Projects per year
Abstract
Promoting the sustainable development of modern society, biorefineries were suggested as an alternative concept to commercial industrial processes for the production of bioenergy, biofuels, and biochemicals through biomass utilization. Lactic acid is a biochemical of interest as it exhibits a wide range of applications due to its unique properties as a platform molecule. It was first used as a preservative and flavor agent in the food and beverage sectors. Nowadays, it is utilized for the production of drugs, cosmetics, and cleaning products, or as a dyeing assistant in the textile industry. The highest demand for lactic acid in the market is attributed to its application as a building block for poly-lactic acid, a biodegradable alternative to plastics.
The industrial production of lactic acid is 90% based on microbial fermentation, a process characterized by low environmental impact, low energy consumption, and generation of products of high purity. Nevertheless, there are challenges to be overcome in both upstream and downstream strategies to obtain a process competitive to petrochemical production. Challenges regarding a successful upstream process include the selection of the fermentation substrate and the microbial factory. The fermentation substrate should be abundant and inexpensive with low pre-treatment requirements. Additionally, the microbial seed should be robust, be able to metabolize a variety of sugars, have low nutritional needs, and have the capacity to resist various stress factors. On the other hand, the downstream process accounts for 20-50% of the total operating costs. This is a result of bulk equipment, hazardous solvents, high energy demand, and generation of waste streams generated in return for high product recovery and purity.
Taking the aforementioned aspects into consideration, the aim of this Ph.D. thesis was to tackle challenges and develop strategies for the optimization of lactic acid production. The study was divided into four individual parts: a. isolation and screening of novel bacterial candidates, b. improvement of the lactic acid fermentation process by applying adaptive laboratory evolution techniques on the bacterial candidates, c. selection and optimization of
fermentation substrate, d. downstream process design for enhanced lactic acid recovery and purity.
First, seaweed, which was suggested as fermentation substrate for biorefinery processes, was used to isolate autochthonous LAB. The bacterial isolates were identified using 16S rRNA sequencing and cultured in a. synthetic media, and b. seaweed hydrolysate. Growth characteristics and lactic acid production were compared for the three different conditions. The study also included carbohydrate metabolism patterns, environmental ranges (i.e., temperature, pH, salinity), substrate inhibition, and enantioselectivity. The findings highlighted the wide spectrum of characteristics, which were even evident between bacteria of the same species or genera. Further research is needed to detect novel microorganisms with unique properties and applications in biorefinery processes.
The second project focused on seaweed as an alternative fermentation biomass to lignocellulosic material. The fact that seaweed lacks lignin results in the application of less harsh pre-treatment techniques. However, seaweed has been characterized by elevated salinity levels that can affect the fermentation process by inhibiting microbial metabolism. Consequently, the aim of this study was to identify seaweed-isolated strains that are resistant to increased salinity concentration. The most resistant candidates were further evolved in hypersaline media, applying Adaptive Laboratory Evolution (ALE) techniques. Genomic data supported the evolution findings, indicating mutations that lead to salt tolerance. The findings of the study suggested that bacteria autochthonous to saline environments can be used as starter cultures for lactic acid production, using substrates with high salt concentration, thereby avoiding a previous desalination step.
The utilization of inexpensive and abundant biomasses in biorefineries is essential to increase competition with petroleum-based processes. Thus, seaweed hydrolysate was compared with a substrate combining an industrial residue with high sugar content (candy-waste) and a municipal residue rich in nutrients (digestate from full-scale biogas plant). Substrates were characterized and improved to increase sugar utilization, lactic acid production, and to minimize the formation of secondary by-products. The bacterial strains isolated in the first project were tested as potential starter cultures. The results highlighted that while seaweed hydrolysate could serve as a fermentation substrate, the low initial sugar content limited the efficiency of the fermentation. In contrast, the co-fermentation of candy-waste and digestate was found to be a promising approach to achieve high lactic acid concentration, while also serving as an alternative waste management strategy.
The final step was to introduce a strategy for maximum recovery of lactic acid with high purity, considering the total energy consumption and the environmental impact of the proposed process. Membrane separation technologies have been suggested for the recovery of organic acids because they avoid the use of hazardous solvents, have relatively low energy demands, and can be easily upscaled. In this project microfiltration, nanofiltration, monopolar-, and bipolar electrodialysis were tested for the separation of lactic acid from the fermentation broth produced in the third project. Lactic acid rejection rate, purity, and energy consumption were considered for the optimization of the process. The findings indicated the importance of pH as a factor affecting lactic acid separation and highlighted the need for a suitable combination of separation strategies designed according to the specific substrate’s characteristics.
To sum up, the present Ph.D. thesis provides answers to scientific questions covering different parts of the lactic acid production process. Robust bacterial strains with promising characteristics were detected for application in fermentation processes. The seaweed-isolated bacteria were then tested for salinity resistance and were further evolved in hypersaline conditions, highlighting the importance of employing ALE methods for strain optimization. Furthermore, the utilization of residual streams as fermentation substrates and the application of a co-fermentation strategy showed promising results. Finally, membrane separation was successfully applied for the recovery of high-purity lactic acid.
