The present invention relates to a process for producing an organic acid by fermentation and to the use of organic polymers as a polymeric plasticizer in the fermentative production of an or- ganic acid.

Organic compounds such as small dicarboxylic acids having 6 or fewer carbons are commercially significant chemicals with many uses. For example, the smail diacids include 1 ,4-diacids, such as succinic acid, malic acid and tartaric acid, and the 5-carbon molecule itaconic acid. Other diacids include the two carbon oxalic acid, three carbon malonic acid, five carbon glutaric acid and the 6 carbon adipic acid and there are many derivatives of such diacids as well.

As a group the small diacids have some chemical similarity and their uses in polymer production can provide specialized properties to the resin. Such versatility enables them to fit into the downstream chemical infrastructure markets easily. For example, the 1 ,4-diacid molecules fulfill many of the uses of the large scale chemical maleic anhydride in that they are converted to a variety of industrial chemicals (tetrahydrofuran, butyrolactone, 1 ,4-butanediol, 2-pyrrolidone) and the succinate derivatives succindiamide, succinonitrile, diaminobutane and esters of succinate. Tartaric acid has a number of uses in the food, leather, metal and printing industries. Itaconic acid forms the starting material for production of 3-methylpyrrolidone, methyl-BDO, methyl -THF and others.

If the above mentioned dicarboxylic acids are produced by fermentation, the pH of the fermentation broth continuously decreases and basic compounds have to be added as neutralizing agents in order to maintain an acceptable physiological environment for the microorganisms. These basic compounds, which may for example comprise Mg(OH)2 or CaC03, are usually added to the fermentation broth in the form suspensions. However, Mg(OH) ₂ is rather insoluble in water and is usually applied in the form of a very viscous suspension containing only 25% of Mg(OH)2. These properties make the fermentation very challenging, since highly viscous mate- rials are difficult to handle and process. The suspension tends to be difficult to pump and may block pipes and valves. Furthermore, the rather dilute suspension leads to an unfavorable dilution of the resulting fermentation broth with low product concentrations. The possibility of utilization of less viscous bases at higher concentrations would avoid the problems described above. It was therefore an object of the present invention to overcome the disadvantages of the prior art.

In particular, it was an object of the present invention to provide a process for producing an organic acid, such as succinic acid, by fermentation, which allows the production of these organic acids in higher concentrations, compared to the processes known in the prior art. A contribution to achieving the abovementioned aims is provided by a process for producing an organic acid by fermentation, comprising the process steps

I) cultivating microorganisms in a culture medium comprising at least one assimilable carbon source to allow the microorganisms to produce the organic acid, thereby obtaining a fermentation broth comprising the organic acid, wherein the pH of the fermentation broth is controlled to be within a range from 3 to 8 by the addition of a basic compound;

II) recovering the organic acid or the salt thereof from the fermentation broth obtained in pro- cess step I); wherein in process step I) the basic compound is added in the form an aqueous suspension comprising a) water, b) the basic compound, and c) a polymeric p!asticizer.

Organic polymers such as polycarboxylate esters, polycarboxyiate ethers, non-ionic polyether- polyester-copolymers, polyphenylene ethers, phosphor-containing polycondensation products, naphtha!insulfonate-formaldehyde-condensation products and melaminesulfonate-formalde- hyde-condensation products have been widely used as polymeric plasticizers with the goal of improving the properties of cement and related materials (see, for example, WOA-

2012/076365). It was found that the same plasticizers used for cement and concrete also improve the properties of bases used in fermentation processes. The addition of polymeric plasticizers to bases such as Mg(OH) ₂ and CaCOs improves the handling properties of these bases and also allows the preparation of more concentrated suspensions that can be pumped. Using the more concentrated bases results in a decreased amount of water that has to be added to the fermenter in the neutralization process. Therefore, dilution is decreased and higher titers can be achieved. Another observation is that these polymeric plasticizers did not affect the growth rate and did not have any negative effect in the microorganisms. in process step I) of the process according to the present invention microorganisms are cultivated in a culture medium comprising at least one assimilable carbon source to allow the mi- crooganisms to produce the organic acid, thereby obtaining a fermentation broth comprising the organic acid, wherein the pH of the fermentation broth is adjusted by the addition of a basic compound.

Suitable miccorganims according to the present invention may be yeasts, fungi or bacteria. Suitable bacteria, yeasts or fungi are in particular those bacteria, yeasts or fungi which have been deposited at the Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSMZ), Brunswick, Germany, as bacterial, yeast or fungal strains. Bacteria which are suitable according to the invention belong to the genera detailed under http://www.dsmz.de/species/bacteria.htm, yeasts which are suitable according to the invention belong to those genera which are detailed under http://www.dsmz.de/species/yeasts.htm, and fungi which are suitable according to the invention are those which are detailed under http://www.dsmz.de/species/fungi.htm. Preferably, the microorganisms used in process step I) are bacterial cells. The term "bacterial cell' as used herein refers to a prokaryotic organism, i.e. a bacterium. Bacteria can be classified based on their biochemical and microbiological properties as well as their morphology. These classification criteria are well known in the art. According to a preferred embodiment of the process according to the present invention the microroganisms belong to the family of Enterobacte- riaceae, Pasteurellaceae, Bacillaceae or Corynebacteriaceae.

"Enterobacteriaceae" represent a large family of bacteria, including many of the more familiar bacteria, such as Salmonella and Escherichia coli. They belong to the Proteobacteria, and they are given their own order (Enterobacteriales). Members of the Enterobacteriaceae are rod- shaped. Like other Proteobacteria they have Gram-negative stains, and they are facultative anaerobes, fermenting sugars to produce lactic acid and various other end products such as succinic acid. Most also reduce nitrate to nitrite. Unlike most similar bacteria, Enterobacteriaceae generally lack cytochrome C oxidase. Most have many flageila used to move about, but a few genera are non-motile. They are non-spare forming, and mostly they are catalase-positive. Many members of this family are a normal part of the gut flora found in the intestines of humans and other animals, while others are found in water or soil, or are parasites on a variety of different animals and plants. Escherichia coli, better known as E. coli, is one of the most important model organisms, and its genetics and biochemistry have been closely studied. Most members of Enterobacteriaceae have peritrichous Type I fimbriae involved in the adhesion of the bacterial cells to their hosts. Examples for the Enterobacteriaceae are E. coli, Proteus, Salmonella and Klebsiella.

" Pasteurellaceae" comprise a large family of Gram-negative Proteobacteria with members ranging from bacteria such as Haemophilus influenzae to commensals of the animal and human mucosa. Most members live as commensals on mucosal surfaces of birds and mammals, especially in the upper respiratory tract. Pasteurellaceae are typically rod-shaped, and are a notable group of facultative anaerobes. They can be distinguished from the related Enterobacteriaceae by the presence of oxidase, and from most other similar bacteria by the absence of flagella. Bacteria in the family Pasteurellaceae have been classified into a number of genera based on metabolic properties and there sequences of the 16S RNA and 23S RNA. Many of the Pasteurellaceae contain pyruvate-formate-lyase genes and are capable of anaerobically fermenting carbon sources to organic acids.

