Publicação
Polyhydroxyalkanoate production process: real-time monitoring, extraction optimization and framework for sustainability assessment
| Resumo: | The advantageous properties of conventional plastics have made them a major commodity worldwide, with yearly global productions currently exceeding 300 million tons. Despite their benefits, their recalcitrancy to biodegradation combined with the inefficient collection and recycling mechanisms have resulted in an alarming accumulation of plastic waste in the environment. Polyhydroxyalkanoates (PHA) arose as a potential alternative to conventional plastics. PHA are biodegradable aliphatic polyesters that accumulate intracellularly in microorganisms as carbon and energy reserves, and present properties similar to those of conventional plastics. Despite their environmental advantages, the widespread use of PHA is conditioned by their high production costs, which, so far, prevented them from being cost-competitive with conventional plastics. Research efforts in the last decades have managed to reduce PHA process production costs, but further action is still required. Considering this, the goal of the present work was to develop strategies to, potentially, make PHA production more sustainable by exploring three different approaches. The first approach focused on developing predictive models based on near-infrared (NIR) spectroscopy for the real-time monitoring of critical operation parameters in a pilot-scale fedbatch PHA accumulation stage of a three-stage PHA production process using mixed microbial cultures (MMC) and fruit waste as feedstock. These parameters were the carbon substrate concentration and the intracellular PHA content, as their real-time monitoring would allow to minimize or even prevent microbial PHA consumption between feed-pulses during PHA accumulation, and, consequently, contribute to productivity increase. Good predictive models were obtained for intracellular PHA content, total fermentation products concentration (present in the fermented fruit waste), and butyrate (one of the main fermentation products) concentration. The best model for intracellular PHA quantification presented an external validation coefficient of determination (R2) of 0.98 and a root mean square error of prediction (RMSEP) of 1.75 wt.% PHA in a range of 16.6-49.8 wt.% PHA, while the best model for total fermentation products presented an external validation R2 of 0.97 and a RMSEP of 0.11 gCOD/L in a range of 0.00-1.98 gCOD/L. The best predictive model for butyrate quantification presented an external validation R2 of 0.97 and a RMSEP of 0.12 gCOD/L in a range of 0.00-1.4 gCOD/L. These results demonstrate the potential of NIR spectroscopy to be used in the real-time monitoring and optimization of PHA production from real and complex feedstocks. In the second approach, efforts were directed towards downstream processing, which represents one of the most cost-intensive steps in PHA production. PHA extraction methods, using chemicals with lower environmental impact than organic solvents, were first optimized for a MMC produced in the pilot-scale system described in the first approach, and then for a MMC produced from paper-mill wastewater, also at pilot-scale. This approach focused on PHA extraction methods based on the chemical digestion of non-PHA cell matter (NPCM), due to their potential to be more sustainable and scalable than other processes. For the fruit wastefed biomass, a central composite rotatable design of experiments was performed to determine the optimal conditions for NPCM digestion with either sodium hydroxide (NaOH) or sodium hypochlorite (NaClO). These conditions were found to be a concentration of 0.28 M and a digestion time of 4.8h for NaOH, and a concentration of 8.97% (w/v) and a digestion time of 3.4h for NaClO, having resulted in high purity polymers without significant molecular weight reduction, but distinct thermal behaviors. Afterwards, these conditions were used to assess the impact of intracellular PHA content, biomass concentration and biomass pre-treatment on the PHA extraction performance. The results suggested that the higher the PHA content in the biomass, the higher the purity of the extracted polymers. Conversely, increasing biomass concentration during extraction negatively affected polymer purity. Additionally, biomass pretreatment by drying at 60 °C resulted in polymer degradation, as evidenced by reduced molecular weight and altered thermal properties, highlighting that the use of fresh biomass is preferable to preserve polymer quality. In the extraction of PHA from biomass produced with paper-mill wastewater, a few challenges were found, namely its low PHA content (< 30%) and its recalcitrant impurities, including calcium carbonate (CaCO3) and lignin-related compounds. After determining that the previously developed chemical digestion-based methods using NaOH, NaClO and hydrogen peroxide (H2O2) were ineffective for this biomass, a modified approach was developed to specifically address its complex impurities. This strategy introduced a