DE4317488C2 - Process for the fermentative production of gluconic acid and microorganisms suitable therefor - Google Patents

Description

The invention relates to a method for Pro production of gluconic acid by fermentation in sugar containing aqueous liquid and it includes ge suitable microorganisms.

Gluconic acid is a multifunctional carboxylic acid due to their physiological and chemical properties As acid itself or as a salt, in particular Sodium salt, extensive application finds: It serves both for cleaning purposes and in the pharmaceutical food and beverage industry in particular as a preservative and in the construction industry as Cement additive for increasing breaking strength and Resi Stenz against frost and water.

Gluconic acid can be obtained by chemical synthesis the. Because of the better selectivity, however, the preferring microbial production, for which a number of Microorganisms such as Aspergillus and Penicillium, many Bacteria u. a. Gluconobacter spec., Pseudomonas, Phyto monas, Achromobacter, Klepsiella, Zymomonas mobilis and Acetobacter methanolicus are known. Especially common are Aspergillus niger and Gluconobacter oxidans looking.

It has long been known that yeast-like mushroom kul tures such as B. Aureobasidium pullulans, which by poly merbildungen are characterized, small amounts Glu  form acid (V.V. Pervozvanskii, Microbiology [U.S.S.R.], 8, 149ff. [1939]). However, the previously be Gluconic acid activities were so low that economic production is not was thought.

As from the summary in Ullmann's Encyclopedia of Technical Chemistry of 1989, Vol. A12, Pages 449 et seq., However, for the fer mentative production of gluconic acid practically only Ace pergillus niger (predominantly) or gluconobacter suboxidans although Aspergillus niger because of Handling difficulties due to the risk of clogging on the one hand and activity decline on the other hand for a continuous production is not suitable and Glu conobacter produces a relatively high amount of keto acid, thereby the work-up is difficult.

It has now surprisingly been found that a ge Commercial gluconic acid production with strains of Aureobasidium pullulans (de bary) Arnaud well before Partly because this yeast is amazingly osmotolerant is, so that in economically interesting Konzentra tion areas can be worked, optionally can be produced continuously as is the yeast under usable conditions in continuous culture as a long-term useful Gluconsäurepro doubts proves.

It was further found that of this ubiquitous coming yeast production strains can be isolated high selectivity and product concentration supply at high glucose conversions. requirement for the economically interesting behavior are ins special optimized iron ion and manganese ion content hold, which are surprisingly high.  

The invention based on these findings fiction Appropriate process for the fermentative production of Gluconic acid is therefore by characterized in that

• - from incubation approaches of crushed flowers and stems collected from spring to summer Central European forest and meadow flowers isolable Aureobasidium pullulans (de bary) Arnaud tribes, which
• - In continuous culture of glucose 200 g / l Gluconic acid at 90% conversion and 90% molar Form selectivity,
• in a Fe and Mn optimized medium with a N-dependent iron and manganese ion content in Feed, which at an N content corresponding to 3 g / l NH₄Cl at 0.5 mM Fe and 0.5 mM Mn;
• - at pH values from 4.5 to 8 and cultured at temperatures of 24-32 ° C.

Appropriately, in the main flowering period - especially from March to June - collected flowers and Stem of Central European forest and meadow flowers put in crushed form in a shaker flask and as nutrient medium, an acidic glucose-containing nutrient solution used. Preferably, with pH values around 2.5 worked and for the acidification expediently Citric acid used. The culture fluid should relatively high glucose concentrations, in particular by 100 g / l, and she will especially use one Wetting agents such as Tween 40 added. Because the gluconic acid  forming strains are strictly aerobic, should be for one good oxygenation, especially by rich shake, be taken care of.

In this way, especially in the German collection of microorganisms and cells  deposited tribes with the accession number DSM 7085 isolated until 88, who are able to in Schüttelkol Gluconic acid concentrations of ≳ 100 g / l within of 2 days to produce.

The sugar solution useful for the reaction should Contain or form glucose. So are starch hydrolytes sate, molasses, maltose, etc. usable.

Conveniently, glucose concentrations are around 150 g / l for the gluconic acid production, whereby in the batch process Berei of 20-400 g / l glucose appear useful in the Fed-batch process in the growth phase also Kon can be present around 150 g / l glucose and Values of 100-200 g / l are preferred by insbe especially highly concentrated solution is replenished, which may contain up to 800 g / l of glucose. In the continuous fermentation is with feed concentration worked about 300-600 g / l, and then Sales of 90% and selectivities of 90 mol%, in favorable cases 95 mol% can be achieved.

Preferably, the pH in the fermentation medium kept between 6.0 and 7. The temperature should be suitably in the range of 29-31 ° C.

The appropriate levels of iron, manganese and magne ions in the feed depend on the nitrogen content of the Medium off: in a medium with nitrogen contents ent speaking 3 g / l NH₄Cl is their concentration in particular in the range of 0.5-3 mM (for iron ions), 2.5- 5 mM (for manganese ions) or 1-2 mM (for magnesium ions).