" Bac/llaceae" is a family of Gram-positive, heterotrophic, rod-shaped bacteria that may produce endospores. Motile members of this family are characterized by perit chous flagellae. Some Bac/llaceae are aerobic, while others are facultative or strict anaerobes. Most are non- pathogenic, but Bacillus species are known to cause disease in humans. This family also com- prieses the genus Bacilli ^' which includes two orders, Bacillales and Lactobacillales. The bacillus species represents a large cylindrical bacteria that can grow under aerobic conditions at 37°C. They are typically nonpathogenic. The genus Bacillales contains the species Alicyclobacillace- ae, Bac/llaceae, Caryophanaceae, Listeriaceae, Paenibacillaceae, Planococcaceae, Sporolac- tobac/i/aceae, Staphylococcaceae, Thermoactinomycetaceae, Turicibacteraceae. Many of the Bacilli contain pyruvate-formate-lyase genes and are capable of anaerobically fermenting carbon sources to organic acids.

" Corynebacteriaceae" is a large family of mostly Gram-positive and aerobic and nonmotile rod- shaped bacteria of the order Eubacteriales. This family also comprises the genus Corynebacte- rium, which is a genus of Gram-positive, rod-shaped bacteria. Corynebacteria are widely distributed in nature and are mostly innocuous. Some are useful in industrial settings such as C. glutamicum. The microorganisms used in process step I) may genetically altered, modified or engineered (e.g., genetically engineered) such that they exhibit an altered, modified or different genotype and/or phenotype (e. g., when the genetic modification affects coding nucleic acid sequences of the microorganism) as compared to the naturally-occurring wildtype microorganism from which they have been derived. The way in which the microoganisms may be genetically altered, modi- fied or engineered of course depends on the organic acid that should predominantly be produced by the microorganism. Preferably, the organic acid is selected from the group consisiting of carboxylic acids such as formic acid, acetic acid, lactic acid, propionic acid, 2- hydroxypropionic acid, 3-hydroxypropionic acid, 3-hydroxybutyric acid, acrylic acid, pyruvic acid or salts of these carboxylic acids, dicarboxylic acids such as malonic acid, succinic acid, malic acid, tartaric acid, glutaric acid, itaconic acid, adipic acid or salts thereof and tricarboxylic acids such as citric acid or salts thereof.

If the organic acid is succinic acid, a particularly preferred microorganism used in process step I) is a modified microorganism that has been derived from a wildtype that belongs to the family Pasteurellaceae. In this context it is furthermore preferred that the wildtype from which modified microorganism has been derived belongs to the genus Basfia and it is particularly preferred that the wildtype from which the modified microorganism has been derived belongs to the species Basfia succiniciproducens.

Most preferably, the wildtype from which the modified microroganism used to produce succinic acid has been derived is Basfia succlnlclproducens-strain DD1 deposited under the Budapest Treaty with DSMZ {Deutsche Sammlung von Mikroorganismen und Zellkuituren, GmbH), Germany, having the deposit number DSM 18541. This strain has been originally isolated from the rumen of a cow of German origin. Pasteurella bacteria can be isolated from the gastro-intestinal tract of animals and, preferably, mammals. The bacterial strain DD1 , in particular, can be isolat- ed from bovine rumen and is capable of utilizing glycerol (including crude glycerol) as a carbon source. A further strain of the genus Basfia thai can be used for preparing the modified microorganism according to the present invention is the Basfia-si ain that have been deposited under the deposit number DSM 22022. According to a preferred embodiment of the modified microoganism used for the production of succinic acid is a microorganisms that has i) a reduced pyruvate formate lyase activity, ii) a reduced lactate dehydrogenase activity, or iii) a reduced pyruvate formate lyase activity and a reduced lactate dehydrogenase activity.

Modified microorganisms being deficient in lactate dehydrogenase and/or being deficient in py- ruvate formate lyase activity are disclosed in WO-A-2010/092155, US 2010/0159543 and WO A-2005/052135, the disclosure of which with respect to the different approaches of reducing the activity of lactate dehydrogenase and/or pyruvate formate lyase in a microorganism, preferably in a bacterial cell of the genus Pasteurella, particular preferred in Basfia succiniciproducens strain DD1 , is incorporated herein by reference.

The assimilable carbon source is preferably selected from from sucrose, maltose, D-fructose, D- glucose, D-xylose, L-arabinose, D-galactose, D-mannose, glycerol and mixtures thereof or compositions containing at least one of said compounds, or is selected from decomposition products of starch, cellulose, hemicellulose and/or lignoce!lulose. Preferably, the assimilable carbon source comprises D-glucose, sucrose, glycerol or a mixture of at least two of these compounds, wherein mixtures of glycerol and D-glucose, glycerol and sucrose, glycerol and D- xylose and D-glucose and D-fructose are particularly preferred. The initial concentration of the assimilable carbon source is, preferably, adjusted to a value in a range of 5 to 100 g/l, preferably 5 to 75 g/l and more preferably 5 to 50 g/l and may be maintained in said range during culti- vation. The microorganisms are preferably incubated in the culture medium at a temperature in the range of about 10 to 60°C or 20 to 50°C or 30 to 45°C.

Preferably, the organic acid such as succinic acid is produced under anaerobic conditions. An- aerobic conditions may be established by means of conventional techniques, as for example by degassing the constituents of the reaction medium and maintaining anaerobic conditions by introducing carbon dioxide or nitrogen or mixtures thereof and optionally hydrogen at a flow rate of, for example, 0.1 to 1 or 0.2 to 0.5 vvm. Aerobic conditions may be established by means of conventional techniques, as for example by introducing air or oxygen at a flow rate of, for ex- ample, 0.1 to 1 or 0.2 to 0.5 vvm. If appropriate, a slight over pressure of 0.1 to 1.5 bar may be applied in the process.

The fermentation step I) according to the present invention can, for example, be performed in stirred fermenters, bubble columns and loop reactors. A comprehensive overview of the possi- ble method types including stirrer types and geometric designs can be found in Chmie!: "Bio- prozesstechnik: Einfuhrung in die BioverfahrenstechniH ', Volume 1. In the process according to the present invention, typical variants available are the following variants known to those skilled in the art or explained, for example, in Chmiel, Hammes and Bailey: " Biochemical Engineering , such as batch, fed-batch, repeated fed-batch or else continuous fermentation with and without recycling of the biomass. Depending on the production strain, sparging with air, oxygen, carbon dioxide, hydrogen, nitrogen or appropriate gas mixtures may be effected in order to achieve good yield (YP/S).

