WO2011042434A1

WO2011042434A1 - 1,3-propanediol production with immobilised cells reactors

Info

Publication number: WO2011042434A1
Application number: PCT/EP2010/064825
Authority: WO
Inventors: Michel Chateau; Philippe Soucaille; Jean-Yves Dubois
Original assignee: Metabolic Explorer
Priority date: 2009-10-05
Filing date: 2010-10-05
Publication date: 2011-04-14
Also published as: AR078525A1

Abstract

The present invention concerns a new method for the fermentative production of 1,3-propanediol from glycerol comprising culturing immobilized microorganism on a culture medium with high glycerine content, wherein the microorganism is a Clostridium species, modified for an improved production of 1,3-propanediol.

Description

1,3-PROPANEDIOL PRODUCTION WITH IMMOBILISED CELLS REACTORS

The present invention concerns a new method for the fermentative production of 1,3-propanediol from glycerol comprising culturing immobilized microorganisms on a culture medium with high glycerine content, wherein the microorganism is a Clostridium species, modified for an improved production of 1,3-propanediol.

BACKGROUND OF THE INVENTION 1,3-Propanediol (PDO) is one of the oldest known fermentation products. It was reliably identified as early as 1881 by August Freund, in a glycerol- fermenting mixed culture obviously containing Clostridium pasteurianum as the active organism. Quantitative analysis of the fermentation of different enterobacteria producing PDO, also known as trimethylene glycol or propylene glycol, started at the microbiology school of Delft as early as 1928 and was successfully continued at Ames, Iowa, in the 1940s. In the 1960s, interest shifted to the glycerol-attacking enzymes, in particular to the glycerol and diol-dehydratases, as these enzymes were peculiar in requiring coenzyme B 12 for there activity. PDO-forming Clostridia were first described in 1983 as part of a process to obtain a specialty product from glycerol-excreting algae (Nakas et al., 1983). PDO is a typical product of glycerol fermentation and has not been found in anaerobic conversions of other organic substrates. Only very few organisms, all of them bacteria, are able to synthesise it. They include enterobacteria of the genera Klebsiella (i.e K pneumoniae), Enterobacter (i.e. E. agglomerans) and Citrobacter (i.e. C. freundii), lactobacilli (i.e. L. brevis and L. buchneri) and Clostridia of the C. butyricum and the C. pasteurianum group.

Analysis of the fermentation products shows that part of the glycerol is converted to the same products as in the sugar fermentation of this different species, namely acetic acid, 2,3-butanediol, butyric acid, lactic acid, ethanol and succinic acid. These conversions provide the necessary energy for growth. However, for many of these products, the conversions released reducing equivalents which are then used in the reductive conversion of glycerol to PDO. Notably, butyrate formation in Clostridia or ethanol formation in Klebsiella, seem to be dependent on growth rate, and lower the PDO yield. Even if substrate is not in excess, decreasing the dilution rate decreases rapidly, the butyrate titre. In any case, changes in the acetate/butyrate ratio do not have an important impact on the PDO yield.

PDO, as a bi-functional organic compound, could potentially be used for many synthesis reactions, in particular as a monomer for polycondensations to produce polyesters, polyethers or polyurethanes. PDO can be produced by different chemicals routes but they generate waste streams containing extremely polluting substances and the cost of production is high. With respect to its production cost chemically produced PDO could not compete with the petrochemically available diols 1 ,2-ethanediol, 1 ,2- propanediol, or 1,4-butanediol. Therefore, in the past PDO was only found in specialised niche applications of negligible market volume. Although an environmentally friendly process for the biological conversion of glucose to PDO exists, it has the disadvantages to use vitamin B 12, an expensive cofactor, and to be a discontinuous process due to the instability of the producing strain. Today, with the availability of large amounts of glycerol issued from bio-diesel industry, a continuous vitamin B12-free process, with higher carbon yields would be industrially advantageous.

