic acid prepared in 1 mL of methanol was also added as the internal standard. The tubes were heated at 100 ◦ C for 140 min and cooled at room temperature. Before vortexing the tubes for 30 s, 1 mL of distilled water was added and then the phases were allowed to separate. The chloroform phase (the bottom phase) was collected and transferred to a vial for chromatography analysis. Samples were frozen at this point at − 70/ − 80 ◦ C until analysis. In addition, commercial PHBV with 9% HV (Sigma-Aldrich, St. Louis, MO, USA) was used as an external standard. A curve was made with a PHBV mass of 0.005 g, 0.010 g, 0.015 g, and 0.020 g. These standards must be subjected to the same methanolysis process as the test samples. Regarding gas chromatography analysis, a Shimadzu gas chromatograph (model 17A) equipped with a flame ionization detector (FID) set at 280 ◦ C was used. The temperature program was set at a temperature of 50 ◦ C for 2 min, subsequently increased from 50 ◦ C to 110 ◦ C at a rate of 20 ◦ C/min, and finally increased to 250 ◦ C at a rate of 20 ◦ C/min. The injector was kept at 250 ◦ C, and the oven was kept at 120 ◦ C. The used column was a VB-WAX (VICI) column, 30 m long, 0.25 mm in diameter, and 0.25 mm thick of the film. The injected volume was 1 µ L, with the helium flow adjusted to 1 mL/min, with a total time of 12 min. To determine the 3HB and 3HV content using the chromatograms, the area values of the 3-hydroxybutyrate methyl ester (3-HBME), 3-hydroxyvalerate methyl ester (3-HVME), and benzoic acid methyl ester (internal standard) peaks were used as follows: Normalized area =3HBME ∨3HVME area Internal standard area A straight-line pattern was created from the normalized areas to determine the equation of the function (y = a × x + b). 3HB or 3HV weight in the sample was calculated using the equation 3HB ∨3HVweight ∈the sample =Normalized area o f the sample −b a The 3HB or 3HV percentage per cell dry weight (CDW) was calculated using the equation 3HB ∨3HVcontent(%per CDW)=3HB ∨3HV weight ∈the sample 100 Cells weight 2.8. Analysis of the Biopolymer by Differential Scanning Calorimetry (DSC) A DSC25 TA calorimeter was used to analyze the glass transition of each sample, melting point, and crystallization temperature. Calibration was carried out using zinc and indium standards. The samples were heated from − 30 to 185 ◦ C at a rate of 10 ◦ C min −1 under N2 flow (50 mL min −1 ). The samples were then held at 185 ◦ C for 3 min before cooling at 10 ◦C min−1to −30 ◦C and the cycle repeated. The percentage of crystallinity of the samples was calculated according to the following equation: Xc(%)=∆Hm ∆Hom ·100 Bioengineering 2024,11, 870 6 of 16 where ∆ xH om is the melting enthalpy of the PHBV 100% crystalline and ∆ H m the melting enthalpy registered during the first heating scan. The extracted polymer has been compared with a commercial PHBV (PHBV Y1000P from Tianam Biopolymer). 2.9. Thermogravimetric Analysis (TGA) Thermogravimetric analysis (TGA) was performed in a Netzsch TG209F1 instrument TGA 2950. The samples were heated from room temperature to 800 ◦ C at a heating rate of 20 ◦ C/min in a nitrogen atmosphere (nitrogen gas flow rate of 20 mL/min), followed by an isothermal step (800 ◦ C over 20 min) and a second heating to 900 ◦ C at a heating rate of 20 ◦ C/min in an oxygen atmosphere. The derivative of TGA curves (DTG) was obtained using Netzsch analysis software (version 7.0). The extracted polymer has been compared with a commercial PHBV (PHBV Y1000P from Tianam Biopolymer). 3. Results 3.1. Monitorization of the Cultures Growth Cultures grown with 1% R1 reached the exponential phase around day 4, as did those grown with 1% R2, while cultures with 1% (w/v; 1 g/100 mL) glucose reached this phase earlier (Figure 1). Faster growth was observed at the beginning of the incubation in the presence of glucose (control media), which is an expected behavior since glucose is a preferred carbon source for H. mediterranei cells and its transport processes are simpler to those that could imply preliminary extracellular degradation of the carbon sources, as occurs with starch [ 31 ]. Thus, it is possible to think that due to the complexity and heterogeneity of both residues (Table 1), extracellular degradation of the carbon sources could be involved, resulting in a similar behavior in terms of kinetical growth. The R1 cultures were the first to reach the stationary growth