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Bio-based C-3 Platform Chemical: Biotechnological Production and -Conversion of 3-Hydroxypropionaldehyde

Rezaei, Roya LU (2013)
Abstract (Swedish)
Popular Abstract in English

Microbes are present everywhere in the environment and have quite an intimate relationship with humans. Much of our perception about microbes is as disease causing agents but on the other hand it is also the microbes that present cure for the disease. Besides using microbes for traditional applications, for example for providing antibiotics, production of fermented foods, etc., humans are increasingly taking advantage of the ”good” microbes as probiotics for improving health, wastewater treatment, mining of metals, cleaning the environment, and for producing bioenergy, chemicals and materials. The micro-sized organisms contain a complex network of metabolic pathways involving a large number of... (More)
Popular Abstract in English

Microbes are present everywhere in the environment and have quite an intimate relationship with humans. Much of our perception about microbes is as disease causing agents but on the other hand it is also the microbes that present cure for the disease. Besides using microbes for traditional applications, for example for providing antibiotics, production of fermented foods, etc., humans are increasingly taking advantage of the ”good” microbes as probiotics for improving health, wastewater treatment, mining of metals, cleaning the environment, and for producing bioenergy, chemicals and materials. The micro-sized organisms contain a complex network of metabolic pathways involving a large number of chemical reactions catalysed by enzymes for utilizing different substances in the environment and converting them to a variety of products.

Lactic acid bacteria comprise an important group of microbes that humans have used for thousands of years to conserve and enhance the nutritional value of sensitive foods. Lactobacillus species are a major part of this group. Some Lactobacillus species are used for the production of yoghurt, cheese, sauerkraut, pickles, beer, wine, cider several fermented foods, as well as animal feeds, such as silage. Lactobacillus reuteri is a major component of the bacteria present in guts of mammals and birds. It has been shown that several different strains of L. reuteri have a positive effect on health, including various types of gastrointestinal disorders and oral health. In the late 1980s, it was discovered that L. reuteri produced a novel broad-spectrum antibiotic substance by fermentation of glycerol, which was named as "reuterin” after Gerhard Reuter. Reuterin can inhibit the growth of some harmful Gram-negative and Gram-positive bacteria, along with yeasts, fungi and protozoa. Reuterin is a mixture of three components, made of 3-hydroxypropionaldehyde (3HPA) and its derivatives.

This thesis is about 3HPA as a molecule of interest for the chemical industry based on renewable resources. Today, as we become increasingly aware of our dependence on fossil resources to fulfill our needs, and the environmental problems associated with the use of these non-renewable resources, there is a growing interest in the use of renewable resources as raw materials and environment-friendly methods for the production of chemicals, materials and energy. 3HPA is currently not a commercial product. If it could be economically produced from glycerol using the bacteria it can potentially be used as a building block or ”platform” for several other chemicals with 3 carbon atoms (C3), e.g. 1,3-propanediol (1,3PDO), 3-hydroxypropionic acid (3HP), acrolein, etc.

Glycerol, commonly known as glycerine, is produced as a side product of hydrolysis of fats, production of ethanol and biodiesel. Over the past decade or more, biodiesel is being produced from several plant oils such as rapeseed-, soybean- and palm oil, and also from used oils. In this thesis, conversion of glycerol to 3HPA using L. reuteri is investigated. When the 3HPA level reaches a certain limit, it starts to affect the cell viability and activity, hence inhibiting its own production. Different strategies to complex 3HPA were studied to improve its production.

L. reuteri has also the ability to convert 3HPA to 1,3PDO and 3HP via different pathways. In the thesis, the pathway for 3HP production has been introduced in standard bacteria, Escherichia coli by recombinant DNA technology and shown to be active. One of the enzymes of the pathway has further been studied.