It is known in the art that PDO can be produced from "industrial glycerol", an unwanted by product of the biodiesel production, which contains roughly 80 to 85% of glycerol mixed mainly with salts and water. For example, C. butyricum was previously described as being able to grow and produce PDO from industrial glycerol in batch and two-stage continuous fermentations (Papanikolaou et al., 2000). However, at the highest glycerol concentration, the maximal PDO titre obtained was 48g/l at a dilution rate of 0.02h-l , meaning a productivity of 0.9g/l/h. WO2006/128381 discloses the use of this industrial glycerol for the production of PDO with batch and fed-batch cultures using natural PDO producing organisms such as K. pneumoniae, C. butyricum or C. pasteurianum. As described in this patent application, the maximal productivity reached is comprised between 0.8 and l . lg/l/h. Finally, the performance of a C. acetobutylicum strain that lacks the megaplasmid pSOLl, necessary to produce the solvents acetone and butanol, and that contains the vitamin B 12-independent glycerol-dehydratase and the PDO- dehydrogenase from C. butyricum, called C. acetobutylicum DG1 pSPD5, has been described in Gonzalez-Pajuelo et al, 2005. At the highest glycerol concentration tested, 120g/l of pure glycerol, the maximal PDO titre obtained was 59.88g/l with a maximal productivity of 3.0g/l/h.

Immobilized cell reactors, in which high cell concentrations can be achieved, are known in the art (See for review Qureshi et al., 2005). Cells can be immobilized by three different techniques: entrapment, covalent bond formation, and adsorption. Entrapment and covalent bond formation require use of chemicals. On the contrary, the technique of 'adsorption' is characterized by the fact that cells bind and adhere to a support, naturally and firmly.

The entrapment technique is characterized by an encapsulation of the cells in gel- cored beads, made of either calcium alginate, polyacrylamide, carrageenan, agarose, gellan gum or polytetrafluoro ethylene. Cells are first mixed with said beads in aqueous solution, and addition of cations produce beads of hardened gum entrapping the cells. A classical matrix is the LentiKats® matrix, made of polyvinyl alcohol (PVA), commercialized by the Company GeniaLab®. This technique includes the microencapsulation method. The covalent bond formation is characterized by the use of chemicals leading to the formation of bonds between a support and the cells.

The adsorption technique is characterized by the formation of a 'biofilm' on a support. A biofilm is a structured community of bacteria enclosed in a self-produced matrix composed of a peptidoglycan polymer, also known as extracellular polymeric substances or EPS. Bio films are of specific medical importance as one origin of persistent infections and, in this case, EPS is often responsible for strong immuno-responses. Industrially, bio films are mostly used in waste water treatment. The advantages of bio films are numerous. First of all, natural biofilms are composed of several bacterial species that allow for the removal of different types of toxic organic compounds and/or heavy metals. Secondly, the composition and network of the polymeric matrix allows for a better resistance of the bacteria to toxic compounds. It is well known for example that the antibiotic resistance of chronic infections is often du to the presence of a bacterial biofilm in the body. This biofilm resists treatment and releases cells causing new rounds of infections. In fine chemical production, Li et al, 2006 have shown that a Zymomonas mobilis biofilm protected the bacteria from the killing effect of benzaldehyde, even allowing a constant bioconversion of benzaldehyde into benzyl alcohol in a continuous system. Finally, the formation of biofilms is a natural process that does not require the use of chemicals.

Biofilm formation of Clostridia species to improve the production of solvents has been described in the literature (See Kobayashi et al, 2005 and Zhang et al, 2009). C. acetobutylicum butanol productivity was improved up to a dilution rate of 1.2 h^"1, but decreased with higher dilution rate (Huang et al, 2004). Butanol titre remained at a maximum of 12,5 g/1 because the biofilm could not protect the cells from the toxic effects of butanol. In 2005, Qureshi et al, reviewed the use of biofilm reactors for industrial bioconversions and reported that in the case of solvents production by C. acetobutylicum biofilms formed in 2 to 4 days and reactors became productive only after four day of continuous operation.

Finally, one example of PDO production by immobilised cells is described in the prior art. Using Citrobacter freundii as PDO producing species, Pflugmacher and Gottschalk (1994) reached a productivity of 8.2g.l.h, with a titre of 16.34g.l and a yield of approximately 0.46g.g of glycerol.