phase, specifically on day 7, followed by the control cultures, which took approximately half a day longer, and finally, the cultures containing R2, which reached it on day 8. In addition, the R1 and control cultures showed a more stable and better-defined stationary phase than the R2 cultures. The OD in this phase was 4.6 for the R1 cultures, 6.5 for the R2 cultures, and 7.5 for the control cultures containing glucose. Considering that the chemical composition of the waste could vary among different stocks, up to five replicates were performed to monitor the reproducibility. Chemical analysis of the waste provided by the candy industry and the cellular growth profiles observed indicate that the cellular growth and the PHA production were stable and reproducible. Bioengineering 2024, 11, x FOR PEER REVIEW 6 of 16 under N2 flow (50 mL min−1). The samples were then held at 185 °C for 3 min before cooling at 10 °C min−1 to −30 °C and the cycle repeated. The percentage of crystallinity of the samples was calculated according to the following equation: 𝛸(%)= 𝛥𝐻 𝛥𝐻 · 100 where ΔxHom is the melting enthalpy of the PHBV 100% crystalline and ΔHm the melting enthalpy registered during the first heating scan. The extracted polymer has been compared with a commercial PHBV (PHBV Y1000P from Tianam Biopolymer). 2.9. Thermogravimetric Analysis (TGA) Thermogravimetric analysis (TGA) was performed in a Netzsch TG209F1 instrument TGA 2950. The samples were heated from room temperature to 800 °C at a heating rate of 20 °C/min in a nitrogen atmosphere (nitrogen gas flow rate of 20 mL/min), followed by an isothermal step (800 °C over 20 min) and a second heating to 900 °C at a heating rate of 20 °C/min in an oxygen atmosphere. The derivative of TGA curves (DTG) was obtained using Netzsch analysis software (version 7.0). The extracted polymer has been compared with a commercial PHBV (PHBV Y1000P from Tianam Biopolymer). 3. Results 3.1. Monitorization of the Cultures Growth Cultures grown with 1% R1 reached the exponential phase around day 4, as did those grown with 1% R2, while cultures with 1% (w/v; 1 g/100 mL) glucose reached this phase earlier (Figure 1). Faster growth was observed at the beginning of the incubation in the presence of glucose (control media), which is an expected behavior since glucose is a preferred carbon source for H. mediterranei cells and its transport processes are simpler to those that could imply preliminary extracellular degradation of the carbon sources, as occurs with starch [31]. Thus, it is possible to think that due to the complexity and heterogeneity of both residues (Table 1), extracellular degradation of the carbon sources could be involved, resulting in a similar behavior in terms of kinetical growth. The R1 cultures were the first to reach the stationary growth phase, specifically on day 7, followed by the control cultures, which took approximately half a day longer, and finally, the cultures containing R2, which reached it on day 8. In addition, the R1 and control cultures showed a more stable and better-defined stationary phase than the R2 cultures. The OD in this phase was 4.6 for the R1 cultures, 6.5 for the R2 cultures, and 7.5 for the control cultures containing glucose. Considering that the chemical composition of the waste could vary among different stocks, up to five replicates were performed to monitor the reproducibility. Chemical analysis of the waste provided by the candy industry and the cellular growth profiles observed indicate that the cellular growth and the PHA production were stable and reproducible. Figure 1. Growth curve (OD600) of H. mediterranei with the different carbon sources. Figure 1. Growth curve (OD600) of H. mediterranei with the different carbon sources. 3.2. Analysis of the PHA Granules within the Cells by Transmission Electron Microscopy (TEM) As shown in Figure 2, TEM of H. mediterranei cells growing in both residues and harvested at the beginning of the stationary phase of growth revealed the presence of PHA granules. Most of the PHA granules synthesized are spherical and their boundaries are well defined. Bioengineering 2024,11, 870 7 of 16 Bioengineering 2024, 11, x FOR PEER REVIEW 7 of 16 3.2. Analysis of the PHA Granules within the Cells by Transmission Electron Microscopy (TEM) As shown in Figure 2, TEM of H. mediterranei cells growing in both residues and harvested at the beginning of the stationary phase of growth revealed the presence of PHA granules. Most of the PHA granules synthesized are spherical and their boundaries are well defined. Residue 1 Residue 2 Control Figure 2. Electron micrographs of H. mediterranei cells grown with candy industry residues demonstrate the accumulation of PHA granules. Cells were harvested at the beginning of the stationary phase of growth. 