FAIR-CT96-1912 Biodegradable polyesters from 1,3-propanediol and succinate produced by fermentation of regrowing resources

Contacts
Summary Information



To find similar Items, click on a keyword below:
Biological Conversion : Biopolymers/Gums : FAIR Area 1.2 - Green Chemicals and Polymers Chain : Paints/Coatings/Plastics : Sugar

	Proposal No:	FAIR-CT96-1912
	Date Prepared:	March 2000, November 1998
	Source:	Final Report Abstract Progress Report

---

Final Report Abstract

Introduction

The overall goal of the present project was the synthesis of biodegradable polyesters from two main monomers, 1,3-propanediol and succinate (produced by fermentation from renewable sources) and from other dicarboxylic acids (like terephthalic acid) used as an auxiliary monomers to modified the chemical and physical properties of the polyester. The main advantage of this approach compared to polyhydroxyalkanoates or polylactides (two biodegradable polyesters that can be produced from renewable sources) was the possibility to easily modify the physical properties of the polyester to the qualities required for articles made of plastic.

In the framework of this project we have demonstrated that Clostridium butyricum can ferment low grade glycerol and produce 1, 3 propanediol. We have isolated and sequenced the genes encoding the butyrate, the glycerol oxidation and the glycerol reduction pathways of C. butyricum

By a metabolic control analysis approach we have identified the glycerol dehydratase as the limiting step in the conversion of glycerol to 1,3 propanediol. Based on the analysis on C butyricum a recombinant strain of C acetobutylicum was developed that converted glycerol to 1,3 propanediol at high yield and high rate. Although not totally completed the basis for a continuous process for 1,3 propanediol production at a high volumetric productivity was established.

A new process for the continuous production of succinic acid at a high volumetric productivity and yield was successfully developed. Compared to conventional fed- batch cultures, the productivity was increased 11 fold.

A new polymer (PEU for polyester-based urethanes) was developed that possesses better properties than BTA44/56, a biodegradable polyester commercially produced by BASF and Eastman.

Finally, through the synthesis of different polyester based urethanes made of 1, 3 propanediol and adipic acid or succinic acid, the factors that influence their hydrolysis by lipase have been identified.

Activities

The main activities of this project were:

• Production of 1,3-propanediol from glycerol coming from the oleochemical industry (fatty acids, fatty alcohols) and rape seed oil production and development of genetically modified microorganisms for maximal yield of 1,3-propanediol including evaluation of the performances of strains in large scale fermenters and in high volumetric productivity membrane bioreactors.
• Production of succinic acid from starch hydrolysates using Anaerobiospirillum succiniproducens in high performance bioreactors (membrane bioreactors with optimised ratio of cell bleeding and effluent flow rates and in situ product recovery by electrodialysis) to increase the productivity and decrease the waste water treatment required by the process.
• Development of polyesters based on 1,3-propanediol and succinic acid from renewable resources, optimised regarding processability and thermo-mechanical properties by means of improved polymerization-copolymerisation methods and blending, followed by manufacture of prototypes of product using the optimised polymers, copolymers and blends. The methods considered for improving the properties of the polymers include the use of additional monomers (higher aliphatic and aromatic acids) and use of chain elongators (bisoxiranes and isocyanates). In addition various methods, from polycondensation mechanisms to ring-opening polymerisation, which offers the possibility of synthesising typical polycondensation polymers with higher molecular weights, and which presents a very broad spectrum of possibilities for the synthesis of aliphatic polyesters, were also used.
• Biodegradation studies using various test systems, including C-balances, isolation and characterisation of degradative microbial consortia and involved enzymes systems (lipases, hydrolases) were carried out.

Results

The main achievements of this project were:

• The isolation and sequencing of the genes for butyrate formation in C. butyricum.
• The isolation and sequencing of the genes for the oxidation and reduction of glycerol in C. butyricum.
• The identification of the glycerol dehydratase as the limiting step in the conversion of glycerol to 1,3-propanediol under high initial glycerol concentrations.
• The confirmation that a B12 independent glycerol dehydratase is present in C. butyricum, which was concluded by genetic and biochemical data.
• The expression of the 1,3-propanediol operon in C acetobutylicum strains, which led to the conversion of glycerol to 1,3-propanediol by this organism (0.58 g/g).
• The high yield of 1,3-propanediol obtained in fed-batch cultures of C butyricum (0.58- 0.69 mol/mol, 54-62 g/l of 1,3-propanediol).
• The definition and evaluation of a downstream processing strategy for 1,3- propanediol purification: separation of biomass by centrifugation, followed by water removal using multiple effect evaporation and 1,3-propanediol rectification by vacuum distillation.
• The fermentation of various types of low-grade glycerol by C butyricum, with results similar to those obtained by fermentation of commercial glycerol (around 0.60 mol 1,3- propanediol /mol glycerol consumed).
• The development of a continuous culture with high productivity of 1,3- propanediol and high yield (10.3 g/l/h, 0.69 mol 1,3-propanediol per mol glycerol consumed).

