High Cell Density Strategies and Adaptive Evolution for Microbial Propionic Acid Production
(2022)- Abstract
- Propionic acid (PA) is a naturally occurring short chain fatty acid produced by microbial fermentation of the indigestible foods in the intestine, having a positive effect on the gastrointestinal health. PA inhibits the growth of pathogenic bacteria and molds; hence it is widely used as preservative for food, feed and grains. It is also used as a component in several industrial products like polymers, cosmetics and pharmaceuticals. Its industrial production still depends on petrochemistry, however microbial fermentation as a route for production from renewable resources has attracted attention but is not yet competitive with the existing route. Dairy propionibacteria, used as starters for the maturation of Swiss-type cheeses and also known... (More)
- Propionic acid (PA) is a naturally occurring short chain fatty acid produced by microbial fermentation of the indigestible foods in the intestine, having a positive effect on the gastrointestinal health. PA inhibits the growth of pathogenic bacteria and molds; hence it is widely used as preservative for food, feed and grains. It is also used as a component in several industrial products like polymers, cosmetics and pharmaceuticals. Its industrial production still depends on petrochemistry, however microbial fermentation as a route for production from renewable resources has attracted attention but is not yet competitive with the existing route. Dairy propionibacteria, used as starters for the maturation of Swiss-type cheeses and also known for their probiotic properties, produce propionic acid from different sugars or glycerol via Wood-Werkman cycle under anaerobic conditions. The fermentation process has a long lag phase and is also subject to product inhibition, resulting in low productivity and yield.
This thesis involves studies on high cell density fermentations and development of acid tolerant strain as ways to improve the efficiency of propionic acid fermentation. Studies have been performed using Propionibacterium acidipropionici DSM 4900 (ATCC 25562), a bacterium that is generally regarded as safe (GRAS). Glycerol, a by-product of biodiesel production process, was used as the carbon source for fermentations.
Different strategies of high cell density fermentations including sequential batch cultivation, cyclic batch cultivation, cell immobilization and cell retention using membrane filter, were evaluated. Propionate yield and productivity of 93 mol % and 0.53 g/L/h were obtained in cyclic batch while maximum productivity of 1.63 g/L/h was obtained from 90 g/L glycerol during sequential batch cultivations with free cells. P. acidipropionici cells were immobilized on plastic based AnoxKaldnes® and recycled glass based Poraver® carriers, either by polyethyleneimine (PEI) treatment or by biofilm formation. Exposing the cells to stress factors like 30 mM citric acid and 1 M NaCl was beneficial for increasing the Biofilm Forming Capacity index (BFC) and for production of exopolysaccharides (EPS), both indicators of biofilm formation. Fermentations in the immobilized packed-bed bioreactors using the bacteria pre-exposed to citric acid and NaCl gave propionic acid productivities of 0.7 and 0.78 g/L/h, respectively, with Poraver® matrix, and the corresponding values for AnoxKaldnes® were 0.39 and 0.43 g/L/h, respectively. Continuous propionic acid fermentations by cell retention in a ceramic membrane filter, gave productivity of 2.35 g/L/h in a medium with 60 g/L glycerol and 20 g/L yeast extract.
A tolerant strain able to grow in a culture medium supplemented with 20 g/L of PA, was obtained by adaptive laboratory evolution. It exhibited 1.4-fold higher growth rate, 3.7-fold higher propionic acid production kinetics, 1.2-fold higher yield and almost 2-fold higher titer in batch cultivations compared to the parental cells in a basal cultivation medium. It was immobilized to PEI-treated Poraver® and used for fermentations in recycled batch mode with increasing glycerol concentration and decreasing pH, respectively, in packed-bed bioreactors. Glycerol up to a concentration of 100 g/L was completely consumed, resulting in 58 g/L propionic acid and product yield of 0.64 mol/mol. Progressive decrease in pH values gave increased propionic acid yield and productivity from 20 g/L glycerol, while maintaining the product titer nearly constant at pH 5.0.
The exposure to stress factors for biofilm formation and to high propionic acid concentration during cultivation of P. acidipropionici showed trehalose to play a protective role. Following the expression of the trehalose synthesis-related genes treY and otsA, and by Real Time-qPCR revealed treY expression (but not otsA) to be significantly increased during biofilm formation under the influence of citric acid. Expression of the biofilm-related luxS gene was also detected by fluorescence in situ hybridization (FISH). Expression of treY was increased 82-fold in the tolerant strain obtained by adaptive evolution with respect to the 16S rRNA gene used as a control.