The pH of the reaction medium is controlled to be in the range from 3 to 8 by addition of a basic compound that is added in the form of an aqueous suspension comprising a) water, b) the basic compound and c) a polymeric plasticizer. Preferably, the pH is controlled to be in the range from 4.0 to 8.0, preferably 5.0 to 7.5 and more preferably 6.0 to 7.0, wherein the particularly preferred pH-value of course also depends on the particular microorganism that is used. The basic compound b) in the aqueous suspension can be any compound that the person skilled in the art would consider as appropriate for adjusting the pH-value of a fermentation broth during cultivation. Suitable basic compounds b) are selected from the group consisting of NH4HCO3, (NH ₄ ) ₂ C0 _3j NaOH, Na ₂ C0 ₃ , NaHC0 ₃ , KOH, K2CO3, KHC0 ₃ , Mg(OH) ₂ , MgC0 ₃ , Mg(HC0 ₃ ) ₂ , Ca(OH) ₂ , CaC0 ₃ , Ca(HC0 ₃ ) ₂ , CaO, CH ₆ N ₂ 0 ₂ , C2H7N and/or mixtures thereof. Par- ticularly preferred basic compounds b) are Mg(OH) ₂ , MgC0 ₃ , Mg(HC0 ₃ ) ₂ , Ca(OH) ₂ , CaC0 ₃ , Ca(HC0 ₃ ) ₂₎ wherein Mg(OH) ₂ , CaC0 ₃ or a mixture thereof are most preferred.

The concentration of the basic compound b) in the aqueous suspension is generally at least 10 wt.-%, wherein the maximal concentration depends on the chemical nature of the basic compound b). If the basic compound is Mg(OH) ₂ , the concentration of the basic compound in the aqueous suspension is preferably at least 15 wt.-%, more preferably at least 30 wt.-% and most preferably at least 40 wt.-%, in each case based on the totai weight of the aqueous suspension, wherein the concentration typically is between 15 wt.-% and 75 wt.-%, more preferably between 30 wt.-% and 60 wt.-%.

If the basic compound is MgCCb, the concentration of the basic compound in the aqueous suspension is preferably at least 10 wt.-%, more preferably at least 20 wt.-% and most preferably at least 30 wt.-%, in each case based on the total weight of the aqueous suspension, wherein the concentration typically is between 0 wt.-% and 75 wt.-%, more preferably between 20 wt.-% and 40 wt.-%.

If the basic compound is Ca(QH)2, the concentration of the basic compound in the aqueous suspension is preferably at least 15 wt.-%, more preferably at least 30 wt.-% and most prefera- bly at least 40 wt.-%, in each case based on the total weight of the aqueous suspension, wherein the concentration typically is between 15 wt.-% and 75 wt.-%, more preferably between 25 wt-% and 55 wt.-%.

If the basic compound is CaC03, the concentration of the basic compound in the aqueous sus- pension is preferably at least 10 wt.-%, more preferably at least 20 wt.-% and most preferably at least 30 wt.-%, in each case based on the total weight of the aqueous suspension, wherein the concentration typically is between 10 wt.-% and 75 wt.-%, more preferably between 25 wt.-% and 55 wt.-%. The polymeric plasticizer c) in the aqueous suspension is preferably an organic polymer selected from the group consisting of polycarboxylate esters, polycarboxylate ethers, non-ionic poly- ether-polyester-copolymers, polypheny!ene ethers, phosphor-containing polycondensation products, naphthalinsulfonate-formaldehyde-condensation products, melaminesulfonate- formaldehyde-condensation products and mixtures of at least two of these organic polymers.

Preferred polymeric plasticizers c) in the above list of compounds are those having a solubility in water at 25°C of at least 0.1 g/L, preferably of at least 0.5 g/L, more preferably of at least 1 g/L, more preferably of at least 5 g/L and most preferably of at least 10 g/L. - A "polycarboxylate estef in the sense of the present invention is polymer the backbone of which carries a plurality of carboxylate groups and a plurality of polyester groups, wherein the polyester groups are preferably polyalkyleneglykol groups that are bound as side chains to the polymer backbone and that are connected to the polymer backbone via an ester bond. These polycarboxylate esters can, for example, be obtained by copolymerising ethylenicaily unsaturat- ed, carboxylate group containing monomers, such as acrylic acid or methacrylic acid, and acry- late esters or methacrylate esters of polyalkylenglykols. Suitable polycarboxylate esters are the "copolymers <¾>" that are disclosed in US 2013/0005861. These copolymers can be obtained by polymerizing a monomer mixture that contains an (alkoxy)polyalkylene glycol mono(meth)acrylate monomer (a) of the general formula (I)

H ₉ C=C— R ¹

COO(R ² 0) _m R ³

(I) in which R represents a hydrogen atom or a CH group, R ² 0 represents one representative or a mixture of at least two oxyalkylene groups having 2 to 4 carbon atoms, R ³ represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms and m represents a number between 1 and 250 and represents the average number of moles of the oxyalkylene group added, additionally, as monomer (b), a (meth)acrylic acid of the general formula (il), H ₉ C=C— R ⁴

COOM

{") in which R ⁴ represents a hydrogen atom or a CH3 group and M ¹ represents a hydrogen atom, a monovalent metal atom, a divalent metal atom, an ammonium group or an organic amine group, and optionally a monomer (c) which is copolymerized with the monomers (a) and (b).

The monomer (a) can be present in an amount of from 5 to 98 wt.-%, the monomer (b) in a pro- portion of from 2 to 95 wt.-% and the monomer (c) in a proportion up to 50 wt.-% in the monomer mixture, wherein the respective proportions of the monomers (a), (b) and (c) add up to 100 wt.-%.

As typical representatives of the monomer (a), hydroxyethyl(meth)acrylate, hydroxypro- pyl(meth)acry!ate, polyethylene glycol mono(meth)acrylate, polypropylene glycol

mono(meth)acrylate, polybutylene glycol mono(meth)acrylate, polyethylene glycol polypropylene glycol mono(meth)acrylate, polyethylene glycol polybutylene glycol mono(meth)acrylate, polypropylene glycol polybutylene glycol mono(meth)acrylate, polyethylene glycol polypropylene glycol polybutylene glycol mono(meth)acrylate, methoxypolyethy!ene glycol

mono{meth)acrylate, methoxypolypropylene glycol mono(meth)-acrylate, methoxypolybuty!ene glycol mono(meth)acrylate, methoxypolyethylene glycol polypropylene glycol

mono(meth)acrylate, methoxypolyethylene glycol polybutylene glycol mono(meth)acryiate, methoxypolypropylene glycol polybutylene glycol mono(meth)-acrylate, methoxypolyethylene glycol polypropylene glycol polybutylene glycol mono-(meth)acrylate, ethoxy polyethylene glycol mono(meth)acrylate, ethoxypolypropylene glycol mono(meth)acrylate, ethoxypolybutylene gly- col mono(meth)acrylate, ethoxypolyethylene glycol polypropylene glycol mono(meth)acrylate, ethoxy polyethylene glycol polybutyiene glycol mono(meth)acrylate, ethoxypolypropylene glycol polybutylene glycol mono(meth)acrylate, ethoxypolyethylene glycol polypropylene glycol polybutylene glycol mono(meth)acrylate or mixtures thereof are suitable.

