WO2007137809A1 - Minimal medium for microorganisms - Google Patents

Abstract

A minimal, yeast extract-free, liquid medium for the cultivation of microorganisms characterized in that removal of the yeast extract from a standard liquid yeast extract- containing medium is compensated by inorganic ammonium salts and glutamate as N-sources and corresponding method of cultivating microorganisms.

Description

Minimal Medium for Microorganisms

The present invention relates to a new minimal medium for microorganisms. More precisely, the present invention relates to a new minimal, yeast-free, liquid medium for the cultivation of microorganisms.

The commoditization of many bulk fine chemicals demands rapid development of new and low cost processes for their preparation on a commercial scale. Therefore, contemporary manufacturing practices of many fine chemicals are increasingly based on fermentation processes using all kinds of microorganisms due to their economic advantages. However, the potential pitfalls of using fermentation for large volume - low value products, e.g. vitamins, resides in the raw materials and nutritional factors used. For example, in the commercial production of vitamin B2 the raw materials account for about 60% of the overall production costs. 30% of that cost is made up by the complex N-source used: Yeast Extract (YE), which is a component of most "rich" industrial culture media. Replacement of such expensive nutrient would further strengthen the economic viability of fermentation technology. While microorganisms are capable of being cultured on minimal media, viz. media which contain well-defined chemical substances necessary for their metabolism (growth, reproduction, formation of metabolic intermediates and end products) such media are normally enriched with less well-defined N-containing substrates, such as, soy bean flour, corn steep liquor, peptones, meat extract and, preferably, yeast extract, when it comes to commercial processes carried out in large fermenters. However, previous attempts in removing YE from the industrial fermentation media, e.g. in the production of vitamin B2 by Bacillus subtilis, resulted in impaired vitamin production. Mez/ER, 30.04.2007 Similar challenges were expected in the development of a fermentation process for the industrial production of another B vitamin, pantothenate (vitamin B5), using Bacillus subtilis. Undoubtedly, YE provides compounds closely related to those which will ultimately be incorporated in the cellular material, thereby promoting growth. However, its removal from industrial media and replacement by less complex N-sources is desirable when accounting for the larger goal of process profitability on a commercial scale. Furthermore, the substitution of YE by defined N-sources may provide additional benefits in the recovery and purification of the desired product, especially when it is meant to be used for food application purposes, since a cleaner fermentation broth is obtained, without additional fiber or non-metabolized compounds that are present when complex N-sources are used.

Therefore, attempts have been made to solve this problem, viz. to remove YE from fermentation media, especially in commercial productions, without affecting negatively the production of a desired product, e.g., a vitamin.

In accordance with the present invention a solution of this problem has been found. A new minimal, yeast-free, liquid medium for the cultivation of microorganisms has been developed which medium is characterized in that removal of the yeast extract from a standard yeast extract-containing liquid medium is compensated by inorganic ammonium salts and glutamate as N-source. In a further embodiment the present invention relates to a method for cultivating microorganisms characterized in that such a minimal medium is used.

In a preferred embodiment of the present invention the inorganic ammonium salts are (NH₄)₂HPO₄, (NH₄)₂SO₄ and/or NH₄Cl, particularly (NH₄)₂HPO₄ and (NH₄)₂SO₄. The glutamate can be glutamic acid or a physiologically acceptable salt thereof, preferably a sodium salt. In a particularly preferred embodiment of the present invention the (NH₄)₂HPO₄ and (NH₄)₂ SO₄ and Na-glutamate concentrations (g/1, ± 25%) are 3.33, 2.22 and 6.66, respectively. In a further particularly preferred embodiment the concentrations (g/1, ± 25%) of other phosphates are: KH₂PO₄ 4.0; K₂HPO₄ 4,0 and Na₂HPO₄ x 12H₂O 1 1.1. Description of the Figures:

Figure 1 shows the growth profile of B. subtilis Pa 49 as a function of the nitrogen source in shake flash cultivations.