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Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/bea74a88-1f0f-4094-a25f-36e949f86f54
- author
- Cavero Olguin, Victor Hugo LU
- supervisor
- opponent
-
- Dr. Venus, Joachim, ATB Potsdam, Germany
- organization
- publishing date
- 2022-08-23
- type
- Thesis
- publication status
- published
- subject
- pages
- 174 pages
- publisher
- Division of Biotechnology, Lund University
- defense location
- Lecture Hall KC:A, Kemicentrum, Sölvegatan 39, Faculty of Engineering LTH, Lund University, Lund
- defense date
- 2022-09-15 14:00:00
- ISBN
- 978-91-7422-896-0
- 978-91-7422-897-7
- language
- English
- LU publication?
- yes
- id
- bea74a88-1f0f-4094-a25f-36e949f86f54
- date added to LUP
- 2022-08-24 01:06:50
- date last changed
- 2022-10-12 06:37:58
@phdthesis{bea74a88-1f0f-4094-a25f-36e949f86f54,
abstract = {{Propionic acid (PA) is a naturally occurring short chain fatty acid produced by microbial fermentation of the indigestible foods in the intestine, having a positive effect on the gastrointestinal health. PA inhibits the growth of pathogenic bacteria and molds; hence it is widely used as preservative for food, feed and grains. It is also used as a component in several industrial products like polymers, cosmetics and pharmaceuticals. Its industrial production still depends on petrochemistry, however microbial fermentation as a route for production from renewable resources has attracted attention but is not yet competitive with the existing route. Dairy propionibacteria, used as starters for the maturation of Swiss-type cheeses and also known for their probiotic properties, produce propionic acid from different sugars or glycerol via Wood-Werkman cycle under anaerobic conditions. The fermentation process has a long lag phase and is also subject to product inhibition, resulting in low productivity and yield.<br/>This thesis involves studies on high cell density fermentations and development of acid tolerant strain as ways to improve the efficiency of propionic acid fermentation. Studies have been performed using Propionibacterium acidipropionici DSM 4900 (ATCC 25562), a bacterium that is generally regarded as safe (GRAS). Glycerol, a by-product of biodiesel production process, was used as the carbon source for fermentations.<br/>Different strategies of high cell density fermentations including sequential batch cultivation, cyclic batch cultivation, cell immobilization and cell retention using membrane filter, were evaluated. Propionate yield and productivity of 93 mol % and 0.53 g/L/h were obtained in cyclic batch while maximum productivity of 1.63 g/L/h was obtained from 90 g/L glycerol during sequential batch cultivations with free cells. P. acidipropionici cells were immobilized on plastic based AnoxKaldnes® and recycled glass based Poraver® carriers, either by polyethyleneimine (PEI) treatment or by biofilm formation. Exposing the cells to stress factors like 30 mM citric acid and 1 M NaCl was beneficial for increasing the Biofilm Forming Capacity index (BFC) and for production of exopolysaccharides (EPS), both indicators of biofilm formation. Fermentations in the immobilized packed-bed bioreactors using the bacteria pre-exposed to citric acid and NaCl gave propionic acid productivities of 0.7 and 0.78 g/L/h, respectively, with Poraver® matrix, and the corresponding values for AnoxKaldnes® were 0.39 and 0.43 g/L/h, respectively. Continuous propionic acid fermentations by cell retention in a ceramic membrane filter, gave productivity of 2.35 g/L/h in a medium with 60 g/L glycerol and 20 g/L yeast extract.<br/>A tolerant strain able to grow in a culture medium supplemented with 20 g/L of PA, was obtained by adaptive laboratory evolution. It exhibited 1.4-fold higher growth rate, 3.7-fold higher propionic acid production kinetics, 1.2-fold higher yield and almost 2-fold higher titer in batch cultivations compared to the parental cells in a basal cultivation medium. It was immobilized to PEI-treated Poraver® and used for fermentations in recycled batch mode with increasing glycerol concentration and decreasing pH, respectively, in packed-bed bioreactors. Glycerol up to a concentration of 100 g/L was completely consumed, resulting in 58 g/L propionic acid and product yield of 0.64 mol/mol. Progressive decrease in pH values gave increased propionic acid yield and productivity from 20 g/L glycerol, while maintaining the product titer nearly constant at pH 5.0.<br/>The exposure to stress factors for biofilm formation and to high propionic acid concentration during cultivation of P. acidipropionici showed trehalose to play a protective role. Following the expression of the trehalose synthesis-related genes treY and otsA, and by Real Time-qPCR revealed treY expression (but not otsA) to be significantly increased during biofilm formation under the influence of citric acid. Expression of the biofilm-related luxS gene was also detected by fluorescence in situ hybridization (FISH). Expression of treY was increased 82-fold in the tolerant strain obtained by adaptive evolution with respect to the 16S rRNA gene used as a control.<br/>}},
author = {{Cavero Olguin, Victor Hugo}},
isbn = {{978-91-7422-896-0}},
language = {{eng}},
month = {{08}},
publisher = {{Division of Biotechnology, Lund University}},
school = {{Lund University}},
title = {{High Cell Density Strategies and Adaptive Evolution for Microbial Propionic Acid Production}},
year = {{2022}},
}