For the monomer (b), representatives of the group consisting of acrylic acid, methacrylic acid, monovalent metal salts, divalent metal salts, ammonium salts and organic amine salts thereof and mixtures of at least two of the said representatives are to be regarded as suitable. For the monomer (c) an ester of an aliphatic alcohol with 1 to 20 carbon atoms with an unsaturated carboxylic acid can be used. As the unsaturated carboxylic acid, in particular maleic acid, fumaric acid, citraconic acid (meth)acrylic acid or monovalent metal salts, divalent metal salts, ammonium salts or organic amine salts thereof are especially suitable. Monoesters or diesters of unsaturated dicarboxylic acids such as maleic acid, fumaric acid or citraconic acid with ali- phatic C1-C20 alcohols, C2-C4 glycols or with (alkoxy)polyalkyiene glycols are suitable representatives of monomer (c).

A " polycarboxylate ethef in the sense of the present invention is polymer the backbone of which carries a plurality of carboxylate groups and a plurality of polyether groups, wherein the polyether groups are preferably polyalkyleneglykol groups that are bound as side chains to the polymer backbone and that are connected to the polymer backbone via an ether bond. These polycarboxylat ethers can, for example, be obtained by copolymerising ethylenically unsaturated, carboxylate group containing monomers, such as acrylic acid or methacrylic acid, and vi- nylmonomers carrying a polya!kylenglykol group, preferably a polyethylenglykol group.

Suitable polycarboxylate ethers are the " copolymers a " that are disclosed in US-A- 2013/0005861. These copolymers preferably consist of two monomer components, the first monomer component being an olefinically unsaturated monocarboxylic acid comonomer or an ester or a salt thereof and/or an olefinically unsaturated sulphonic acid comonomer or a salt thereof (component 1), and the second monomer component (component 2) a comonomer of the general formula (III)

(Ml) wherein R ⁶ represents a structural unit of the formula (IV)

(C _m H _2m O) _x (C _n H _2n O) _y — (CH ₂ — C— O)— R8

R ⁷ (IV) and R ⁵ represents H or an aliphatic Ci-C ₅ -hydrocarbon residue, R ⁷ represents a unsubsti- tuted or substituted aryl residue and preferably phenyl, and R ⁸ represents H or an aliphatic hydrocarbon residue with 1 to 20 C atoms, cycioaiiphatic hydrocarbon residue with 5 to 8 C atoms, a substituted aryl residue with 6 to 14 C atoms or a member of the series

O O O

I I II I I

— O-C— R ⁹ — O-C— R ¹⁰ — C— OH

O

— O-C— (NH)R ¹¹ wherein R ⁹ and R ¹ each represent an alky!, aryl, aralkyi, or alkaryl residue and R ⁰ for an alkyli- dene, arylidene, aralkylidene or alkarylidene residue, and p = 0, 1 , 2, 3 or 4 m, n mutually independently mean 2, 3, 4 or 5 x and y mutually independently denote an integer≤ 350 and - z = 0 to 200.

In the preferred embodiment of the polycarboxylate ethers the copolymer contains the comon- omer component 1 in proportions of 30 to 99 mol. % and the comonomer component 2 in proportions of 70 to 1 mol. %. The comonomer component 1 can preferably be selected from the group consisting of acrylic acid, methacrylic acid, crotonic acid, isocrotonic acid, allylsulphonic acid, vinylsulphonic acid and suitable salts thereof and alkyl or hydroxyalkyl esters thereof.

In addition, the copolymer can have additional structural groups in copolymerized form. In this case, the additional structural groups may be styrenes, acrylamides and/or hydrophobic compounds, ester structural units, polypropylene oxide and polypropylene oxide/polyethylene oxide units being particularly preferred. The copolymer should contain the said additional structural groups in proportions up to 5 mol. %, preferably from 0.05 to 3.0 mol. % and in particular from 0.1 to 1.0 mol. %.

Also suitable as polycarboxylate ethers are the copolymers that are disclosed in EP-A-736 553. These copolymers are copolymers of derivates of unsaturated dicarboxylic acids and vinyleth- ers carrying polyalkyleneglykol groups, in particular polyethylenmeglykol groups. Furthermore, the copolymers disclosed in EP-A-736 553 may also comprise monomer units based on acrylic acid or methacrylic acid or based on derivatives, in particular esters, of acrylic acid or methacrylic acid. The disclosure of EP-A-736 553 is incorporated herein by reference. - A " ¹ polyether-polyestef in the sense of the present invention is polymer the backbone of which carries a plurality of polyether groups and a pluralityof polyester groups, wherein the pol- yether groups are preferably polyalkyleneglykol groups that are bound as side chains to the polymer backbone and that are connected to the polymer backbone via an ether bond. These polyether-polyesters can, for example, be obtained by copolymerising esters of ethylenically unsaturated, carboxy!ate group containing monomers, such as acrylic acid esters or methacrylic acid esters, and vinylmonomers carrying a polyalkylenglykol group, preferably a poiyeth- ylenglykol group.

Suitable non-ionic polyether-polyester-copolymers are the "copolymers a? that are disclosed in US-A-2013/0005861. In this context it is particularly preferred that the non-ionic polyether- polyester-copolymers is of the general formula (V)

(V) wherein G means O, C(O)— O or O— (Chbjp— O with p = 2 to 8, wherein mixtures of the modifications of G in one polymer are possible; R stands for H or Ch , R ¹² and R ¹³ mutually independently mean at least one C2-Csalkyl; R ⁴ comprises (CH2)c, where c is a whole number between 2 and 5 and where mixtures of the representatives of R ¹⁴ in the same polymer molecule are possible; R ¹⁵ means at least one representative selected from the series H, a linear or branched, saturated or unsaturated C1-C20 aliphatic hydrocarbon residue, a C ₅ -C ₈ cycloaliphatic hydrocarbon residue or a substituted or unsubstituted Ce-Cw aryl residue; m = 1 to 30, n = 31 to 350, w = 1 to 40, y = 0 to 1 and z = 0 to 1, where the sum (y + z) > 0. R ⁶ means at least one d- C20 alkyl or C2-C20 hydroxyalkyl radical.