Figure 2 shows conversion efficiency indexes as a function of the N-source used. Y p/s

(yield of pantothenate on glucose; open circles) and YX/NH4 (yield of biomass on the NH₄-content of the N-source; closed circles).

Figure 3 shows biomass (μ; open circles) and pantothenate specific production rates (qp_/s; closed circles) as a function of the N-user applied, qp_/s units are given in mg/g/h.

Figure 4 shows glucose feeding profiles of fed-batch cultivations using complex (CM) and minimum medium (MM2).

Figure 5 shows time-course profiles of Biomass development as function of complex (CM) and minimum (MM2) medium formulations using fed-batch cultivations.

Figure 6 shows comparative time-course profiles of pantothenate production using complex medium (CM) and minimum medium (MM) formulations.

Figure 7 shows qp_/χ->μ relationship for B. subtillis PA49 using complex (CM) and defined (MM2) N-sources. Feeding profile in MM2 was adjusted to compensate for the slower growth in the batch phase

Figure 8 shows the result of a chemostat experiment for Ca-pantothenate production performed with complex and minimum medium (as described below) at dilution rate (D = 0.5 h^"1) and feeding solution containing 100 g/1 glucose. It shows clearly that complex medium restricts the synthesis of pantothenate as well as the conversion efficiency of glucose into pantothenate when Bacillus subtilis PA49 is used. With the minimum medium more than 3 times higher pantoate synthesis could be achieved. The terms "minimal" and "rich" or "complex" in connection with media are used herein in the sense normally used by the person skilled in the art; particularly they mean "without" and "with YE. hi a preferred embodiment of the present invention the minimal medium of the present invention is a medium for industrial scale fermentation.

The wording "removal of yeast extract from a standard liquid yeast extract-containing medium is compensated by unorganic ammonium salts and glutamate as N-source" means that the minimal medium (MM) of the present invention does not contain YE. With respect to its preparation it does not mean that the new MM is prepared by removing YE from a rich or complex medium (which may be difficult if not impossible to achieve). The new medium is prepared from its components in a manner known per se by a person skilled in the art.

To satisfy the demands of the microorganisms the normally used carbon sources, iron and magnesium salts as well as trace elements are components of the liquid culture medium. These can be sugars and carbohydrates such as glucose, saccharose, lactose, fructose, maltose, molasses, starch and cellulose; oils and fats such as soy oil, sunflower oil, peanut oil, coconut fat, fatty acids such as palmitic acid, stearic acid and linoleic acid or other organic acids such as acetic and citric acid, and alcohols such as ethanol or glycerol. The normally required metal sources comprise, e.g., magnesium sulphate, iron sulphate, iron chloride, sodium molybdate, boric acid, cobalt chloride, copper sulphate, manganese chloride and zinc sulphate.

The term "microorganism" comprises naturally occurring as well as genetically engineered Gram positive and Gram negative eubacteria and fungi. Examples of Gram positive eubacteria are organisms from the genera Bacillus (e.g. B. subtilis, lentimorbus, lentus, firmus, pantothenicus, amyloliquefaciens, cereus, circulans, coagulans, licheniformis, megaterium, pumilus, thuringiensis, brevis and stearothermophilus), Lactobacillus, Clostridium and Corynebacterium (e.g. C. glutamicum). Examples of Gram negative eubacteria are organisms from the genera Salmonella (e.g. S. typhimurium), Escherichia (e.g. E. coli), Paracoccus (e.g. P. zeaxanthinifaciens), Rhodobacter (e.g. R. sphaeroides), Gluconobacter (G. oxydans), Klebsiella, Serratia and Proteus. An example of the fungi group is Saccharomyces (e.g. S. cerevisiae). The preferred microorganism is B. subtilis.

The term "culturing" or "cultivating" means maintaining and/or growing living microorganisms in a liquid medium including the process of their producing metabolites (particularly endogenous and secondary metabolites). The metabolites comprise the "chemical compounds of interest", viz. compounds which under the culturing conditions are overproduced by the microorganism.