The work in this thesis was done in collaboration with Perstorp AB, and was supported by Vinnova, the Swedish Governmental Agency for Innovation Systems. (Less)
Abstract
Demands for efficient, greener, economical and sustainable production of chemicals, materials and energy have led to development of industrial biotechnology as a key technology area to provide such products from bio-based raw materials from agricultural-, forestry- and related industrial residues and by-products. For the bio-based industry, it is essential to develop a number of building blocks or platform chemicals for C2-C6 chemicals and even aromatic chemicals. 3-hydroxypropionaldehyde (3HPA) and 3-hydroxypropionic acid (3HP) are potential platform chemicals for C3 chemistry and even for producing polymers.

This thesis presents investigations on the biotechnological routes for the production of a C3 platform chemical, 3HPA... (More)
Demands for efficient, greener, economical and sustainable production of chemicals, materials and energy have led to development of industrial biotechnology as a key technology area to provide such products from bio-based raw materials from agricultural-, forestry- and related industrial residues and by-products. For the bio-based industry, it is essential to develop a number of building blocks or platform chemicals for C2-C6 chemicals and even aromatic chemicals. 3-hydroxypropionaldehyde (3HPA) and 3-hydroxypropionic acid (3HP) are potential platform chemicals for C3 chemistry and even for producing polymers.

This thesis presents investigations on the biotechnological routes for the production of a C3 platform chemical, 3HPA from glycerol and its conversion to 3HP. Glycerol, was used as the raw material for production of 3HPA using resting cells of the probiotic bacteria, Lactobacillus reuteri, as the biocatalyst. The antimicrobial effect of the bacteria is attributed to the secretion of “reuterin” that is an equilibrium mixture of 3HPA with its dimer and hydrate forms. Glycerol dehydratase, a Vitamin B12-dependent enzyme, presents in L. reuteri, catalyses the dehydration of glycerol to 3HPA. Production of 3HPA at high concentration results in strong inhibition of the enzyme activity and cell viability, which in turn limits the product yield and -productivity. Different means of in situ capture of 3HPA from the reaction were studied. Complexation of 3HPA with bisulfite in a fed-batch biotransformation of glycerol and subsequent removal through binding to an anion exchange resulted in increase in the production of 3HPA to 5.33 g/g biocatalyst from 0.45 g/g in a batch process. In another approach, in situ removal of 3HPA using semicarbazide-functionalized resin in a batch process, productivity was enhanced 2 fold than that without the resin.