Preferred polyphenylene ethers are compounds having the general formula (Vi)

(VI) wherein R represents an identical or different group selected from the group consisting of hydrogen atom, halogen atom, hydrocarbon group, substituted hydrocarbon group, hydrocarbon- oxy group and substituted hydrocarbon-oxy group. Examples of the substituent in the substitut- ed hydrocarbon group and substituted hydrocarbon-oxy group include thermally stable groups such as halogen atom, hydroxy! group, amino group, nitro group, cyano group, ester group, am- ido group, ether group, sulfide group, sulfone group and the like. Concrete examples of suitable polyphenylene ethers include poly(2,6-dimethyl-1 ,4-phenyiene ether), poly(2,6-diethyl-1 ,4- phenylene ether), poly (2-methyl-6-ethyl-1,4-pheny-lene ether), poly(2-methyl-6-propyl- ,4- phenylene ether), poly(2,6-dipropyl-1 ,4-phenylene ether), poly(2-ethyl-6-propyl-1 ,4-phenylene ether), poly(2,6-dibutyl-1 ,4-phenylene ether), poly(2,6-diproponyl-1 ,4-phenylene ether), poly(2,6-dilauryl-1,4-phenylene ether), poly(2,6-diphenyl-1 ,4-phenylene ether), poly(2 _f 6- dimethoxy-1 ,4-phenylene ether), poly(2,6-diethoxy-1,4-phenyiene ether), poly (2-methoxy-6- ethoxy-1 ,4-phenylene ether), poly(2-ethy!-6-stearyloxy-1 ,4-phenylene ether), poly(2-methyl-6- phenyl-1 ,4-phenylene ether), po!y(2-methy!-1 , 4-pheny[ene ether), poly(2-ethoxy-1 ,4-phenylene ether), poly(2-chloro-1 ,4-phenylene ether), poly(3-methy!-6-t-butyl-1 ,4-phenylene ether), poly(2,6-dichloro-1 ,4-phenylene ether), poly(2,5-dibromo-1 ,4-phenylene ether), poly(2,6- dibenzyl-1 ,4-pheny!ene ether), and various copolymers having plural kinds of recurring units constituting these polymers. The copolymers also include copolymers formed between poly- substituted phenols such as 2,3,6-trimethy!phenoi, 2,3,5,6-tetramethylphenol and the like and 2,6-dimethyl-phenol, and the like. - Preferred phosphor-containing polycondensation products are those copolymers which in US-A-201 1/0054053 are referred to as "EPPR". These copolymers are polycondensations products comprising

(i) a first polycondensation repeating unit having a polyether side chain and one of the group consisting of an aromatic sub-unit and a heteroaromatic sub-unit;

(ii) a second polycondensation repeating unit having a OP(OH)2 group and one of the group consisting of an aromatic sub-unit and a heteroaromatic subunit; (iii) a third polycondensation repeating unit having one of the group consisting of an aromatic subunit and a heteroaromatic sub-unit, wherein said second polycondensation repeating unit and said third polycondensation repeating unit differ exclusively in that the OP(OH)2 groups of said second polycondensation repeating unit are replaced by H in said third polycondensation repeating unit, and said third polycondensation repeating unit is not the same as said first polycondensation repeating unit.

The first polycondensation repeating unit of the is described by the general formula (VII)

(VII) wherein A units are identical or different and are represented by a substituted or unsubstituted aromatic or heteroaromatic compound having 5 to 10 C atoms;

where B units are identical or different and are represented by N, NH or O; where n = 2, if B = N and n = 1 , if B = NH or O; wherein R ¹ and R ² , independently of one another, are identical or different and are represented by a branched or straight-chain d to Cio alkyl radical, C ₅ to C ₈ cycloalkyi radical, aryl radical, heteroaryl radical or H; wherein "a" values are identical or different and are represented by an integer from 1 to 300; wherein X units are identical or different and are represented by a branched or straight-chain C to Cio alkyl radical, Cs to Ce cycloalkyi radical, aryl radical, heteroaryl radical H.

The second polycondensation repeating unit is described by the genera! formula (VIII)

(VIII) and the third repeating unit is described by the general formula (IX)

For general formulas (VIII) and (IX) in each case:

D units are identical or different and are represented by a substituted or unsubstitut- ed heteroaromatic compound having 5 to 10 C atoms;

E units are identical or different and are represented by N, NH or O; m = 2 if E = N and m = 1 if E = NH or O;

R ³ and R ⁴ , independently of one another, are identical or different and are represented by a branched or straight-chain Ci to Cio alkyl radical, C ₅ to C ₈ cycloalkyi radical, aryl radical, heteroaryl radical or H;

"b" values are identical or different and are represented by an integer from 0 to 300; M groups, independently of one another, are an alkaline metal ion, alkaline earth metal ion, ammonium ion, organic ammonium ion and/or H; and - c is 1 or in the case of alkaline earth metal ions ½.

The polycondensation component may further contain a repeating unit of the general formula (X)

wherein Y groups, independently of one another, are identical or different and are represented by general formulae (VII), (VIII), (IX) or further constituents of the polycondensate; wherein R ⁵ groups are identical or different and are represented by H, CH ₃ , COOM _c or a substituted or unsubstituted aromatic or heteroaromatic compound having 5 to 10 C atoms; and wherein R ⁶ groups are identical or different and are represented by H, CH3, COOM _c or a substituted or unsubstituted aromatic or heteroaromatic compound having 5 to 10 C atoms.

Preferably, R ⁵ and R ⁶ in general formula (X), independently of one another, are represented by H, COOM _c and/or methyl.

The molar ratio of the units of general formulae (VII), (VIII), (IX) and (X) of the polycondensation component varies within wide ranges. In some embodtments wherein the molar ratio of the first, second, third and fourth polycondensation repeating units are represented by their formula number, then [(VII) + (VIII) + (IX)] : (X) is 1 : 0.8 to 3, preferably 1 : 0.9 to 2 and particularly preferably 1 : 0.95 to 1.2. The molar ratio of the first, second and third polycondensation repeating units (Vli) : [(VIII) + (IX)] in the polycondensation component is usually 1 : 15 to 15 : 1 , preferably 1 : 10 to 10 : 1 and more preferably 1 : 5 to 3 : 1. In a preferred embodiment, the molar ratio of the second and third repeating units (VII!) : (IX) is adjusted to 1 : 0.005 to 1 : 10, preferably 1 : 0.01 to 1 : 1, in particular 1 : 0.01 to 1 : 0.2 and more preferably 1 : 0.01 to 1 : 0.1.

The groups A and D in the repeating units of general formulae (VII), (VIII) and (IX) of the polycondensation component are preferably represented by phenyl, 2-hydroxyphenyl, 3- hydroxyphenyl, 4-hydroxyphenyl, 2-methoxyphenyl, 3-methoxyphenyl, 4-methoxyphenyl, naph- thyl, 2-hydroxynaphthyl, 4-hydroxynaphthyl, 2-methoxynaphthyl, 4-methoxynaphthyl, preferably phenyl. It is possible for A and D to be chosen independently of one another and also in each case to consist of a mixture of said compounds. The groups B and E, independently of one another, are preferably represented by an oxygen atom, O.

The radicals ¹ , R ² , R ³ and R ⁴ can be chosen independently of one another and are preferably represented by H, methyl, ethyl or phenyl, particularly preferably by H or methyl and especially preferably by H. Value a in the first polycondensation repeating unit of general formula (VII) is preferably represented by an integer from 5 to 280, in particular 0 to 160 and particularly preferably 12 to 120. Value b in the second and third repeating units (VIII) and (IX) is an integer from 0 to 10, preferably 1 to 7 and particularly preferably 1 to 5. The respective radicals, the length of which is defined by a and b, respectively, may consist of uniform building blocks, but a mixture of different building blocks may also be expedient. Furthermore, the radicals of the first, second and third repeating units of Formulae (VII) or (VIII) and (IX), independently of one another, may each have the same chain length, a and b each being represented by a value.