The present invention provides means for the production of 1 ,3 -propanediol with high concentration of glycerol and where a high PDO titre and productivity can be reached. BRIEF DESCIPTION OF THE INVENTION

The present invention concerns a new method for the fermentative production of 1,3-propanediol from glycerol, comprising culturing a microorganism of a Clostridium species producing the 1,3-propanediol from glycerol in an appropriate culture medium comprising glycerol, and recovery of the 1 ,3-propanediol, wherein the culture is made in an immobilized cell reactor. In the method of the invention, the reactor is advantageously filled with inert support material on which a biofilm immobilizing the microorganisms is formed.

In the method of the invention, the production is advantageously done in a batch, fed-batch or continuous process, preferably in a continuous process.

During the fermentation of glycerol, a biofilm is formed early and increases with increased concentration of glycerol in the fed medium. In the presence of the biofilm, high concentrations of glycerol can be fed to the C. acetobutylicum strain. The role of the biofilm is to protect the bacterial cells from high glycerol and high fermentation products concentrations, and any toxic compound that could be introduced during the fermentation process.

Optionally, 1,3-propanediol is further purified. DETAILED DESCIPTION OF THE INVENTION

Clostridium species producing 1,3-propanediol from glycerol

Clostridium species producing 1,3-propanediol from glycerol are known in the art and include Clostridium butyricum and Clostridium pasteuricum

In a preferred embodiment, the microorganism of a Clostridium species producing

1,3-propanediol from glycerol is a recombinant Clostridium species modified for an improved production of 1,3-propanediol, preferably Clostridium acetobutylicum.

"Clostridium species modified for an improved production of 1 ,3-propanediol " means that the strain has been transformed in the aim to change its genetic characteristics towards an improved production of 1,3-propanediol from glycerol compared to the untransformed microorganism. Endogenous genes can be attenuated, deleted, or over- expressed. Exogenous genes can be introduced, carried by a plasmid, or integrated into the genome of the strain, to be expressed into the cell.

The term "plasmid" or "vector" as used herein refers to an extra chromosomal element often carrying genes which are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA molecules. The modified Clostridium species used in the method of the invention are advantageously transformed for directing the glycerol metabolism towards production of 1,3-propanediol.

Such microorganisms are known in the art (see for instance WO2006/128381 , Gonzalez-Pajuelo & al. 2006, which contents are incorporated herein by reference).

In one embodiment of the invention, the producing microorganism presents an increased flux of 1 ,3-propanediol production by introducing extra copies of the 1 ,3- propanediol operon from C. butyricum, (coding for enzymes involved in the vitamin B12- independent 1 ,3-propanediol pathway) either over-expressed by a plasmid or integrated into the chromosome of the microorganism. For example the pSPD5 plasmid can be used for an over-expression of the 1,3-propanediol operon.

In a preferred embodiment, the glycerol dehydratase activity in the producing microorganism is independent of the presence of coenzyme B12 or one of its precursors and is derived from Clostridium butyricum.

In one embodiment, the Clostridium species may be further adapted to grow on high concentrations of industrial glycerine (see US 61/175,564 filed on 5 May 2009, which content is incorporated herein by reference).

Culture medium comprising glycerol

An "appropriate culture medium" or a "culture medium" refers to a culture medium optimized for the growth and the diol-production of the Clostridium strain. The fermentation process is generally conducted in reactors with a synthetic, particularly inorganic, culture medium of known defined composition adapted to the Clostridium species used and containing glycerol.

The term "synthetic medium" means a culture medium comprising a chemically defined substrate upon which organisms are grown. In the culture medium of the present invention, glycerol is advantageously the single source of carbon.

In a particular embodiment, the glycerol is added to the medium in the form of glycerine, said glycerine comprising at least 50% of glycerol, preferably at least 85% of glycerol.

Advantageously, the glycerine used in the culture medium of the invention is industrial glycerine. "Industrial glycerine" means a glycerine product obtained from an industrial process without substantial purification. Industrial glycerine can also be designated as "raw glycerine". Industrial glycerine comprises more than about 70% of glycerol, preferably more than about 80 %, water and impurities such as mineral salts and fatty acids.