The microorganisms can be cultivated in accordance with methods described in textbooks and well-known to the person skilled in the art, i.e., continuously or discontinuously by the batch or feed batch or repeated feed batch method.

The term "overproducing" is well-known in the art and to be interpreted accordingly. To achieve overproduction of a metabolite or chemical compound of interest a number of different measures exist, e.g., increasing the number of copies of the corresponding genes, increasing the strength of promoters, introducing stronger promoters or extending the life of mRN A. Overproducing also comprises production of a foreign chemical compound by a microorganism, i.e. a compound which it does not produce normally but upon genetic engineering.

Any chemical compound which has a commercially interesting utility or is an intermediate for such a compound, e.g., a pharmaceutical or cosmetic product or a dairy supplement, can be "a chemical compound of interest". Examples, without limitation, of such products are vitamins (such as ascorbic acid, riboflavin, phylloquinones), carotenoids (astaxanthin, zeaxanthin), ubiquinones (CoQlO) and panto compounds, which latter group is of particular interest. The term panto compound includes a compound (a substrate, intermediate or product) in the pantothenate biosynthetic pathway which is downstream from a particular pantothenate biosynthetic enzyme. Therefore, each of ketopantoate/ketopantoic acid, pantoate/pantoic acid, beta-alanine and pantothenate/pantothenic acid are panto compounds. The cultivation process can be carried out in the usual way with control of pH (normally in the range of 6 - 8), temperature (normally approximately 20 - 50⁰C, preferably 25 - 45°C), nutrients and duration (normally 10 - 160 hours).

The cultivation of the microorganisms by using the novel minimal medium can be effected in different ways which are well known per se. For example batch, fed-batch and continuous processes or methods of fermentation can be used. "Batch process" or "batch fermentation" refers to a system in which the composition of the media, nutrients, supplemental additives and the like is set at the beginning of the fermentation and not subject to alteration during the fermentation, however attempts may be made to control such factors as pH and oxygen concentration to prevent growth and/or production deterioration. "Fed-batch process" or "fed-batch fermentation" refers to a batch fermentation with the exception that one or more substrates (e.g., C-source, minimum medium components) or supplements are added (e.g., added in increments or continuously) as the fermentation develops. "Continuous fermentation" refers to a system in which a defined fermentation medium is added continuously to a fermentor and an equal amount of used medium is simultaneously removed, preferably for recovery of the desired product. A variety of such processes have been developed and are well known in the art.

A particularly preferred embodiment of the present invention is the manufacture of pantothenate by B.subtilis. hi the following Example the present invention is illustrated in more detail wherein B. subtilis PA49 is used, a genetically engineered, pantothenate overproducing strain, described in WO 2004/113510.

Many other specific genetically engineered and transformed microorganisms are described in the literature which are capable of overproducing pantothenate which can be cultured in the present minimal medium. Examples of publications of such microorganisms in the patent literature are:

WO 97/10340, EP 1 001 027, EP 1 006 189, WO 01/21772, WO 01/92556, EP 1 167 520, WO 02/24936, WO 02/29020, WO 02/055711, WO 02/057474, WO 02/057476, WO 02/061 108, WO 02/064806, WO 02/072838, WO 02/072840, WO 02/072854, WO 02/072855, EP 1 247 868, WO 03/004672, WO 03/006664, DE 102 01 540 Al, WO 03/029476, WO 2004/005525, and WO 2004/005527. E x a m p 1 e

1. Materials and Methods

1.1 Strain Bacillus subtilis overproducer of pantothenate strain PA49 (Pi₂panBCD Pi₅PanE) was used.

1.2 Minimal Medium (MMl; g/1)

Glucose 20; Na-glutamate, 0.6; KH₂PO₄, 4; K₂HPO₄, 4; Na₂HPO₄- 12H₂O, 7; Antifoam Basildon, 0.2; trace elements as described in Section 1.3.