1µm Figure 2. Electron micrographs of H. mediterranei cells grown with candy industry residues demonstrate the accumulation of PHA granules. Cells were harvested at the beginning of the stationary phase of growth. 3.3. PHA Quantification Total PHA isolation and quantification were conducted as described in the materials and methods section. Cells grown in culture media prepared with each of both residues produced more biopolymer than cells grown in the standard culture; total biomass recovered at the stationary phase of growth was also higher in cultures containing candy residues than in standard cultures. By comparing results obtained from cultures containing the residues (Table 2), it is possible to conclude that cells grown in R2 cultures produced more biopolymer than cells grown in the presence of R1, although the H. mediterranei biomass obtained from cultures containing R1 was slightly higher than that the obtained Bioengineering 2024,11, 870 8 of 16 from cultures containing R2. The results related to biopolymer quantification displayed in Table 2are expressed as grams of PHBV per liter because the analysis summarized in Section 3.4 demonstrated that the biopolymer produced by the cells under the tested conditions was PHBV. R2 is more suitable for PHBV production than R1. H. mediterranei can synthesize more polymer in less time, even using glucose as the carbon source. R2 has a lower content of carbohydrates than R1, but a higher content of proteins than the other two substrates, which influences the ratio C/N, thus changing the stress pressure, which affects the cells. In this case, having less stress for growing helps the PHBV accumulation, although this does not happen with the CDW production. Table 2. Cell Dry Weight gram per liter, PHBV grams per liter, PHBV yield, and 3HV% content in H. mediterranei cells growing with different residues from the confectionery industry. Sample gCDW/L gPHBV/L gPHBV/gCDW 3HV% by GC a3HV% by NMR bmgPHBV/L Y100P - - - - 1 cResidue 1 2.840 0.256 ±0.166 0.236 ±0.075 8.630 ±0.350 12.129 ±0.849 21.323 ±3.864 Residue 2 2.600 0.983 ±0.330 0.378 ±0.112 9.36 0 ±0.430 8.832 ±0.371 66.943 ±10.912 Control 1.890 0.350 ±0.095 0.111 ±0.030 10.150 ±0.740 11.901 ±0.853 35.018 ±3.044 a the analysis was carried out on the biomass; b the analysis was carried out on the PHBV; c information provided by the manufacturer. On the other hand, R1 shows the lowest yields of PHBV production but the highest CDW production. Under this condition, H. mediterranei cells have suffered a higher stress due to significant changes in the C/N ratio. Also, R1 does not contain free sugars, which increases cellular stress (the cells must segregate enzymes able to degrade complex polysaccharides like starch to make the sugars more accessible [ 31 ]). This process required more time, so H. mediterranei took more to produce PHBV. 3.4. Characterization of the Biopolymer Produced by H. mediterranei Considering that PHA constitutes a big group of biopolymers of slightly different structures and compositions, different techniques have been used to identify and characterize the major biopolymer produced by the cells under the tested conditions. Thus, using FTIR, the results revealed that 100% of the biopolymer isolated was a PHBV type. Then, its structure and HV content were confirmed by NMR. Subsequently, HV determination by GC was performed as an additional confirmatory test. Once the chemical composition was elucidated, thermal characterization of PHBV was carried out by differential scanning calorimetry and thermogravimetric analysis. The detailed results obtained from each analysis are described. 