The maximum content of glycerol in industrial glycerine is generally 90%, more generally about 85%. Industrial processes from which industrial glycerine is obtained are, inter alia, manufacturing methods where fats and oils, particularly fats and oils of plant origin, are processed into industrial products such as detergent or lubricants. In such manufacturing methods, industrial glycerine is considered as a by-product.

In a particular embodiment, the industrial glycerine is a by-product from biodiesel production and comprises known impurities of glycerine obtained from biodiesel production, comprising about 80 to 85% of glycerol with salts, water and some other organic compounds such as fatty acids. Industrial glycerine obtained from biodiesel production has not been subjected to further purification steps.

Preferably, the culture medium comprises high concentrations of glycerine.

The terms "high glycerine content" or "high concentration of glycerine" means more than 90 g/1 of glycerol in the culture medium. In preferred embodiments, the glycerol concentration is comprised between 90 and 200 g/L of glycerine, more particularly between 90 and 140 g/L of glycerine, preferably about 120g/L of glycerine.

Immobilized cell reactors

Immobilized cell reactors are known in the art (See for example the review Qureshi et al., 2005). Cells can be immobilized by three different techniques: entrapment, covalent bond formation, and adsorption on an inert support.

The entrapment method presents numerous disadvantages, in particular the use of expensive and toxic chemicals.

In a particular embodiment of the invention, the cell reactor comprises with inert support material, on which microorganisms will adsorb and then form a biofilm. Inert supports and how to fill the reactor with them are known in the art. Said inert support material is generally selected among the group consisting of metal, glass, ceramic, polypropylene, or polyvinyl chloride. A preferred support is non-treated glass, metal, ceramic, polypropylene, or polyvinyl chloride. A most preferred support of the invention is glass.

Other material to offer support to bacterial cell are fibrous materials. Many different fibrous materials may be used as scaffold in the bioreactor such as tangled fibres, porous particles, sponge, and sponge-like material. Fibres are prepared using a wide variety of materials including natural polymers such as polysaccharides and fibrous proteins, synthetic polymers such as polyamides (nylon), polyesters, polyurethanes and minerals including ceramics and metals, coral, gelatine, polyacrylamide, cotton, glass fibre, corrageenans, and dextrans. Examples of tangled fibres include glass wool, steel wool, and wire or fibrous mesh. Examples of porous particles include pumice stone, vermiculite or pozzolan. The inert support material is shaped as Pall ring, Rashig ring, Lessing ring, Intalox saddle, Berl saddle, railings grid or other industrial biomass support such as Cloisonyl™, Kaldnes™, Mutag BioChip™. In another embodiment of the invention, the support is treated to improve the bacterial attachment. Treatments of the support are done using chemical compounds with the aim of modifying the natural physico-chemical properties of the material. Such known treatments are washes with acids or bases to improve respectively hydrophilic or hydrophobic characters of supports.

Culturing the microorganisms

Setting up continuous processes with cells immobilized in a reactor is known in the art. The cultures are first grown batch- wise. Before the end of the exponential growth phase continuous feeding starts by continuously adding fresh medium, and recovering residual nutrients and cells at the same rate, thus maintaining the culture in the reactor at a constant volume.

Dilution rate is defined as the volume of nutrient medium supplied per hour divided by the volume of the culture. In particular, during chemostat cultivation, an equilibrium or

"steady state" is established at which the growth rate of the cells equals the dilution rate.

Indeed, with the immobilization of the cells, the dilution rate may be higher than the growth rate of the cells.

In a particular embodiment of the invention, the dilution rate applied in the continuous process is comprised between 0.05 and 0.5 h^"1. For this rate of dilution, observed productivity is between 3 to 30 g/l/h of 1,3-propanediol.

In a particular embodiment of the invention, the dilution rate applied in the continuous process is comprised between 0.1 and 0.2 h^"1. For this rate of dilution, observed productivity is between 6 to 12 g/l/h of 1,3-propanediol.