1.3 Culture Media used as a Control

The composition of VYS medium is as follows (g/1): Veal infusion broth, 30; Yeast extract, 5; Sorbitol, 10; K₂HPO₄ 2.5. VYS medium was used in the first stage of the inoculum.

In the second stage inoculum and in the main culture the following media formulation was applied (g/1): glucose, 27.3; Yeast extract, 12; Na-glutamate, 0.75; KH₂PO₄ , 4.71 ;

K₂HPO₄, 4.71 ; Na₂HPO₄- 12H₂O, 8.23; NH₄Cl, 0.23; (NH₄)₂SO₄, 1.41, Antifoam Basildon, 0.2; MnSO₄ H₂O, 0.017; CoCl₂-O H₂O, 0.005; (NH₄)₆Mo₇O₂₄ 4H₂O, 0.0018; AlCl₃ 6

H₂O, 0.0012; CuCl₂-2H₂O, 0.0009; MgSO₄ 7H₂O, 1.2; ZnSO₄ 7 H₂O, 0.005; CaCl₂ 2 H₂O,

0.0625; FeSO₄ 7H₂O, 0.05. Glucose and the trace elements were sterilized separately.

The basal feeding solution contains (g/1): Glucose, -750-800; MgSO₄ 7 H₂O, 2;

MnSO₄ H₂O, 0.015; ZnSO₄ 7 H₂O, 0.004.

J solution is as follows (g/1): KH₂PO₄, 90; K₂HPO₄, 90; Na₂HPO₄- 12H₂O, 164; NH₄Cl, 5;

(NH₄)₂SO₄, 28, and a mixture of trace elements similar to that used in the main culture.

1.4 Inoculation Procedure Inoculum preparation for PA49 was carried out in two stages. In the first stage 1 ml stock culture, previously prepared and preserved at -25°C, was inoculated into 25 ml VYS broth supplemented with tetracycline (15 μg/ml) in a 100 ml Erlenmeyer flask, which was then incubated for 7 h at 39⁰C on a rotary shaker at 200 rpm. hi the second stage. 3 ml of this culture (1 % v/v) was transferred to 300 ml of the production medium containing 15ug/ml chloramphenicol. This second pre-culture was carried out in a 2 L Erlenmeyer flask and was incubated again at 39°C for 16 ± Ih, time at which an OD > 14 was achieved. For the final stage, the content of such flask was transferred aseptically to the stirrer tank reactor (fermenter) to give approximately 5% w/w inoculum concentration.

1.5 Shake Flasks Cultivations for N-screening

Nitrogen screening was carried out in 2 L Erlenmeyer Flasks containing 300 ml of reaction volume. For this particular set of experiments the oversimplified minimum medium (MMl) was used. Glucose and trace elements were sterilized separately. This medium was supplemented with the N-source in question to a final concentration of 3 g/1. To this end stock solution of (NHU)₂HPO₄, Na-glutamate, NH₄Cl, (NH₄)₂SO₄ and YE were prepared separately. MMl was also used in the second stage of the innoculum development, which procedure was similar to that of section 1.4. Experiments were performed in duplicate for each type of nitrogen source. After addition of 6 ml of exponentially grown cells to give approximately 2% inoculum concentration, the flasks were incubated at 39°C in a rotary shaker with a speed of 200 rpm for 24h. Periodically, 1 ml sample was taken to follow-up the major metabolic events.

1.6 Reactor and Operating Conditions Fermentations were carried out in either 20 L New Brunswick or 15 L stirred tank reactor Fa. Braun (Germany). The starting volume was at 6 L while the maximum volume depends upon the experiment in question. The temperature was controlled at 39°C, and the pH was kept at 6.8 ± 0.1 by controlled addition Of NH₄OH 27%. Air was sparged at > 1 wm (volume/volume/minute), as required. The dissolved oxygen partial pressure (pθ₂) was measured with polarographic oxygen probes. A back pressure of 500 mbar was applied. The initial agitation speed was set at 400 rpm with stepwise increase as required by the pθ₂ control.