3.4.1. Analysis of the Biopolymer by FTIR The purified polymer was analyzed in the solid state by FTIR spectroscopy using ATR sampling methodology and compared with a commercial PHBV (Figure 3). FTIR spectra of the different samples showed an absorption peak centered at 1721 cm −1 ascribed to the stretching band of the ester carbonyl bond (C=O). Other absorption bands for the polymer sample obtained under the conditions of this study were found in the range 2924–2856 cm−1 (CH, CH 2 symmetric and asymmetric stretching), 1450–1380 cm −1 (C-C stretching), at 1132 cm −1 (C-O stretching), and in the range 978–821 cm −1 corresponding to C-C deformation. The spectra corresponded to the typical profile of a copolymer PHBV, previously reported in H. mediterranei [ 32 ]. Unfortunately, the comonomer molar ratio (3HB/3HV) could not be determined by this technique. Bioengineering 2024,11, 870 9 of 16 Bioengineering 2024, 11, x FOR PEER REVIEW 9 of 16 stretching), at 1132 cm −1 (C-O stretching), and in the range 978–821 cm −1 corresponding to C-C deformation. The spectra corresponded to the typical profile of a copolymer PHBV, previously reported in H. mediterranei [32]. Unfortunately, the comonomer molar ratio (3HB/3HV) could not be determined by this technique. Figure 3. Attenuated total reflectance–Fourier transform infrared (ATR–FTIR) spectra of the PHBV purified from H. mediterranei and of the commercial PHBV ENMAT Y1000P (3HV content ≈ 1 mol %). 3.4.2. NMR and GC To complete the structural analysis of the PHBV, the polymer composition was investigated by nuclear magnetic resonance (spectra in supporting information), and the results are gathered in Table 2. The results corroborate FTIR analysis conclusions, i.e., the biopolymer synthesized corresponded to the PHBV type. The PHBV produced by the cells grown in the control cultures and cultures containing R1 showed higher content of the comonomer 3HV (11.90 ± 0.85% and 12.13 ± 0.85%, respectively) than the biopolymer produced by the cells grown in cultures containing R2 (8.83 ± 0.37%). The concentration of 3HV was also analyzed by gas chromatography. These results differ from GC results because they are complementary and were not performed with the same substrate. In the case of GC, the methodology was applied directly over the biomass, without isolation of the polymer. Conversely, the content in 3HV measured by NMR was inferred from the spectra of the purified materials. The biopolymer synthesized by cells grown in control cultures accumulated 33.17 mg of 3HB (66.34%, in % per CDW), while the PHBV synthesized by the cells grown in cultures with R1 contained 31.03 mg of 3HB (62.07% in % per CDW). The PHBV obtained Figure 3. Attenuated total reflectance–Fourier transform infrared (ATR–FTIR) spectra of the PHBV purified from H. mediterranei and of the commercial PHBV ENMAT Y1000P (3HV content ≈ 1 mol %). 3.4.2. NMR and GC To complete the structural analysis of the PHBV, the polymer composition was investigated by nuclear magnetic resonance (spectra in Supporting Information), and the results are gathered in Table 2. The results corroborate FTIR analysis conclusions, i.e., the biopolymer synthesized corresponded to the PHBV type. The PHBV produced by the cells grown in the control cultures and cultures containing R1 showed higher content of the comonomer 3HV (11.90 ± 0.85% and 12.13 ± 0.85%, respectively) than the biopolymer produced by the cells grown in cultures containing R2 (8.83 ± 0.37%). The concentration of 3HV was also analyzed by gas chromatography. These results differ from GC results because they are complementary and were not performed with the same substrate. In the case of GC, the methodology was applied directly over the biomass, without isolation of the polymer. Conversely, the content in 3HV measured by NMR was inferred from the spectra of the purified materials. The biopolymer synthesized by cells grown in control cultures accumulated 33.17 mg of 3HB (66.34%, in % per CDW), while the PHBV synthesized by the cells grown in cultures with R1 contained 31.03 mg of 3HB (62.07% in % per CDW). The PHBV obtained from cells grown in the presence of R2 showed the highest amount of 3HB, making up to 48.02 mg of 3HBweight (96.05% of 3HB content in % per CDW). Regarding the 3HVcomonomer content, the PHBV produced by the cells grown in control cultures showed a higher content of 3HV (5.07 mg of 3HVweight (10.15%)), followed by the PHBV synthesized by cells grown in the presence of R2 cultures (4.68 mg (9.36%)) Bioengineering 2024,11, 870 16 of 16 50. Cai, S.; Wu, Y.; Li, Y.; Yang, S.; Liu, Z.; Ma, Y.; Lv, J.; Shao, Y.; Jia, H.; Zhao, Y.; et al. Production of polyhydroxyalkanoates in unsterilized hyper-saline medium by halophiles using waste silkworm excrement as carbon source. Molecules 2021,26, 7122. [CrossRef] 51. 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