It is understood that prior to starting the continuous culture, the cells are cultured in the reactor to form a biofilm and are immobilized on the inert material. In the first phase, cells are cultured for 12 to 96 hours, preferably 36 hours, at an appropriate temperature, generally between 24 and 39°C, preferably 35°C, with a pH comprised between 6 and 7, preferably 6.5. For homogeneity, the medium is recycled, usually at a speed comprised between 0.05 and 3.5 h^"1, preferably 3 h^"1. At the end of the first phase, the continuous culture is started with a dilution rate comprised between 0.01 and 0.1 h^"1, preferably 0.02 h^"1, until the presence of a bio film is visually confirmed on the supporting material.

Once the biofilm is settled, the process of the invention can be performed with dilution rates comprised between 0.05 and 0.5 h^"1 preferably more than 0.1 h^"1, particularly comprised between 0.1 and 0.2 h^"1.

Feed medium with glycerol concentration comprised between 10 and 200g/L, preferably 120g/L, is used and glycerol from any origin is converted to 1,3-propanediol.

1,3-propanediol recovery

Methods for recovering and eventually purifying 1 ,3-propanedio l from a fermentation medium are known to the skilled person. 1,3-propanediol may be isolated by distillation. In most embodiments, 1 ,3-propanediol is distilled from the fermentation medium with a by-product, such as acetate, and then further purified by known methods.

DESCRIPTION OF THE DRAWING

Figure 1 describes the continuous bioreactor for 1 ,3-propanediol production with cells immobilization.

Figure 2 describes the evolution of a non-immobilized continuous cell reactor which undergoes increase in glycerol concentration in the fed medium. Periods of feeding with various glycerol concentrations are indicated by braces above the graph. GI: Industrial Glycerine.

Figure 3 describes the evolution of a non-immobilized cell reactor which undergoes increase in raw glycerol concentration in the fed medium. Periods of feeding with various glycerol concentrations are indicated by braces above the graph.

Figure 4 describes the evolution of an immobilized cell reactor which undergoes increase in raw glycerol concentration in the fed medium. Periods of feeding with various glycerol concentrations are indicated by braces above the graph. EXAMPLES

Example 1- Immobilization of cells

The setup of an immobilized cell reactor in the laboratory was done with a reactor filled with inert support material for biofilm formation (Figure 1). The reactor volume of 1 litre of medium is seeded with a culture of the PDO producing C. acetobutylicum DG1 pSPD5 strain.

Bacterial strain:

C. acetobutylicum ATCC824 cured from pSOLl (DG1 strain) and transformed with plasmid pSPD5 with following characteristics: MLS^r (erythromycin resistance), Ap^r (ampiciline resistance), 1,3-propanediol operon from C.butyricum witch contains dhaBl, dhaB2 and dhaT genes as described in patent application WOO 1/04324.

Culture media:

The synthetic media used for Clostridia batch cultivations contained per litre of deionised water: glycerol, 30g; KH₂P0₄, 0.5g; K₂HP0₄, 0.5g; MgS0₄ x 7H₂0, 0.2g; CoCl₂ x 6H₂0, O.Olg, H₂S0₄ (96%), 0.1ml; NH₄C1, 1.5g, biotin, 0.16mg; /?-amino benzoic acid, 32mg and FeS0₄ x 7H₂0, 0.028g, resazurin, lmg; cysteine, 0.5g. Resazurin and cysteine could be eliminated without any problem. Their use allows viewing oxygen entry in the system. The pH of the medium was adjusted to 6.3 with NH₄OH 3N. For batch cultivation we used a commercial glycerol purchased from Sigma (purity 99.5%). The feed medium for continuous cultures contained per litre of tap water: raw glycerol, different amounts (105, 120, 140g); KH₂P0₄, 0.5g; K₂HP0₄, 0.5g; MgS0₄ x 7H₂0, 0.2g; CoCl₂ x 6H₂0, 0.026g; NH₄C1, 1.5g, biotin, 0.16mg; /?-amino benzoic acid, 32mg; FeS0₄ x 7H₂0, 0.04g, anti-foam (Struktol), 0,05ml; ZnS0₄ x 7H₂0, 8mg; CuCl₂ x 2H₂0, 4mg; MnS0₄ x H₂0, 40mg, H₃BO₃, 2mg; Na₂Mo0₄ x 2H₂0, 0.8mg. Medium pH was not adjusted in this case. Raw glycerol, from the trans-esterification process for biodiesel was supplied by SAIPOL (Le Meriot, France) and has the following purity: glycerol 83% (w/w).