The pθ₂ was controlled at values > 15% by a combination of aeration and agitation using a PID controller action. This controller has one inner loop (a slave) involving the measurement of agitation speed and an outer loop (the master) involving the pθ₂ measurement. The output of the pθ₂ loop controller serves as the set point for the agitation loop. Once the agitation speed reached the upper limit (1000 ± 50 rpm), the control is switched to the aeration loop. The aeration flow was adjusted in a stepwise form from 6 NL/min to 12 NL/min as the lower limit of pθ₂ was approached.

1.7 Determination of Pantothenate and Pantothenate-Related Metabolites Chromatography of samples was performed on a Phenomenex LUNA C8 column, using an Agilent 1100 HPLC system equipped with a thermostatted autosampler and a diode array detector. The column dimensions are 150 x 4.6 mm, particle size 5 micron. The column temperature was kept constant at 20°C. The mobile phase is a mixture of 0.1% acetic acid (A) and methanol (B). Gradient elution is applied, ranging from 1% B to 45% B in 15 minutes. The flow rate is 1 ml/min. Pantothenate was monitored using UV absorption at 220 nm, and is eluted at approximately 9.6 min. The calibration range of the method is from 1 to 100 mg/1 pantothenate. Other pantothenate related metabolites are also separated and measured by this method.

1.8 Cell Dry Weight

Cell dry weight concentration was determined in duplicate using gravimetric methods. The culture broth was diluted as required and then centrifuged in 2 mL Eppendorf tubes at 14000 rpm at 4°C for 10 min to settle cells. The supernatant was decanted and saved for other analytical determinations. The cell pellet was washed and then centrifuged again under the same conditions described for the sample centrifugation. After this second centrifugation the washing water was discarded and the cells pellet was dried under vacuum at 40°C and weighed.

1.9 Determination of Ammonium The determination of ammonium ions was done with an automatic analyser 'Vitros DT II' that works with special coated reaction slides. The slides are multilayered and contain immobilized/coated analytical elements on a plastic support. 10 μL of sample containing ammonium in the range of 1-500 μMol was deposited on the slide and evenly distributed by the spreading layer. The ammonia in the sample then diffuses through a semi-permeable membrane into a layer containing bromphenol blue as the indicator dye. By measuring the amount of light reflected from the dyed layer after a fixed incubation period, the analyzer calculates the amount of ammonia present in the sample. 1.10 Abbreviations

F [g/h] Flow t [ h ] Time

S [ g/i] Substrate (glucose)

N [ g/i] N-source substrate

Y [ - ] Yield (conversion efficiency)

P [ g/i] Product (Pantothenate)

X [ g/i] Biomass

Pv [ g/l/h] Volumetric productivity

C [ - ] Carbon source

N [ - ] Nitrogen source

OD [ - ] Optical Density

DCW [ g/i] Dry Cell weight (Biomass)

CM [ - ] Complex medium

MM [ - ] Minimum Medium (defined N-source)

V [ L ] Volume

YE [ - ] Yeast Extract μ Specific growth rate

Yp/s [ - ] Product Yield (conversion efficiency of glucose into product)

Yχ/s [ - ] Biomass Yield (conversion efficiency of glucose into biomass)

Yχ/NH4 [ - ] Biomass Yield (conversion efficiency of ammonia into biomass) qp/x [g/g/h] Specific productivity (g product/g biomass/h) qs/x [g/g/h] Specific substrate uptake rate (g substrate/g biomass/h)

PID [ - ] Partial Integral Derivative

2. Results

2.1 N-screening

Different N-sources were assessed on their role as N-provider for biomass production and pantothenate synthesis by Bacillus subtilis PA49. To this end MMl medium (Section 2.4) containing 20 g/1 glucose was applied with several single N-sources. (NH₄)₂HPO₄, Na- Glutamate, NH₄Cl, (NH₄)₂SO₄ and Yeast extract (YE) were evaluated independently at constant initial concentration (3 g/1). The selection of glutamate as the only single amino acid N-source evaluated here was due mainly to its low cost in the market and its nutritional link between carbon and nitrogen metabolism. Figure 1 summarizes the time- course profiles of bacterial growth as a function of the N-source used, while Figures 2 and 3 depict the average growth and production indicators such as yields and specific rates.