pre-treatment step aimed at the removal of CaCO₃ and lignin-derived compounds, based on incubation with acetic acid (CH₃COOH) and H₂O₂. The in situ formation of peracetic acid enables lignin degradation, enhances cell disruption, and generates low-toxicity byproducts, while the acidic conditions simultaneously promote the dissolution of CaCO₃. This method was developed and optimized at lab-scale and, afterwards, successfully applied at pilot-scale, where ca. 7 kg of fresh biomass, containing 1.7 kg of solids (with ca. 29% of PHA and 53% of inorganics) were subjected to a first incubation with CH3COOH at 20% and H2O2 at 6% (w/v), at room temperature for 2 hours, and then a second incubation with NaClO at 0.5 g/gorganics, at room temperature for 3 hours. At these conditions, 0.3 kg of dry polymer with a PHA purity of 70% and 4% of inorganics were obtained. This polymer presented properties comparable to those of the polymer extracted using the benchmark protocol (PHA dissolution in chloroform and precipitation in ethanol), in terms of molecular weight and thermal behavior. These results show that the developed PHA extraction method has the potential to efficiently recover PHA from biomass produced with complex wastes, and to be used in large scale. The third approach entailed the development of a framework to assess the sustainability of PHA, and other bioplastics, as well as conventional plastics, in the environmental, economic and societal dimensions, so that these materials can be fairly compared. This life-cycle sustainability assessment (LCSA) framework was built based on information present in the literature and information provided by a group of stakeholders directly involved in the life-cycles of bioplastics and conventional plastics, who responded to a survey elaborated in this context. This work, particularly the insights gathered from stakeholders, made it evident that the economic dimension cannot be the only vector for evaluating the sustainability of plastics/bioplastics. Additionally, an integrated analysis such as LCSA has the benefit of providing a broader overview of the advantages/disadvantages of products and potential trade-offs between the three sustainability dimensions and between its life-cycle stages. The results of this approach provide guidance to perform an overall sustainability assessment of plastics/bioplastics. |
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| Autores principais: | Rodrigues, Ana Marta da Silva |
| Assunto: | Polyhydroxyalkanoates (PHA) Mixed microbial culture (MMC) Real-time monitoring Near-infrared (NIR) spectroscopy PHA extraction chemical digestion |
| Ano: | 2026 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade Nova de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório Institucional da UNL |
| Resumo: | The advantageous properties of conventional plastics have made them a major commodity worldwide, with yearly global productions currently exceeding 300 million tons. Despite their benefits, their recalcitrancy to biodegradation combined with the inefficient collection and recycling mechanisms have resulted in an alarming accumulation of plastic waste in the environment. Polyhydroxyalkanoates (PHA) arose as a potential alternative to conventional plastics. PHA are biodegradable aliphatic polyesters that accumulate intracellularly in microorganisms as carbon and energy reserves, and present properties similar to those of conventional plastics. Despite their environmental advantages, the widespread use of PHA is conditioned by their high production costs, which, so far, prevented them from being cost-competitive with conventional plastics. Research efforts in the last decades have managed to reduce PHA process production costs, but further action is still required. Considering this, the goal of the present work was to develop strategies to, potentially, make PHA production more sustainable by exploring three different approaches. The first approach focused on developing predictive models based on near-infrared (NIR) spectroscopy for the real-time monitoring of critical operation parameters in a pilot-scale fedbatch PHA accumulation stage of a three-stage PHA production process using mixed microbial cultures (MMC) and fruit waste as feedstock. These parameters were the carbon substrate concentration and the intracellular PHA content, as their real-time monitoring would allow to minimize or even prevent microbial PHA consumption between feed-pulses during PHA accumulation, and, consequently, contribute to productivity increase. Good predictive models were obtained for intracellular PHA content, total fermentation products concentration (present in the fermented fruit waste), and butyrate (one of the main fermentation products) concentration. The best model for intracellular PHA quantification presented an external validation coefficient of determination (R2) of 0.98 and a root mean square error of prediction (RMSEP) of 1.75 wt.% PHA in a range of 16.6-49.8 wt.