Experimental set-up:

Figure 1 show the bioreactor used. Continuous cultures were performed in a 1,71 bioreactor made of a jacketed glass column. The bioreactor is packed with an inert support. The inert support used is made of different material like glass, ceramic, metal, polypropylene, tissue, fabric or other inert material. The support could have different form: Raschig ring, Pall ring, Lessing ring, saddle Berl or saddle Intalox, etc and with different size. According to the support used for immobilization, the working volume of the bioreactor will be variable between 1 to 1.5 1. The culture volume was kept constant by automatic regulation of the culture level. For homogeneity, the medium is continuously circulated between 0.05 and 4.5 h^"1, preferably 3 h^"1, with a peristaltic pump (Masterflex Type 7550-50). The temperature was set between 24 and 39°C, preferably 35°C and pH maintained constant between 6 and 7, preferably at 6.5 by automatic addition of NH₄OH 3N. The oxidation-reduction potential (ORP) (mV) was monitored during the entire culture. To create anaerobic conditions, the sterilized medium in the vessel was flushed with sterile 0₂-free nitrogen for two hours at 60°C and flushed again at 35°C during fifteen hours. The bioreactor gas outlet was protected form oxygen entry by a pyrogallol arrangement (Vasconcelos et al., 1994). After inoculation, the bioreactor is maintained under nitrogen at 200mbar to avoid 0₂ entry. After sterilisation the feed medium was also flushed with sterile 0₂-free nitrogen until room temperature was attained and maintained under nitrogen at 200mbar to avoid 0₂ entry.

Batch and continuous cultures process:

For inoculation, a sample of a continuous culture made in a bioreactor was used as inoculum (5% v/v). The bioreactor is under the same conditions and contains the same medium as describe above. A growing culture in 100ml flasks on synthetic medium (the same as described above for batch culture but with the addition of acetic acid, 2.2g.l^_1 and MOPS, 23.03g.l^~1) taken at the end of the exponential growth phase can also be used as inoculum (5% v/v).

The cultures were first grown batch-wise. At the early exponential growth phase we performed a pulse of commercial glycerol: the pulse is defined by the addition of synthetic medium (the same as described for batch culture) with commercial glycerol 120g.l^_1 at a flow rate of 50ml. h^"1 during 2 hours (i.e. an addition of 12g of glycerol). Then the growth continued batch-wise and before the end of the exponential growth phase the continuous feeding started with a dilution rate of 0.005 to 0.02h^_1. The feed medium contained 105 g.l^"1 of raw glycerol. After 3 to 9 days under these conditions (i.e. 6 days after inoculation of the bioreactor), until the presence of the bio film is visually confirmed on the support material, the dilution rate was increased from 0.005h^_1 to the range of 0.1 Oh^"1 to 0.50h^_1, preferably to more than 0.1 Oh^"1. The reactor performance at various dilution rates was studied. To evaluate the performances, we waited for a stabilisation of the culture at the dilution rate tested. Stability of the culture was followed by product analysis using the HPLC protocol described below. Particularly we waited for a residual glycerol as low as possible to limit glycerol accumulation when extra glycerol was introduced in the fed medium. Using this approach, we tested various raw glycerol concentrations (105, 120, 140 g.l^"1) to evaluate the reactor performances.

Analytical procedures:

Glycerol, 1,3-propanediol, ethanol, butanol, acetic and butyric acid concentrations were determinate by HPLC analysis. Separation was performed on a Biorad Aminex HPX- 87H column and detection was achieved by refractive index. Operating conditions were as follows: mobile phase sulphuric acid 0.5mM; flow rate 0.5ml/min, temperature, 25°C. Example 2: Culture with high concentration of raw glycerol - Non- immobilized cells

The synthetic media used were the same as described in example 1, except that the feed medium contained at the beginning increasing concentrations of pure glycerol, from 90 to 120g.l^_1, and later 120g.l^_1 of raw glycerol. Pre-culture, inoculation and batch-wise growth were performed under the same conditions as described above.