• The cost assessment for 1,3-propanediol (PDO) biotechnological production: 1.77 Euro/kg PDO; a PDO capacity of 75000 t/a is a good compromise between increasing plant size and decreasing production cost; the price of raw glycerine (or alternative carbon sources) has the strongest impact on the PDO economics (PDO [Euro/t] = 950 + 2.14 - Glycerine [Euro/t]).
• The development of an efficient continuous process (flat membrane bioreactor) for succinic acid production by Anaerobiospirillum succiniciproducens at a high volumetric productivity: 15 g/l/h for a dilution rate of 0.93 h^-1, which represents a 20 fold increase compared to a batch mode.
• The definition of a soluble medium for succinic acid production by Actinobacillus succinogenes; the development of an immobilised cells bioreactor for succinic acid production by this organism.
• The development of a purification process for succinic acid recovery: conventional electrodialysis to concentrate sodium succinate; chelating resin to extract Ca, Mg and Fe; bipolar electrodialysis to transform sodium succinate into succinic acid; ion exchange resin to extract all the impurities; and crystallisation. Dried crystals of succinic acid have a purity of 98.5%.
• The synthesis and characterisation of aliphatic polyesters, aliphatic-aromatic copolyesters and polyesters-based urethane from 1,3-propanediol and succinic acid; polyesters- based urethane, PEU, were obtained and possess better properties than BTA44/56 a biodegradable polyester commercially produced by BASF and Eastman.
• The synthesis of cyclic esters (lactones) deriving from diacid and diol units, followed by polymerisation of the same.

• The synthesis of low molecular weight aliphatic homo-polyester by polycondensation of either 1,3-propanediol or 1,6-cyclohexyldimethanol with succinic acid; the segmented copolymerisation of oligomeric polypropylensuccinates with end terminated poly(L- lactic acid), resulting in polyesters containing small amounts of degradable carbonate functions.
• The development of four different tests for the biodegradability of polyesters: C0₂ determination in liquid medium or compost, enzyme test with bacterial lipases, composting simulation test, accelerated agar plate test with pure thermophilic actinomycetes.
• The demonstration of biodegradability of the aliphatic polyesters made exclusively of 1,3 propanediol and succinic acid.

• The synthesis and the identification of the factors that influence lipase hydrolysis of polyester-based urethanes made of 1, 3 propanediol and adipic acid.

---

Progress Report November 1998

Summary

Introduction The overall goal of the present project is, the synthesis of biodegradable polyesters from two main monomers, 1,3-propanediol and succinate (produced by fermentation from renewable sources) and from other dicarboxylic acids (such as terephthalic acid) used as an auxiliary monomers to modified the chemical and, physical properties of the polyester. The main advantage of this approach compared to polyhydoxyalkanoates or polylactides (two biodegradable polyesters that can be produced from renewable sources) is the possibility to easily modified the physical properties of the polyester to the qualities required for the article made of plastic.

Objectives The main aims of this project are listed below.

• Production of 1,3-propanediol out of glycerol coming from the oleochemical industry (fatty acids, fatty alcohols) and rape seed oil production. Development of genetically modified micro-organisms for maximal yield of 1, 3-propanediol production and evaluation of their performances in fermenters of up to the M³ scale and in high volumetric productivity membrane bioreactor. Although several studies have shown the feasibility of glycerol conversion to 1,3-propanediol, nobody has tried to improve the economy of the process by genetic modification of strain or by process intensification using membrane bioreactors.

• Production of succinic acid from starch hydrolysates using Anaerobiospirillum succiniproducens in high performance bioreactor (membrane bioreactors with optimised ratio of cell bleeding and effluent flow rates and in situ product recovery by electrodialysis), to increase the productivity and decrease the waste water treatment of the process.

• Development of polyesters based on 1, 3-propanediol and succinic acid from renewable resources in terms of processability and thermo-mechanical properties by use of improved polymerization-copolymerization methods and blending, followed by manufacture of prototypes of product using the optimised polymers, copolymers and blends. The methods considered for improving the properties of the polymers include the use of additional monomers (higher aliphatic and aromatic acids), use of chain elongators (bisoxiranes and isocyanates) and variation from polycondensation mechanisms to ring-opening polymerization (method which offers the possibility of synthesising typical polycondensation polymers with higher molecular weights, and which gives polymers with a very broad spectrum of possibilities for the synthesis of aliphatic polyesters).

• Biodegradation studies using various test systems including C-balances., isolation and characterisation of degradative microbial consortia and involved enzymes systems (lipases, hydrolases).

Activities These were carried out within the following main tasks.

Task 1 Conversion of glycerol (coming from the oleochemical industry and rape seed oil production) to 1,3-propanediol using Clostridium butyricum.

Sub-task 1.1 Development of genetically modified C butyricum for maximal conversion yield of glycerol to 1, 3-propanediol.