The gluconic acid formation according to the invention can be used as a batch Run or fed-batch process, particularly appropriate However, a continuous fermentation,  especially with residence times of 10-40 hrs.

Important for the production according to the invention is a good supply of oxygen: In a continuous production of dissolved oxygen should linger time dependent z. At 80-150% saturation for 13 hrs. Residence time, with higher residence times hö here oxygen saturations are appropriate.

Although a fermentation in the fluidized bed reactor with supported biomass procedurally appropriate can be a fermentation with unsupported Microorganisms and biomass retention preferred. because at higher dissolved oxygen contents of ≳ 200% Saturation sought.

For the continuous production proves one Cascade-shaped sequence of at least two fermenters as Cheap. It is in the first stage with 120-170% Oxygen saturation and 10-25h, especially 12-18h dwell time and in the second stage with <360% oxygen saturation and a total residence time of 15 h, in particular of 18-30 h expediently worked.

In addition to the already mentioned metal ion contents in Me dium is also the phosphate content of importance: purpose moderately is in the feed for 0.7 g / l KH₂PO₄ (at N- maintained according to 3 g / l NH₄Cl) taken care of. At N content in accordance with 1.5 g / l NH₄Cl is the KH₂PO₄ content in Feed usefully at 0.34-2.4 g / L, in particular at 0.7-2.1 g / l.

Zinc, cobalt, copper, calcium, iodine, chlorine, boric acid and Sulphate and molybdenum are present in the nutrient medium see, as well as vitamins such as thiamine, biotin, panto thenat and nicotinic acid as well as pyridoxine.

By the series connection of at least two Fermenters in which the same reaction conditions prevail, one receives particularly high conversions and Glu  consäureausbeuten. This way, in the first Fer menter achieved a high space-time yield and in the or the subsequent fermenter (s) a maximum order set and a maximum yield, wherein in the 2. High oxygen saturation levels are advantageous.

Other special features arise from the following the description by way of examples with reference on the attached drawings.

The attached drawings serve as an example Illustration of the dependencies of gluconic acid produk tion of individual parameters, and that became special investigated the continuous gluconic acid fermentation using the aureobasidium strain DSM 7085 with the highest activity. Show in detail

Fig. 1 and 2, the pH dependence;

Fig. 3 to 5 the influence of the dissolved oxygen concentration;

FIGS. 6 and 7 the influence of the residence time;

Fig. 8 and 9 tion of Mangankonzentra tion;

Fig. 10 to 13 tion of the Eisenkonzentra tion;

Fig. 14 and 15 the influence of the phosphate content;

Fig. 16 and 17, the influence of the Mg⁺⁺ content or the temperature and

Fig. 18 curves for a fed-batch experiment.

The strain DSM 7085 is identical to the "isolate No. 70 ".

The subsequent experiments were largely at 30 ° C. carried out. However, the organisms are also in the Able to wake up at lower temperatures, below 30 ° C sen.  

example 1 Isolation of Aureobasidium pullulans (de bary) Arnaud

Forest and field flowers were used to isolate the trunks from Jülich, Düsseldorf, Heidelberg and Holland zer cut down and filled in shake flasks, which is a Me contained the following composition:

Glucose | 100 g / l yeast extract 3 g / l citric acid 10 g / l Tween 40th 2 drops / l Autoclaved at 121 ° for 15 minutes

The pH was adjusted to pH 2.5-2.6 with citric acid posed. The Schüttelkolbenansätze were at 30 ° C. a shaker (at 200 rpm) for 1 or 2 days in cubed. Once a cloudiness in the isolation medium was seen, samples were taken with the loop, and diluted samples with the Trikalsky spatula on agar-plat isolated. The agar plates contained one of the Screening media of the following composition:

isolation media I. Medium Glucose | 100 g / l yeast extract 5 g / l MgSO₄ · 7aq 0.5 g / l KH₂PO₄ 1 g / l CaCO₃ 5 g / l or 10 g / l agar 20 g / l Thiamine HCl 0.1 g / l (sterile filtered)

II. Medium Glucose | 100 g / l YBN (yeast base nitrogen) 6.7 g / l (sterile filtered)   CaCO₃ 7 g / l agar 20 g / l

The plates were incubated at 30 ° C for 2-5 days. säu reproducing microorganisms can be easily detected be, because the acid formed dissolves the CaCO₃ and forms a clear Lysehof. The acid-producing Microorganisms were grown in pure culture on the o.a. Medium and then on a strain medium (yeast Malt extract agar for mushrooms and yeasts) isolated and incubated at 30 ° C for 1 or 2 days. Subsequently were the recovered isolates are stored at 4 ° C and added in a medium containing 30% glycerol Frozen at -20 ° C and -80 ° C. The main isolates were lyophilized.