Upon increase of the glycerol concentration in the feed medium, we modified the dilution rate in order to slowly adapt the cells to the increasing entry of glycerol (Figure 2). When we reached 120g.l^_1 of pure glycerol with a dilution rate of 0.05h^_1, we then shifted the glycerol supply from pure to raw glycerol. Rapidly, 1,3-propanediol titre dropped and, even by decreasing the dilution rate, we could not recover the culture performance. The culture stopped accumulating glycerol only at a dilution rate of 0.015h^-1, which results in a very low productivity of 0.84 ± 0.03 g/l/h compared to 2.8 ± 0.06 g/l h obtained on pure glycerol.

Example 3: Culture with high concentration of raw glycerol - Comparative example without cells immobilization

Culture media:

The synthetic media used for Clostridia batch cultivations contained per litre of deionised water: glycerol, 30g; KH₂P0₄, 0.5g; K₂HP0₄, 0.5g; MgS0₄ x 7H₂0, 0.2g; CoCl₂ x 6H₂0, O.Olg, H₂S0₄ (96%), 0.1ml; NH₄C1, 1.5g, biotin, 0.16mg; /?-amino benzoic acid, 32mg and FeS0₄ x 7H₂0, 0.028g, resazurin, lmg; cysteine, 0.5g. Resazurin and cysteine could be eliminated without any problem. Their use allows viewing oxygen entry in the system. The pH of the medium was adjusted to 6.3 with NH₄OH 3N. For batch cultivation we used a commercial glycerol purchased from Sigma (purity 99.5%). The feed medium for continuous cultures contained per litre of tap water: raw glycerol, different amounts (105, 120, 140g); KH₂P0₄, 0.5g; K₂HP0₄, 0.5g; MgS0₄ x 7H₂0, 0.2g; CoCl₂ x 6H₂0, 0.026g; NH₄C1, 1.5g, biotin, 0.16mg; /?-amino benzoic acid, 32mg; FeS0₄ x 7H₂0, 0.04g, anti-foam (Struktol), 0,05ml; ZnS0₄ x 7H₂0, 8mg; CuCl₂ x 2H₂0, 4mg; MnS0₄ x H₂0, 40mg, H₃BO₃, 2mg; Na₂Mo0₄ x 2H₂0, 0.8mg. Medium pH was not adjusted in this case. Raw glycerol, from the trans-esterification process for biodiesel was supplied by SAIPOL (Le Meriot, France) and has the following purity: glycerol 83%> (w/w).

Upon increase of the glycerol concentration in the feed medium, we did not modify the dilution rate as in example 2. Upon stabilisation of the chemostat at a dilution rate of 0.06h-l and 105g/L raw glycerol in the fed medium, we switched the feed medium to a concentration of 120g.l-l of raw glycerol with a dilution rate of 0.055h-l . Rapidly, 1,3- propanediol titre dropped and the overall performances of the reactor deteriorated (Table 1). We then switched the feeding glycerol concentration to 140g/L. Performances at the early stage of the switch decreased rapidly (Figure 3). The reactor with non- immobilized cells did not adjust to this last modification and cells were nearly completely washed out within five days (Figure 3). Table 3 reports average performances after one residence time following the introduction of the 140g/l fed medium that is the point where we usually start the measurement of the performances.

Table 1: Overall performance of a chemostat with non- immobilized cells with different raw glycerol concentration in the feed medium. Y: Yield; Q: Productivity

Example 4: Culture with high concentration of raw glycerol - Production of 1,3- propanediol with immobilized cells

The synthetic media used were the same as described in example 3. The feed medium contained increasing concentration of raw glycerol, from 105 to 140g.l-l . Pre- culture, inoculation and batch-wise growth were performed in the same conditions as described above.