The industrial production of lactic acid is 90% based on microbial fermentation, a process characterized by low environmental impact, low energy consumption, and generation of products of high purity. Nevertheless, there are challenges to be overcome in both upstream and downstream strategies to obtain a process competitive to petrochemical production. Challenges regarding a successful upstream process include the selection of the fermentation substrate and the microbial factory. The fermentation substrate should be abundant and inexpensive with low pre-treatment requirements. Additionally, the microbial seed should be robust, be able to metabolize a variety of sugars, have low nutritional needs, and have the capacity to resist various stress factors. On the other hand, the downstream process accounts for 20-50% of the total operating costs. This is a result of bulk equipment, hazardous solvents, high energy demand, and generation of waste streams generated in return for high product recovery and purity.
Taking the aforementioned aspects into consideration, the aim of this Ph.D. thesis was to tackle challenges and develop strategies for the optimization of lactic acid production. The study was divided into four individual parts: a. isolation and screening of novel bacterial candidates, b. improvement of the lactic acid fermentation process by applying adaptive laboratory evolution techniques on the bacterial candidates, c. selection and optimization of
fermentation substrate, d. downstream process design for enhanced lactic acid recovery and purity.
First, seaweed, which was suggested as fermentation substrate for biorefinery processes, was used to isolate autochthonous LAB. The bacterial isolates were identified using 16S rRNA sequencing and cultured in a. synthetic media, and b. seaweed hydrolysate. Growth characteristics and lactic acid production were compared for the three different conditions. The study also included carbohydrate metabolism patterns, environmental ranges (i.e., temperature, pH, salinity), substrate inhibition, and enantioselectivity. The findings highlighted the wide spectrum of characteristics, which were even evident between bacteria of the same species or genera. Further research is needed to detect novel microorganisms with unique properties and applications in biorefinery processes.
The second project focused on seaweed as an alternative fermentation biomass to lignocellulosic material. The fact that seaweed lacks lignin results in the application of less harsh pre-treatment techniques. However, seaweed has been characterized by elevated salinity levels that can affect the fermentation process by inhibiting microbial metabolism. Consequently, the aim of this study was to identify seaweed-isolated strains that are resistant to increased salinity concentration. The most resistant candidates were further evolved in hypersaline media, applying Adaptive Laboratory Evolution (ALE) techniques. Genomic data supported the evolution findings, indicating mutations that lead to salt tolerance. The findings of the study suggested that bacteria autochthonous to saline environments can be used as starter cultures for lactic acid production, using substrates with high salt concentration, thereby avoiding a previous desalination step.
The utilization of inexpensive and abundant biomasses in biorefineries is essential to increase competition with petroleum-based processes. Thus, seaweed hydrolysate was compared with a substrate combining an industrial residue with high sugar content (candy-waste) and a municipal residue rich in nutrients (digestate from full-scale biogas plant). Substrates were characterized and improved to increase sugar utilization, lactic acid production, and to minimize the formation of secondary by-products. The bacterial strains isolated in the first project were tested as potential starter cultures. The results highlighted that while seaweed hydrolysate could serve as a fermentation substrate, the low initial sugar content limited the efficiency of the fermentation. In contrast, the co-fermentation of candy-waste and digestate was found to be a promising approach to achieve high lactic acid concentration, while also serving as an alternative waste management strategy.
The final step was to introduce a strategy for maximum recovery of lactic acid with high purity, considering the total energy consumption and the environmental impact of the proposed process. Membrane separation technologies have been suggested for the recovery of organic acids because they avoid the use of hazardous solvents, have relatively low energy demands, and can be easily upscaled. In this project microfiltration, nanofiltration, monopolar-, and bipolar electrodialysis were tested for the separation of lactic acid from the fermentation broth produced in the third project. Lactic acid rejection rate, purity, and energy consumption were considered for the optimization of the process. The findings indicated the importance of pH as a factor affecting lactic acid separation and highlighted the need for a suitable combination of separation strategies designed according to the specific substrate’s characteristics.
To sum up, the present Ph.D. thesis provides answers to scientific questions covering different parts of the lactic acid production process. Robust bacterial strains with promising characteristics were detected for application in fermentation processes. The seaweed-isolated bacteria were then tested for salinity resistance and were further evolved in hypersaline conditions, highlighting the importance of employing ALE methods for strain optimization. Furthermore, the utilization of residual streams as fermentation substrates and the application of a co-fermentation strategy showed promising results. Finally, membrane separation was successfully applied for the recovery of high-purity lactic acid.
Original language | English |
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Place of Publication | Kgs. Lyngby |
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Publisher | Technical University of Denmark |
Number of pages | 209 |
Publication status | Published - 2023 |
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- 1 Finished
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Process optimization for enhanced lactic and amino acid production, AgRefine ITN
Papadopoulou, E. (PhD Student), Garde, A. (Examiner), Venus, J. (Examiner), Angelidaki, I. (Main Supervisor) & Tsapekos, P. (Supervisor)
01/06/2020 → 16/11/2023
Project: PhD