As shown in Figure 1, yeast extract (YE) and glutamate promoted the faster growth as compared to the ammonium salt forms; however that later group of N-sources resulted in higher cell densities. The clear differences in final cell density should be considered as a consequence of the N content rather than conversion efficiencies. The N-content of both, glutamate and YE is less than half of the N-charge in the salt forms [(NH-O₂HPO₄, NH₄Cl, and (NH₄)₂SO₄]. Consequently, the Yχ/N_H4 instead of Yp/s was considered as the biomass yield indicator. It is worth noting that glutamate showed the highest N-conversion into biomass Y_X/NH4 (Fig- 2), which was -26% higher than YE and on average 44% better than the ammonium salts.

In terms of pantothenate production, (NH₄)₂HPO₄ showed the best performance indicators Yp_/s, qp/_S) and [P]. This may be associated with lower cell density and longer growth phase because of its lower μ, high substrate utilization and a better buffering condition than its other NH₄-SaIt counterparts (see final pH in Tablel). The cultivations containing NH₄Cl, and (NH₄)₂SO₄ showed relatively low pH at the end of the 24h and pantothenate accumulation was -20% lower than those achieved using (NH₄)₂HPO₄. In general, all ammonium salts showed superior performance as compared to YE (Table 1, Fig.3). Although cells grown with glutamate showed lower cell density and hence slower glucose uptake, interestingly this condition also showed a pantothenate production rate (qp/s) comparable to that of the ammonium salts (Fig. 3). Table 1. Cultivation of B. subtilis PA49 in different N-sources: growth and production indexes. YE refer to yeast extract based medium.

[X] [P] [S (M] Yp/s ^X/S ^X/NH4 PH

(g/i) (g/i) (g/i) (-) (") (-) (-)

(NH₄J₂HPO₄ 3.0 1.0 0 0.049 0.15 3.66 6

Na-glutamate 1.9 0.5 2.5 0.024 0.11 6.60 6.4

NH₄CI 3.5 0.8 0 0.039 0.17 3.44 5.2

(NHJ₂SO₄ 3.3 0.8 0 0.038 0.16 3.28 5.4

When comparing Yp_/s with Yχ_/NH4 (Fig. 2) for each nitrogen source a clear inverse relationship can be observed, which confirms that biomass and pantothenate biosynthesis compete for the same C-source and intermediates from the central metabolism. The data in Fig. 2 together with Fig. 3 suggest interesting synergies, for instance a mixture of glutamate and NH₄-salts might in principle improve the low Yp/s of glutamate while providing high production rates, among a reasonable growth rate.

As judged by the results in these experiments, media formulations based on defined single N-source is able to support growth and pantothenate production. Under given conditions, ammonium salts like (NH₄)₂HPO₄ showed superior production performance as compared to complex medium based on YE. Glutamate seems to present interesting options for minimum media formulation: it boosted growth up to a similar range as YE, showed the highest Y_X/NH4 among the N-sources tested, and provided reasonable pantothenate production rate (qp_/s). Combinations of these chemically well-defined N-sources would be an effective alternative to YE.

2.2 Minimal Medium MM2 Formulation

Based on the above result and starting from the commercial reference medium of section 1.3, an alternative minimum medium (MM2) was formulated according to the following procedure: > A breakdown of the main media components of complex medium containing YE into their respective ions (Table T). The NH4-content of YE was calculated based on the N-content resulting from all 18 amino acids known to be present in this complex N- source. The other elements are at negligible levels and hence not included in the overall balance.