Sub-task 1.2 Evaluation of recombinant strains in fermentor up to M³ scale and purification of 1, 3 propanediol; metabolic pathway analysis of C butyricum to identified the limiting step in the conversion of glycerol to 1, 3 propanediol.

Task 2 Development of efficient membrane bioreactors for continuous production of 1, 3 propanediol and succinate.

Sub-task 2.1 Glycerol from various grade will be continuously converted to 1,3 propanediol by C butyricum in high cell density membrane bioreactors.

Sub-task 2.2 Continuous production of succinic acid by Anaerobiospirillum succiniproducens will be done in high performance membrane bioreactors (with in situ product recovery by electrodialysis), to increase the productivity and decrease the waste water treatment of the process.

Task 3 Development of polyesters based on 1, 3-propanediol and succinic acid optimised regarding processability and thermo-mechanical properties by means of utilisation of improved polymerization-copolymerization methods and blending, followed by manufacture of prototypes of product using the referred optimised polymers, copolymers and blends.

Task 4 Biodegradation studies using various test systems including C-balances, isolation and characterisation of degradative microbial consortia and related enzymes systems (lipases, hydrolases).

Results

The following achievements were reached.

Task 1 Sub-task 1.1 The genes coding for the butyrate pathway in Clostridium butyricum VPI 1718 were cloned and sequenced in the first reporting period. They have been used to obtain a knockout strain for the butyrate kinase gene. To further improve the process the genes coding for the oxidation and the reduction of glycerol were cloned and sequenced.

Sub-task 1.2 A MCA approach was developed to identify the limiting step(s) in 1,3 propanediol production. The glycerol dehydratase was identified as the limiting enzyme under high initial glycerol concentrations. A process for 1, 3 propanediol purification after fed batch fermentation was also defined and evaluated. It includes the separation of biomass, the concentration by evaporation and the final rectification of 1, 3 propanediol.

Task 2 Sub-task 2.1 All the low grade glycerolise have been successfully converted to 1, 3 propanediol by C. butyricum. A high cell density membrane bioreactor for continuous cultures was developed and compared to chemostat cultures. Optimisation of dilution rates and cell bleeding will be done for maximising 1,3-propanediol productivities.

Sub-task 2.2 Continuous production of succinic acid by Anaerobiospirillum succiniproducens was optimised in high cell density bioreactor with cell bleeding. At a dilution rate of 0.93 h^-1 and a cell bleeding rate of 0.023 h^-1 a 20 times increase in volumetric productivity was obtained compared to batch cultures. Continuous concentration of sodium succinate, (from a fermentation broth) by monopolar electrodialysis was optimised. After conversion to the acid form by bipolar electrodialysis, succinic acid was finally purified by crystallisation.

Task 3 Based on the interesting results obtained with polyesters-based urethane from 1, 3 propanediol and succinic acid (PEU) more PEU polymers have been synthesised and characterised in term of molecular weight as well as thermal and mechanical properties. A lot of them possesses better properties than BTA44/56 a biodegradable polyester commercially produced by BASF.

Task 4 Polyester based urethanes made of 1, 3 propanediol and adipic acid were synthesised and the factors that influence there hydrolysis by a lipase identified. Biodegradation tests will now be run on the promising PEU polymers (polyester based urethanes made of 1, 3 propanediol and succinic acid).

Discussion

The main achievements of the second reporting period are:

• the fermentation of low grade glycerol by C butyricum
• obtaining a knockout C butyricum strain for the butyrate kinase gene

• the isolation and sequencing of the genes for the oxidation and reduction of glycerol in C butyricum
• the identification of the glycerol dehydratase as the limiting step in the conversion of glycerol to 1, 3 propanediol
• the development of a continuous process for succinic acid production at a high volumetric productivity
• the first developments of a continuous process for 1, 3 propanediol production at a high volumetric productivity
• the obtaining of PEU polymers (polyester based urethanes) that possess better properties than BTA44/56 a biodegradable polyester commercially produced by BASF and Eastman
• the synthesis and the identification of the factors that influence lipase hydrolysis of polyester based urethanes made of 1, 3 propanediol and adipic acid

Future activities

The future actions planned in the coming year are the following:

• the complete deletion of the butyrate pathway genes in order to obtain butyrate negative strains that convert glycerol to 1, 3 propanediol in higher yield
• the construction and evaluation of recombinant C butyricum strains that over expressed the glycerol dehydratase or the whole pathway for 1, 3 propanediol production
• the optimisation of bleeding and effluent rates in a high cell density membrane bioreactor to maximise 1,3-propanediol productivities
• the optimisation of the continuous process for high concentration of succinate production in high cell density membrane bioreactors with in situ extraction of sodium succinate by monopolar electrodialysis
• the evaluation of biodegradability of the promising PEU polymers (polyester based urethane)
• the investigation of factors which influence the properties of polyester-based urethanes
• the synthesis of high molecular weight aliphatic polyesters by the use of lactone and ring opening polymerization