L. reuteri metabolizes 3HPA further to 1,3-propanediol (1,3PDO) and 3-hydroxypripionic acid (3HP) by reductive and oxidative pathways, respectively. The oxidative pathway, comprises 3 enzymes named propionaldehyde dehydrogenase (PduP), phosphotransacylase (PduL) and propionate kinase (PduW). Kinetic characterization and molecular modelling of the first enzyme, PduP, expressed in Escherichia coli was performed. The enzyme had a specific activity of 28.9 U/mg using propionaldehyde as substrate and 18 U/mg with 3HPA as substrate which is the highest specific activity reported up to date. All the Pdu enzymes were then expressed in E. coli in different combinations and used for bioconversion of 3HPA produced by native L. reuteri. Growing cells of the recombinant bacteria with all the three enzymes, E. coli pdu:P:L:W in a fed-batch mode gave 3HP yield of 0.5 mole/mole 3HPA with 1,3PDO as the co-product, while the resting cells gave 3HP yield of 1 mole /mole 3HPA. This showed the possibility of using of Pdu pathway of L. reuteri for production of 3HP. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • Associate Professor Straathof, Adrie, Technical University of Delft, Room 2.511, Julianalaan 67 Postbus 5,2600 AA Delft, The Netherlands
organization
publishing date
type
Thesis
publication status
published
subject
keywords
Lactobacillus reuteri, 3-hydroxypropionic acid, biotransformation, Glycerol, 3-hydroxypropionaldehyde, In situ complexation
pages
176 pages
publisher
Lund University
defense location
Lecture Hall A, Center for Chemistry and Chemical Engineering, Sölvegatan 39, Lund University Faculty of Engineering
defense date
2013-12-11 10:30
ISBN
978-91-89627-96-3
language
English
LU publication?
yes
id
cd8cd4a1-efbd-4f07-a13a-76cefcfcfbbd (old id 4153392)
date added to LUP
2013-11-18 09:30:07
date last changed
2016-09-19 08:45:01
@phdthesis{cd8cd4a1-efbd-4f07-a13a-76cefcfcfbbd,
  abstract     = {Demands for efficient, greener, economical and sustainable production of chemicals, materials and energy have led to development of industrial biotechnology as a key technology area to provide such products from bio-based raw materials from agricultural-, forestry- and related industrial residues and by-products. For the bio-based industry, it is essential to develop a number of building blocks or platform chemicals for C2-C6 chemicals and even aromatic chemicals. 3-hydroxypropionaldehyde (3HPA) and 3-hydroxypropionic acid (3HP) are potential platform chemicals for C3 chemistry and even for producing polymers. <br/><br>
This thesis presents investigations on the biotechnological routes for the production of a C3 platform chemical, 3HPA from glycerol and its conversion to 3HP. Glycerol, was used as the raw material for production of 3HPA using resting cells of the probiotic bacteria, Lactobacillus reuteri, as the biocatalyst. The antimicrobial effect of the bacteria is attributed to the secretion of “reuterin” that is an equilibrium mixture of 3HPA with its dimer and hydrate forms. Glycerol dehydratase, a Vitamin B12-dependent enzyme, presents in L. reuteri, catalyses the dehydration of glycerol to 3HPA. Production of 3HPA at high concentration results in strong inhibition of the enzyme activity and cell viability, which in turn limits the product yield and -productivity. Different means of in situ capture of 3HPA from the reaction were studied. Complexation of 3HPA with bisulfite in a fed-batch biotransformation of glycerol and subsequent removal through binding to an anion exchange resulted in increase in the production of 3HPA to 5.33 g/g biocatalyst from 0.45 g/g in a batch process. In another approach, in situ removal of 3HPA using semicarbazide-functionalized resin in a batch process, productivity was enhanced 2 fold than that without the resin.<br/><br>
L. reuteri metabolizes 3HPA further to 1,3-propanediol (1,3PDO) and 3-hydroxypripionic acid (3HP) by reductive and oxidative pathways, respectively. The oxidative pathway, comprises 3 enzymes named propionaldehyde dehydrogenase (PduP), phosphotransacylase (PduL) and propionate kinase (PduW). Kinetic characterization and molecular modelling of the first enzyme, PduP, expressed in Escherichia coli was performed. The enzyme had a specific activity of 28.9 U/mg using propionaldehyde as substrate and 18 U/mg with 3HPA as substrate which is the highest specific activity reported up to date. All the Pdu enzymes were then expressed in E. coli in different combinations and used for bioconversion of 3HPA produced by native L. reuteri. Growing cells of the recombinant bacteria with all the three enzymes, E. coli pdu:P:L:W in a fed-batch mode gave 3HP yield of 0.5 mole/mole 3HPA with 1,3PDO as the co-product, while the resting cells gave 3HP yield of 1 mole /mole 3HPA. This showed the possibility of using of Pdu pathway of L. reuteri for production of 3HP.},
  author       = {Rezaei, Roya},
  isbn         = {978-91-89627-96-3},
  keyword      = {Lactobacillus reuteri,3-hydroxypropionic acid,biotransformation,Glycerol,3-hydroxypropionaldehyde,In situ complexation},
  language     = {eng},
  pages        = {176},
  publisher    = {Lund University},
  school       = {Lund University},
  title        = {Bio-based C-3 Platform Chemical: Biotechnological Production and -Conversion of 3-Hydroxypropionaldehyde},
  year         = {2013},
}