% PHA, while the best model for total fermentation products presented an external validation R2 of 0.97 and a RMSEP of 0.11 gCOD/L in a range of 0.00-1.98 gCOD/L. The best predictive model for butyrate quantification presented an external validation R2 of 0.97 and a RMSEP of 0.12 gCOD/L in a range of 0.00-1.4 gCOD/L. These results demonstrate the potential of NIR spectroscopy to be used in the real-time monitoring and optimization of PHA production from real and complex feedstocks. In the second approach, efforts were directed towards downstream processing, which represents one of the most cost-intensive steps in PHA production. PHA extraction methods, using chemicals with lower environmental impact than organic solvents, were first optimized for a MMC produced in the pilot-scale system described in the first approach, and then for a MMC produced from paper-mill wastewater, also at pilot-scale. This approach focused on PHA extraction methods based on the chemical digestion of non-PHA cell matter (NPCM), due to their potential to be more sustainable and scalable than other processes. For the fruit wastefed biomass, a central composite rotatable design of experiments was performed to determine the optimal conditions for NPCM digestion with either sodium hydroxide (NaOH) or sodium hypochlorite (NaClO). These conditions were found to be a concentration of 0.28 M and a digestion time of 4.8h for NaOH, and a concentration of 8.97% (w/v) and a digestion time of 3.4h for NaClO, having resulted in high purity polymers without significant molecular weight reduction, but distinct thermal behaviors. Afterwards, these conditions were used to assess the impact of intracellular PHA content, biomass concentration and biomass pre-treatment on the PHA extraction performance. The results suggested that the higher the PHA content in the biomass, the higher the purity of the extracted polymers. Conversely, increasing biomass concentration during extraction negatively affected polymer purity. Additionally, biomass pretreatment by drying at 60 °C resulted in polymer degradation, as evidenced by reduced molecular weight and altered thermal properties, highlighting that the use of fresh biomass is preferable to preserve polymer quality. In the extraction of PHA from biomass produced with paper-mill wastewater, a few challenges were found, namely its low PHA content (< 30%) and its recalcitrant impurities, including calcium carbonate (CaCO3) and lignin-related compounds. After determining that the previously developed chemical digestion-based methods using NaOH, NaClO and hydrogen peroxide (H2O2) were ineffective for this biomass, a modified approach was developed to specifically address its complex impurities. This strategy introduced a pre-treatment step aimed at the removal of CaCO₃ and lignin-derived compounds, based on incubation with acetic acid (CH₃COOH) and H₂O₂. The in situ formation of peracetic acid enables lignin degradation, enhances cell disruption, and generates low-toxicity byproducts, while the acidic conditions simultaneously promote the dissolution of CaCO₃. This method was developed and optimized at lab-scale and, afterwards, successfully applied at pilot-scale, where ca. 7 kg of fresh biomass, containing 1.7 kg of solids (with ca. 29% of PHA and 53% of inorganics) were subjected to a first incubation with CH3COOH at 20% and H2O2 at 6% (w/v), at room temperature for 2 hours, and then a second incubation with NaClO at 0.5 g/gorganics, at room temperature for 3 hours. At these conditions, 0.3 kg of dry polymer with a PHA purity of 70% and 4% of inorganics were obtained. This polymer presented properties comparable to those of the polymer extracted using the benchmark protocol (PHA dissolution in chloroform and precipitation in ethanol), in terms of molecular weight and thermal behavior. These results show that the developed PHA extraction method has the potential to efficiently recover PHA from biomass produced with complex wastes, and to be used in large scale. The third approach entailed the development of a framework to assess the sustainability of PHA, and other bioplastics, as well as conventional plastics, in the environmental, economic and societal dimensions, so that these materials can be fairly compared. This life-cycle sustainability assessment (LCSA) framework was built based on information present in the literature and information provided by a group of stakeholders directly involved in the life-cycles of bioplastics and conventional plastics, who responded to a survey elaborated in this context. This work, particularly the insights gathered from stakeholders, made it evident that the economic dimension cannot be the only vector for evaluating the sustainability of plastics/bioplastics. Additionally, an integrated analysis such as LCSA has the benefit of providing a broader overview of the advantages/disadvantages of products and potential trade-offs between the three sustainability dimensions and between its life-cycle stages. The results of this approach provide guidance to perform an overall sustainability assessment of plastics/bioplastics. |
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