> YE was then completely replaced by (NH₄)₂HPO₄, Na-glutamate, and (NtU)₂SO₄.

> With the introduction of the above N-sources the concentrations of the other media components were adjusted accordingly, so as to be able to manage overall ions balance similar to that of CM medium (compare Table 3 with Table 2).

Table 2. Key media components of complex medium (CM) for B. subtilis. Trace elements and C-source as indicated in section 1.3

B2 Complex Medium

(CM) g/i NH4 PO4 Na SO4 K Cl

Yeast Extract 12 1.68

Na-Glutamate 0.75 0.07 0.09

KH₂PO₄ 4.71 3.29 1.35

K₂HPO₄ 4.71 2.57 1.05

Na₂HPO₄- 12 H₂O 8.23 2.18 0.53

NH₄Cl 0.23 0.08 0.15

(NH₄)₂SO₄ 1.41 0.38 1.03

Total 2.21 8.04 0.62 1.03 2.4 0.15

Table 3. Formulation of minimal medium (MM) for Bacillus cultivation. The yeast extract of Table 2 medium was replaced by (NHt)₂HPO,!, Na-glutamate, and increased

elements and C-source were kept the same.

MM2 g/i NH4 PO4 Na SO4 K Cl

(NH4)2HPO4 3.33 0.91 0.91

Na-Glutamate 6.66 0.64 0.82

KH₂PO₄ 4 2.79 1.15

K₂HPO₄ 4 2.18 0.90

Na₂HPO₄ H H₂O 11.1 2.94 0.71

(NH₄J₂SO₄ 2.22 0.61 1.61 2.2 Fed-batch cultivating using B. subtilis PA49

A first attempt to test the operability of MM2 focused on evaluating kinetic relationships that could give a hint of the response of Bacillus subtilis to changes in the N-source under production conditions. To this end two fed-batch fermentations containing MM2 and CM were performed in 20 L New Brunswick stirred tank reactors using the strain PA49. The production conditions were a short initial batch phase (~6h) followed by C-limited fed- batch operation (~42h). Consequently, during the initial stage of cultivation the growth rates are determined by the N-source in use, while the glucose feeding pattern overtakes such control during the fed-batch mode. An important feature of this process is that μ reduces gradually towards the end of the fermentation and that conditions of N-excess are maintained by the pH control.

B. subtilis PA49 showed a particular pattern when grown in MM2. The average specific growth rate (μ) in the batch phase was ~37% lower than in complex medium (0.33 h^"1 in MM2 compared to 0.55 h^'1 in CM). Such a lower value of MM2 was predicted with -94% approximation when applying the kinetic constants found in Section 3.1 (Table 1, Fig. 3). The lower μ resulted not only in slower biomass development but also in slower glucose uptake rate, which prompted the readjustment of the feeding rate in MM2 to avoid glucose accumulation in the culture broth. The feeding pattern was reduced by -40% at the beginning of the fed-batch mode and gradually increased to achieve the same C-charge as provided in CM (Fig. 4). Such adjustment resulted unavoidably in higher μ values in MM2 during the fed-batch mode. The average μ in MM2 was 30% higher than in CM. These growth patterns plus the higher Y_X/NH4 of the defined N-mixture (Table 1) resulted in significantly higher biomass accumulation in MM2 (Fig. 5). For the same C-charge and within the same period of cultivation time MM2 showed to have utilized N- and C-sources more efficiently. The cumulative biomass yields were 0.33 and 0.24 for MM2 and CM, respectively. The total biomass produced was 763g in MM2 and 569g in CM. It should also be stressed that such efficient biomass production demanded high oxygen transfer rates. To maintain the established set-point of pθ₂ > 15%, the stirred speed was doubled. An examination of the dynamics of pantothenate synthesis revealed that MM2 is able to support pantothenate production with performance indexes comparable and slightly better than complex medium. Overall product yield on glucose (Yp_/s), product output, and hence productivity was 10% higher than in CM (Figure 6). Accordingly the harvested fermentation broth contained ~27 and ~31.5 g/1 of pantothenate in CM and MM2 medium, respectively.