Frequently, the phosphated polycondensate has a weight average molecular weight of 4000 g/mol to 150 000 g/mol, preferably 10 000 to 100 000 g/mol and particularly preferably 20 000 to 75 000 g/mol.

Preferred naphthalinsulfonate-formaldehyde-condensation products ("BNS") are the condensation products that are disclosed in EP-A-0 214 412 and DE-PS-2 007 603. The effect and properties of BNS can be modified by changing the molar ratio between formaldehyde and the naphthalene component that usually is from 0.7 up to 3.5. The ratio between formaldehyde and the sulphonated naphthalene component preferably is from 0.8 to 3.5 to 1.

Preferred melaminesulfonate-formaldehyde-condensation products ("MFS") are the con- densation products that are disclosed in DE-A-196 09 6 4, DE-A-44 1 797, EP-A-0 059 353 and DE-A- 95 38 821.

In the process according to the present invention the concentration of the polymeric plasticizer in the aqueous suspension is in the range from 0.01 to 10 wt.-%, preferably in the range from 0.1 to 5 wt.-%, in each case based on the total weight of the aqueous suspension.

According to a particular embodiment of the process according to the present invention, the polymeric plasticizer, when being present in a concentration of 1 wt.%, reduces the viscosity of a 20 wt.-% Mg(OH ₂ ) aqueous solution at 23°C by at least 10 %, preferably at least 25 wt.-% and most preferably at least 50 wt.-%. Thus, if the viscosity of the 20 wt.-% Mg(OH2) aqueous solution at 25°C is ηι, the viscosity of the 20 wt.-% gfOhb) aqueous solution at 25°C, which additionally comprises 1 wt.-% of the polymeric plasticizer, is not more than 0.9 ^χ ηι (reduction of at least 10 %) preferably not more than 0.75 * ηι (reduction of at least 25 %) and most preferably not more than 0.5 ^χ ηι (reduction of at least 50 %). The viscosities of the 20 wt.-% Mg(OH2) aqueous solution with and without the polymeric plasticizer are determined at 23°C with a Haake rotational viscometer at a shear rate of 00 ^{s 1} .

The aqueous suspension that is used in process step I) to control the pH of the fermentation broth can be prepared by simply mixing the components (i. e. water, basic compound and polymeric plasticizer) in any order. However, it has shown to be particularly advantageous if in a first step a solution or suspension of the polymeric plasticizer in water is formed and if the basic compound is subsequently added to this solution or suspension in a second step. After the components have been combined, the mixture is homogenized by stirring, preferably by stirring with an ultra-turrax.

In process step II) the organic acid, preferably succinic acid, or the salt thereof is recovered from the fermentation broth obtained in process step I).

Usually, the recovery process comprises the step of separating the recombinant microorganims from the fermentation broth as the so called "biomass". Processes for removing the biomass are known to those skilled in the art, and comprise filtration, sedimentation, flotation or combinations thereof. Consequently, the biomass can be removed, for example, with centrifuges, separators, decanters, filters or in a flotation apparatus. For maximum recovery of the product of value, washing of the biomass is often advisable, for example in the form of a diafi!tration. The selection of the method is dependent upon the biomass content in the fermentation broth and the properties of the biomass, and also the interaction of the biomass with the organic acid (i. e. the product of value). In one embodiment, the fermentation broth can be sterilized or pasteurized. In a further embodiment, the fermentation broth is concentrated. Depending on the requirement, this concentration can be done batch wise or continuously. The pressure and temperature range should be selected such that firstly no product damage occurs, and secondly minimal use of apparatus and energy is necessary. The skillful selection of pressure and temperature levels for a multistage evaporation in particular enables saving of energy.

The recovery process may further comprise additional purification steps in which the organic acid, preferably succinic acid, is further purified. If, however, the organic acid is converted into a secondary organic product by chemical reactions as described below, a further purification of the organic acid is, depending on the kind of reaction and the reaction conditions, not necessarily required. For the purification of the organic acid obtained in process step II), preferably for the purification of succinic acid, methods known to the person skilled in the art can be used, as for example crystallization, filtration, electrodia!ysis and chromatography. In the case of succinic acid as the organic acid, for example, succinic acid may be isolated by precipitating it as a cal- cium succinate product by using calcium hydroxide, -oxide, -carbonate or hydrogen carbonate for neutralization and filtration of the precipitate. The succinic acid is recovered from the precipitated calcium succinate by acidification with sulfuric acid followed by filtration to remove the cal- cium sulfate (gypsum) which precipitates. The resulting solution may be further purified by means of ion exchange chromatography in order to remove undesired residual ions. Alternatively, if magnesium hydroxide, magnesium carbonate or mixtures thereof have been used to neutralize the fermentation broth, the fermentation broth obtained in process step l)may be acidified to transform the magnesium succinate contained in the medium into the acid form (i. e. succinic acid), which subsequently can be crystallized by cooling down the acidified medium. Examples of further suitable purification processes are disclosed in EP-A-1 005 562, WO-A-2008/010373, WO-A-201 1/082378, WO-A-201 1/043443, WO-A-2005/030973, WO-A-201 1/123268 and WO- A-2011/064151 and EP-A-2 360 137.

A contribution to solving the problems mentioned at the outset is furthermore provided by the use of an organic polymer selected from the group consisting of polycarboxylate esters, poly- carboxylate ethers, non-ionic polyether-polyester-copolymers, polyphenylene ethers, phosphor- containing polycondensation products, naphthalinsulfonate-formaldehyde-condensation prod- ucts, melaminesulfonate-formalde-hyde-condensation products and mixtures of at least two of these organic polymers as a polymeric plasticizer in the fermentative production of an organic compound. Preferred polymeric plasticizers are those that have been mentioned in connection with the process according to the present invention. Preferred organic compounds are the organic acids that have already been mentioned in connection with the process according to the present invention, in particular succinic acid.

The invention is now explained in more detail with the aid of non-limiting examples. EXAMPLES

Example 1 : Determination of the viscosity of different base concentrations prepared with or without polymers

For the determination of the viscosity and stability, the different concentrations of bases were prepared and the tests were performed as described beiow. a) Base preparation and suspension characterization

There are different possibilities for preparing an aqueous solution comprising water, the basic compound and the polymeric plasticizer. In one approach water and the base at increasing concentrations varying from 10% to 70% were added in either 250 or 500 mL flasks. The polymer was then added at concentrations varying from 0.3 to 0.6% {Table 1) in order to find the best amount for every suspension concentration. The suspensions were stirred vigorously for 5 minutes for homogenization. Another way to prepare the suspensions is by adding water and the polymer previous to the addition of the bases. The mixture of water and polymer are stirred vigorously and the base powder is slowly added to this mixture. Intense agitation is continued until the complete homogenization of the suspension. The latter is the preferred way to prepare the suspension for each combination of base and polymer. This procedure was used to prepare the suspensions for the fermentation experiments which will be described in a further section. b) Viscosity

Tests were performed to check the applicability of three different classes of polymers (MVA, EPPR and poly PPE) to decrease the viscosity of base suspensions. The base suspensions were prepared as described above and stirred for 24 hours. The viscosity was then measured at a temperature of 23°C by the HAAKE viscosity test using Thermo Scientific ^® HAAKE viscotester 7 plus. c) Results

The results of the viscosity experiments with different bases and polymers are shown in Table 1. The addition of polymers in the bases decreased the viscosity in all cases. The higher concentrations were observed for Mg(OH) ₂ and CaC03 but the decrease in viscosity is observed for different bases The possibility of pumping and handling of concentrated suspensions are only possible in the presence of the polymers. The addition of these polymers is beneficial, allowing the preparation of high concentrated and homogeneous suspensions. The homogenization and pumping of high concentrated suspensions without plasticizer is not possible. For this reason the plasticizers represent a new solution for the preparation of more concentrated suspensions, consequently decreasing the dilution rate of the fermentation broth.

