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Recovery of volatile terpenes produced by genetically engineered Escherichia coli strains using fluidized bed


Amiri, Azin (2020) Recovery of volatile terpenes produced by genetically engineered Escherichia coli strains using fluidized bed. Doctoral thesis, Universiti Putra Malaysia.


Terpenes are a highly diverse class of naturally occurring metabolites with more than 40000 structures known to date with traditional and modern applications in various industries such as pharmaceuticals, flavoring compounds, fragrance, biofuel and agriculture. Monoterpenes, as a group of terpenes, are biogenic organic volatile compounds comprised of two isoprene units (C5H8) possessing crucial defence and communication roles in plants. However, being a secondary metabolite, terpene production from natural sources (plants) is not reliable for novel bulk applications as it could be low-yielding and costly and fluctuate in availability and quality. Therefore, heterologous production of terpenes by Escherichia coli and Saccharomyces cerevisiae has been sought as an alternative approach. Although most of the attempts have been focused on strain and pathway engineering to maximize yields, titers and productivities of the chassis, little effort has been made on downstream processing. As more microbial processes mature to the pilot and commercial scale, this aspect of bioprocessing needs to be studied. Current practice for extraction of volatile monoterpenes is liquid-liquid extraction using dodecane. This technique is not feasible in industrial scale due to health, economic and technical concerns. Different techniques such as traditional solvent extraction, accelerated solvent extraction, steam distillation technology and supercritical CO2 extraction have been tested for volatile monoterpene extraction from plants, none of which extracted enough monoterpenes to reach quantification level. Alternative separation modalities are needed in order to facilitate extraction process for better recovery and ease of scaleup of microbial terpene production. In this study, a suitable method to capture microbially produced monoterpenes prior to release of these volatile compounds from the system into environment was developed. The model monoterpene was carene, which was produced by Escherichia coli SACar in a 2 L stirred tank bioreactor. The carene escaped due to its high volatility, proven by headspace analyses. After testing several different prototypes, a new bed column was designed and 3D printed. Several resins were tested and a moderately hydrophobic polyacrylic divinylbenzene resin, Purosorb PAD610, with 84% recovery was chosen as the best resin to capture the non-polar carene molecules with 2h desorption time. Interaction of carene to the resin followed a freundlich isotherm model in which a monolayer adsorbtion pattern occurs, directly relating the binding efficiency and availability of vacant surface area. A fluidized bed system comprising Purosorb PAD610 was successfully implemented to capture monoterpenes continuously as they were produced and volatilized within the bioreactor. Extraction conditions including air flow rate (vvm), resin volume (mL) and agitation speed (rpm) were optimized using fractional factorial experimentation to enhance the capture efficiency of the volatilized carene. The newly-developed fluidized capture device resulted in in-situ capture of 6μL/L of the carene, which was 12 fold higher than carene recovered using dodecane overlay method. This system was successfully set up and tested for three other monoterpenes; linalool, limonene and myrcene and resulted in capture of 19 μL/L, 1 μL/L and 3 μL/L of each monoterpene, respectively. Therefore, superiority of the new extraction method in capturing volatile monoterpene is proved. This novel technique is the first method for in-situ capture of microbial volatile monoterpene with potential to be applied to other volatile compounds in different scales.

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Additional Metadata

Item Type: Thesis (Doctoral)
Call Number: FBSB 2020 14
Chairman Supervisor: Professor Rosfarizan Mohamad, PhD
Divisions: Faculty of Biotechnology and Biomolecular Sciences
Depositing User: Mas Norain Hashim
Date Deposited: 17 Aug 2021 00:17
Last Modified: 17 Aug 2021 00:17
URI: http://psasir.upm.edu.my/id/eprint/90503
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