Direct introduction of 105g.l-l of raw glycerol was supported by the cell culture and was used to establish the biofilm. Upon increase of the glycerol concentration from 105 to 120g.l in the feed medium, residual glycerol did not accumulates and the 1,3- propanediol titre increased. Reactor performances reached ~60g.l^"1 of 1,3-propanediol, with a yield of 0.53g.g^_1 of glycerol and a productivity of 3g.l.h^_1 at a dilution rate of 0.05h-\

When the glycerol concentration was again increased up to 140g.l^_1 in the fed medium, an adaptation phase was observed, characterized by fluctuating 1 ,3-propanediol and residual glycerol concentration. After a period of 2 weeks, we reached the same performances as with the fed medium containing 120g.l^_1 of raw glycerol (Figure 4). Notably, the extra 20g.l^_1 of raw glycerol added did not appear to be consumed by the cells, suggesting we attained the maximum glycerol import rate in these conditions (Table 2). Table 2: Overall performance of a chemostat with immobilized cells with different raw glycerol concentration in the feed medium. Y: Yield; Q: Productivity

Cell immobilisation improved resistance to the industrial glycerine and PDO production with a high titre (60.65 +/- 1.08g/l) and the maintenance of high productivity (3.29 +/- 0.13g/l/h) when compared to the results of free cells (PDO titre: 1 1.47 +/- 5.55g/l; PDO productivity: 0.64 +/- 0.30g/l/h. See example 3). Example 5: Culture with high concentration of raw glycerol - Production of 1,3- propanediol with immobilized cells at high dilution rate

The same synthetic media were used as described in example 3. The feed medium contained increasing concentration of raw glycerol, from 105 to 120g.l-l . Pre-culture, inoculation and batch-wise growth were performed in the same conditions as described above.

Direct introduction of 105g.l^_1 of raw glycerol was supported by the cell culture and was used to establish the bio film. Reactor performances reached ~50g.l^_1 of 1,3- propanediol, with a yield of 0.53g.g^_1 of glycerol and a productivity of 3.2g.l-l .h^_1 at a dilution rate of 0.05h^_1. Dilution rate was increased up to 0.09. PDO titre and yield did not vary, remaining at 50.4 lg/1 and 0.54g/g of glycerol respectively, but productivity increased to 4.56g/l/h.

Table 3: Overall performance of a chemostat with immobilized cells with different raw glycerol concentration in the feed medium. Y: Yield; Q: Productivity

In immobilized cells fermenter PDO productivity could be improved 1.5 times on this example. Notably, no glycerol accumulation was observed at any time during the increase of the dilution rate. REFERENCES

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Claims

1. A method for the fermentative production of 1,3-propanediol from glycerol, comprising culturing a microorganism of a Clostridium species producing the 1,3- propanediol from glycerol in an appropriate culture medium comprising glycerol and recovery of the 1,3-propanediol, wherein the culture is made in an immobilized cell reactor.

2. The method of claim 1, wherein the microorganism of a Clostridium species producing the 1,3-propanediol from glycerol is a recombinant Clostridium species modified for an improved production of 1 ,3-propanediol.

3. The method of one of claims 1 or 2, wherein Clostridium species producing the 1,3- propanediol from glycerol is a Clostridium acetobutylicum strain.

4. The method of one of claims 1 to 3, wherein the reactor is filled with inert support material on which a bio film immobilizing the microorganisms is formed.

5. The method of claim 4, wherein the inert support material is selected among the group consisting of metal, glass, ceramic, polypropylene and polyvinyl chloride.

6. The method of claim 4, wherein the inert support material is non-treated glass.

7. The method of anyone of claims 4 to 6, wherein the inert support material is shaped as a Pall ring, a Rashig ring, a Lessing ring, a Intalox saddle, a Berl saddle, a railing or a grid.

8. The method of one of claims 1 to 7, wherein the production is done in a batch, fed- batch or continuous process.

9. The method of claim 8, wherein the production is done in a continuous process.

10. The method of one of claims 1 to 8, wherein the glycerol concentration fed to the culture is comprised between 90 and 200g.l^~1

11. The method of claim 10, wherein the glycerol concentration fed to the culture is comprised between 90 and 140g.l^_1.

12. The method of claim 10, wherein the dilution rate applied in the continuous process is comprised between 0.05 and 0.5 h^"1.

13. The method of claim 12, wherein the dilution rate is comprised between 0.1 and 0.2 h^"1.

14. The method of one of claims 1 to 13, wherein the 1,3-propanediol is further purified.