HAAKE viscosity test

Concentration

Concentration of viscosity [mPa*s] at 23°C of the base susplasticizer plasticizer added

pension

[%] Rotor

[%] Viscosity

Rotor speed [mPas]

[rpm]

Mg(OH) ₂

None 30 0.0 7200 R2 4

None 44 0.0 * * *

Poly PPE 160 > 30 0.3 26 R2 200

Poly PPE 160 44 0.3 55 R2 200

Poly PPE 160 65 0.3 48000 R5 5

MVA 2500 L1 ¹ » 30 0.3 26 R2 200

MVA 2500 L 44 0.3 49 R2 200

MVA 2500 L 65 0.3 2740 R5 20

VP EPPR 312 U> 30 0.3 26 R2 200

VP EPPR 312 L 44 0.3 51 R2 200

VP EPPR 312 L 65 0.3 2980 R2 12

MgC0 ₃

None 20 0.0 4100 R2 6

None 25 0.0 * * *

Poly PPE 160 20 0.3 63 R2 200

Poly PPE 160 25 0.3 187 R2 100

Poly PPE 160 30 0.3 1610 R2 20

MVA 2500 L 20 0.3 58 R2 200

MVA 2500 L 25 . 0.3 143 R2 100

MVA 2500 L 30 0.3 840 R2 30

VP EPPR 312 L 20 0.3 63 R2 200

VP EPPR 312 L 25 0.3 155 R2 100

VP EPPR 312 L 30 0.3 750 R2 30

Ca(OH) ₂

None 39 0.0 1400 R2 2.5

None 47 0.0 * * *

Poly PPE 160 39 0.3 120 R2 200

Poly PPE 160 47 0.3 300 R2 100

Poly PPE 160 55 0.6 360 R2 100

MVA 2500 L 39 0.3 80 R2 200

MVA 2500 L 47 0.3 180 R2 00 MVA 2500 L 55 0.3 380 R2 60

VP EPPR 312 L 39 0.3 65 R2 200

VP EPPPv 312 L 47 0.3 150 R2 200

VP EPPR 312 L 55 0.6 26000 R2 1

CaCC

None 33 0.0 1 1200 R2 3

None 44 0.0 * _* *

Poly PPE 160 33 0.3 28 R2 200

Poly PPE 160 44 0.3 51 R2 200

Poly PPE 160 65 0.3 950 R2 20

MVA 2500 L 33 0.3 28 R2 200

MVA 2500 L 44 0.3 53 R2 200

MVA 2500 L 65 0.3 2500 R2 12

VP EPPR 312 L 33 0.3 29 R2 200

VP EPPR 312 L 44 0.3 54 R2 200

VP EPPR 312 L 62.5 0.32 2100 R2 12

Table 1 : Concentration of suspensions prepared with the plasticizers and their viscosities

"Viscosity is too high and not possible to be measured. At these concentrations, homogeniza- tion is very difficult and the suspension is not liquid.

> The polymers are commercially available from BASF SE, Germany

Example 2: Determination of viscosity of Mg(OH) ₂ with different polymers

The viscosity of the Mg(OH)2 suspension was also tested with additional polymers. The results are shown in Table 1. The addition of polymers to the Mg(OH)2 suspension decreased the vis- cosity.

HAAKE viscosity test

Concen¬

Concen-tration of viscosity [mPaxs] at 23°C tration of the base

plasticizer plasticizer added Rotor suspension Viscosity

[%] Rotor speed

[%1 [mPas]

[rpm]

None 25 0.0 1620 R2 4

4 and

MVA 2062LD 50 0.1 0 R2

30

4 and

MVA 2510U ⁾ 50 0.1 0 R2

30

MVA 1319L ¹ > 50 0.1 682 R2 4

MVA 1319L 50 0.1 391 R2 30

MVA 2425LJ ⁾ 50 0.1 2112 R2 4

MVA 2425L 50 0.1 1131 R2 30 4 and

MVA 3164LD 50 0.1 0 R2

30

MVA 2321 L ¹⁾ 50 0.1 1054 R2 4

MVA 2321 L 50 0.1 605 R2 30

4 and

MVA 0417 ¹⁾ 50 0.1 0 R2

30

MVA 2454L ¹⁾ 50 0.1 1333 R2 4

MVA 2454L 50 0.1 781 R2 30

MVA 6016U ⁾ 50 0.1 750 R2 4

MVA 6016L 50 0.1 389 R2 30

Poly PPE 160 60 0.3 350 R2 50

MVA 5025 L ¹ > 60 0.3 570 R2 50

MVA 2500 L 60 0.3 300 R2 50

VP EPPR 312 L 60 0.3 600 R2 50

MVA 2062L ¹⁾ 60 0.3 270 R2 50

MVA 2510L 60 0.3 320 R2 50

MVA 1319L 60 0.3 720 R2 50

MVA 2425L 60 0.3 630 R2 50

MVA 3 64L 60 0.3 320 R2 50

MVA 2321 L 60 0.3 630 R2 50

MVA 0417 60 0.3 320 R2 50

MVA 2454L 60 0.3 660 R2 50

MVA 6016L 60 0.3 570 R2 50

MVA 2062L 65 0.3 2110 R5 20

MVA 2510L 65 0.3 5650 R5 20

MVA 3164L 65 0.3 1980 R5 20

MVA 0417 65 0.3 1720 R5 20

Poly PPE 160 64.3 0.6 1304 R2 4

MVA 2500 L 64.6 0.6 1820 R2 4

MVA 2062L 64.6 0.6 1736 R2 4

MVA 2510L 64.6 0.6 2180 R2 12

MVA 3164L 64.6 0.6 1720 R2 4

MVA 0417 64.6 0.6 1430 R2 4

4, 30

MVA 5025 L 25 0.3 0 R2

and 50

4, 30

MVA 5025 L 30 0.3 0 R2

and 50

4, 30

MVA 5025 L 50 0.3 0 R2

and 50

4 and

MVA 5025 L 55 0.3 0 R2

30 MVA 5025 L 55 0.3 90 R2 50

MVA 5025 L 60 0.3 410 R2 4

MVA 5025 L 60 0.3 370 R2 30

MVA 5025 L 60 0.3 300 R2 50

MVA 5025 L 65 0.3 1910 R2 4

MVA 5025 L 65 0.3 1180 R2 30

MVA 5025 L 65 0.3 870 R2 50

Table 2: Concentration of Mg(OH)2 suspension prepared with different plasticizers and their viscosities

¹⁾ The polymers are commercially available from BASF SE, Germany

Example 3: Comparison between succinic acid titer of fermentations using Mg(OH)2 25% and 50% as base

The comparison between fermentations using Mg(OH)2 25% and 50% was done using the me- dia and fermentation parameters described below. d) Medium preparation

The composition and preparation of the cultivation medium used for seed culture is as de- scribed in the following table 3 and 4. For the main culture in the fermenter a medium is used as described in table 5.