MM2 not only promoted good biomass yield but also improved the conversion efficiency of glucose into pantothenate. Further assessment of the qp_/s -> μ relationship revealed interesting insights (Figure 7). It became evident that yeast extract (YE) repressed pantothenate synthesis. At high μ values (mostly batch phase of the process), qp_/s showed values that were as low as one fifth of what was achieved with MM2. As YE is being consumed these values recovers slowly indicating selective use of amino-acids and variable repression power. Liebs et al. (Folia Microbiol. 31, 88-95, 1988) reported hierarchy in the utilization of amino-acids. Glutamate, aspartate, serine, and alanine were depleted from the growth medium at similar rates through exponential growth. The majority of the extracellular arginine, glycine and proline were taken up only during late exponential growth, while histidine, isoleucine, threonine and valine were not used significantly until the onset of the stationary phase.

Once the YE was depleted the kinetics profiles of CM resembled the MM2-grown cells. The other common point is that at this stage of the process both are being supplied with the same N-source, derived from the pH control (Section 1.6).

Based on these results, it is apparent that the only distinguishable benefit of YE is the rapid biomass build-up at the beginning of the fermentation. Such benefit can easily be offset by using either a feeding approach as above or a higher seed density. Alternatively, MM2 could be provided by external feeding, separately or together with glucose. These are encouraging results, since it releases pressure on the economics of the process by removing an expensive N-source. Moreover, the negative impact of YE on pantothenate synthesis demonstrates that there is no need of complex medium for a commercial pantothenate process, at least with the pantothenate overproducing strains tested. An overall assessment of the results in this study suggests that the believed "benefit" of complex medium in vitamins production should be taken carefully. Undoubtedly, yeast extract is not necessary for pantothenate production and this work managed to remove it from media formulation.

Claims

Claims

1. A minimal, yeast extract-free, liquid medium for the cultivation of microorganisms characterized in that removal of the yeast extract from a standard liquid yeast extract- containing medium is compensated by inorganic ammonium salts and glutamate as N- source.

2. A minimal medium as claimed in claim 1 wherein the inorganic ammonium salts are (NH₄)₂HPO₄, (NH₄)₂SO₄ and/or NH₄Cl.

3. A minimal medium as claimed in claim 1 or claim 2 wherein the inorganic ammonium salts are (NH₄)₂HPO₄ and (NH₄)₂SO₄.

4. A minimal medium as claimed in any one of claims 1-3 wherein the phosphate, sulfate and glutamate concentrations (g/1, ±25%) have the following values:

(NH₄)₂ HPO₄ 3.33

(NH₄)₂ SO₄ 2.22 Na-glutamate 6.66

5. A minimal medium as claimed in any one of claims 1-4, characterized in that the microorganism is a microorganism capable of overproducing a chemical compound of interest.

6. A minimal medium as claimed in any one of claims 1-5, wherein the microorganisms is a member of the genus Bacillus.

7. A minimal medium as claimed in any one of claims 1-6, wherein the microorganism is Bacillus subtilis.

8. A minimal medium as claimed in any one of claims 1-7, wherein the microorganism overproduces a product of interest which is a normal product of its biochemical pathway.

9. A minimal medium as claimed in any one of claims 1-7, wherein the microorganism overproduces a panto compound.

10. A minimal medium as claimed in any one of claims 1-7, wherein the microorganism overproduces pantothenic acid.

1 1. A minimal medium as claimed in any one of claims 1-10 for the cultivation of microorganisms on industrial scale.

12. A method for cultivating microorganisms characterized in that a minimal medium is used as claimed in any one of claims 1 - 11.

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