3: Medium composition for cultivation of the seed culture in the first step

trace metals table 7

Osmolytes table 8

Table 4: Medium composition for cultivation of the seed culture in the second step

Table 5: Medium composition for the main culture in fermenters

Table

Table 7: Trace elements added in the defined lean medium Compound Concentration in the medium (mM)

Prolin 1

Betain 1

Carnitin 1

Glutamate 1

(2-carboxyethyl)dimethylsuifonium chloride 1

Table 8: Osmolytes used in the second step seed culture and in the main culture e) Cultivations

For the culture experiments, Basfia succiniciproducens DD1 AldhAApfID cells have been used which correspond to the Basfia succiniciproducens cells "LU 15224" as disclosed in WO-A- 2010/092155. The main culture is inoculated from a seed train consisting of two seed cultures. For the first seed culture 2.5% of cryo stocks was inoculated in a 100 ml-serum bottle with gas tight butyl rubber stopper containing 50 ml of the liquid medium described in Table 3 with a CO2 atmosphere with 0.8 bar overpressure. The starting pH of the medium is in the range of 7.5 to 8 due to the gCC>3 used as base and due to the CO2 gassing. The incubation was overnight at 37°C under anaerobic conditions. This first seed culture was used to inoculate the second one to OD600 = 0.75 in a 100 ml-serum bottle with gas tight butyl rubber stopper containing 50 ml of the liquid medium described in table 4. C0 ₂ atmosphere with 0.8 bar overpressure was also applied and the starting pH is in the range of 7.5 to 8. The bottles were incubated at 37°C, and 160 rpm (shaking diameter: 2.5 cm).

For growing the main culture bacteria, the second seed culture (incubated overnight at 37°C under anaerobic conditions) was used as inoculum in a concentration of 10%. The medium used is described in Table 5. The fermentation was done in 7L fermenters containing an initial volume of 3L. Glycerol and glucose were used as carbon sources and were provided by both batch and feed. The base utilized was magnesium hydroxide 25% or a higher concentration (50% or 62%) containing the plasticizer MV5025L and the pH was kept constant at 6.5. CC½ was applied in the fermenter at flow of 0.1 vvm and the steering rate is 500 rpm. The feeding rates were 0.67 g/(Lh) of glucose and between 2.5 - 5 g/(Lh) for glycerol.

The analytics of the seed culture and the main culture are described in the next section. f) Analytics

The production of succinic acid was quantified via HPLC (HPLC methods are described in Table 9) after 24h. Cell growth was measured by measuring the absorbance at 600nm (OD600) using a spectrophotometer (Ultrospec3000, Amersham Biosciences, Uppsala Sweden).

Table 9: HPLC method (2X-THF50) for analysis of glucose, succinic acid, formic acid, lactic acid, acetic acid, pyruvic acid and ethanol g) Results

The titers of succinic acid obtained with g(OH) ₂ suspensions 25% and 50% are shown in Ta- b!e 10. The results show that the concentration of the base suspension has a direct impact in the succinic acid titer. Higher titers are achieved when more concentrated suspensions of bases are used.

Table 10: Influence of the base concentration on the succinic acid titer

Example 4: Comparison between succinic acid titer of fermentations using base 25% or 62%

The comparison between the main cultures with 25% and 62% bases were done as described previously for example 3. h) Results

The succinic acid titer with 25% and 62% Mg(OH) ₂ are shown in Table 11. The succinic acid titer in the fermentations with higher concentrations of Mg(OH)2 was higher than with lower con- centrations. The result shows that higher base concentrations provide fermentations with higher succinic acid titers.

Table 1 1 : influence of the base concentration on the succinic acid titer

Example 5: Growth of different microorganisms in the presence of the different classes of plasti- cizers i) Cultivations and analytics

For the culture experiments, Aspergillus niduians, Saccharomyces cerevisiae and Lactobacillus case/were used as examples to test if the different classes of polymers used in this study would be toxic for these organisms. We have already shown that Basfia succiniciproducens can grow and produce succinic acid in the presence of the polymers.

For the cultivation of A. niduians, 100 ml_ of potato dextrose broth was used in 500 mL shaking flasks. Three different classes of polymers (MVA2500L, Poly PPE160 and EPPR312L) were tested and compared with a control without polymer addition. The polymers were autoclaved separated and added in a sterile way to the medium right before inoculation. 0.5g/L and 1g/L of the polymers were tested. The cultures were incubated at 27°C with agitation of 170 rpm. The visual growth was observed every day for a period of 3 days.

Yeast Extract Peptone Dextrose (YEPD) broth and MRS broth were used for S, cerevisiae and L casei respectively. 500 mL shaking flasks containing 100 mL of each medium with or without polymers were used. The cultivations were performed in Three different classes of polymers (MVA2500L, Poly PPE160 and EPPR312L) were tested and compared with a controi without polymer addition. The polymers were autoclaved separated and added in a sterile way to the medium right before inoculation. Q.5g/L and 1 g/L of the polymers were tested. The cultures were incubated at 30°C with agitation of 70 rpm. The visual growth of the cultures was ob- served every day for a period of 3 days. The growth of the cultures of S. cerevisiae and L casei were also determined after 3 days of incubation by measuring the absorbance at 600nm (OD600) using a spectrophotometer (Ultrospec3000, Amersham Biosciences, Uppsala Sweden). j) Results The growth of Aspergillus nidulans, Saccharomyces cerevisiae and Lactobacillus case/were not affected by the addition of the different classes of polymers to the cultivation nnedia as shown in table 12. The concentrations used in this experiment are concentrations equal or higher to the final concentrations of these polymers in a fermenter when the polymers are used in the base suspensions. The addition of the different polymers does not affect the growth of A nidulans, S. cerevisiae and L. case/.

Table 12: influence of different classes of polymers on the growth of A nidulans, S. cerevisiae and L. case/. (-): no growth, +: low growth, ++: growth as the control

Example 6: Influence of polymers on the on measured growth of S. cerevisiae and L. case/

The different classes of polymers tested did not influence the growth measured as optical densi- ty of S. cerevisiae and L. case/ (table 13). The results confirm that the polymers tested are not toxic for the cells tested.

Table 13: influence of different classes of polymers on the optical density (OD600) of S. cerevisiae and L. easel