The isolated strains have a characteristic Pink colour. They are strictly aerobic, d. they are not able to grow without oxygen.

colony features

On malt extract medium yeast-like smooth, margin fringed through mycelium; initially pale pink, later in places  dark brown. Colony diameter after 7 days at 25 ° C about 40 mm.

morphology

Hyphen colorless, often septate, initially thin-walled, later in places also dark brown, thick-walled. Koni dienbildende cells undifferentiated, intercalar or terminal. Blastokonidien arise simultaneously in groups, ellipsoid, size very variable (3-7 × 7-16 μm), mostly with short hilum at the attachment site. Partial education of endoconidia (3 × 6 μm).

The isolated microorganisms can in addition to the glucose also awake on maltose as sole carbon source sen. This property can be exploited when using maltose-containing cheap partial starch hydrolysates of In be interesting. On mannitol they grow slowly. you can be easily immobilized on porous sintered glass and offer themselves for the continuous Gluconsäu reproduction with immobilized cells.

Example 2 gluconic acid

The microorganisms isolated as above were used in Shake flask examined. To the medium was CaCO₃ added to neutralize the acid formed. The Screening medium contained the following components:

glucose 100 or 150 g / l NH₄Cl 0.3 g / l MgSO₄ · 7AQ 0.2 g / l KH₂PO₄ 0.3 g / l yeast extract 0.05 g / l Thiamine HCl 0.1 mg / l CaCO₃ 20 or 40 g / l

500 ml shake flask containing 50 ml of the o.g. Fermenta were from fresh cultures of isolated strains inoculated with the loop and 48 Hours at 30 ° C on a shaker 200 rpm incubated.

The acid concentrations were quantified with a HPLC apparatus (RP₁₈ column, 2% acetonitrile and 5 mM TBA as eluent, with 1 ml / min running speed) Room temperature measured. The detection took place with a UV detector at 210 nm.

With the Aureobasidium pullulans isolates could half of 2 days with 0.3 g / l NH₄Cl as a nitrogen source depending on the CaCO₃ concentration used Gluconic acid concentrations between 22 and 140 g / l he be enough. The best results were with 40 g / l CaCO₃ achieved at 150 g / l initial glucose concentration. A selection of the isolates has been deposited under the no. DSM 7085, DSM 7086, DSM 7087 and DSM 7088.

Example 3 pH influence on gluconic acid production with aureobasis dium strains

The isolated strains produce gluconic acid in one pH range between 3.5 and 8, preferably pH Values between 5.5 and 7.5. The PH optimum is included pH 6.5.

The strain DSM 7086 produced intermittently in Small fermenter after 27.5 hours 13.74 g / l at pH 4, 22.63 g / l at pH 5, 42.51 g / l at pH 6 and 67.71 g / l Gluconic acid at pH 7.  

table

pH influence on gluconic acid production with DSM 7086

Example 4 pH influence on the continuous gluconic acid fermentation tion with DSM 7085

The PH optimum for growth and production DSM 7085 was used in continuous chemostat experiments (in the same small fermenter) under otherwise the same Fermentation conditions examined. The residence time was at a constant substrate supply dependent on the ver needed NaOH between 21.86 and 25.51 hours. Around To reach a steady state condition became more needed as five residence times.

fermentation medium Glucose | 360 g / l NH₄Cl 3 g / l (measured 761 mg / l nitrogen) KH₂PO₄ 0.7 g / l MgSO₄ · 7 aq 0.35 g / l CuSO₄ · 5 aq 100 μg / l Na₂MoO₄ · 2 aq 200 μg / l ZnSO₄ · 7 aq 0.01 g / l CoSO₄ · 7 aq 4 mg / l H₃BO₃ 0.04 g / l MnSO₄ · 4a q 5mM FeSO₄ · 7 aq 100 μM CaCl₂ 0.1 g / l NaCl 0.1 g / l KJ 0.1 mg / l citric acid 2.5 g / l Thiamine HCl 2 mg / l biotin 0.25 mg / l Pyridoxine HCl 0.625 mg / l Ca D-pantothenate 0.625 mg / l nicotinic acid 0.5 mg / l temperature 30 ° C pH correction 22.5% NaOH solution NH₄Cl and vitamin solutions were sterile filtered

Gluconic acid can be continuously produced in a pH range between 3.5 and 8 with the strain DSM 7085 ( FIG. 1). The highest space-time yield was achieved at pH 6.5 and was at a residence time of 21.86 hours 11.64 g / l · h. The biomass-specific productivity increased with increasing pH and was at pH 7 2.08 g / g · h. The selectivity (moles of product / mole of converted glucose) was above 90% with the exception of pH 4.5, at pH 5.5 to 6 it was almost 100%. The highest conversion (g glucose / g glucose used) and the highest yield (moles of product / mole of glucose used) were achieved at pH 6.5 ( Figures 1 and 2).

At lower pH's (pH 4.5) the selectivi was efficiency, space-time yield and biomass-specific product very poor, but the highest biomass to observe concentration. The high viscosity of Fer ment solution is based on the formation of polymers recirculate. Aureobasidium strains are known for Pullulan. With pH values above 4.5, the poly be forced back.

Example 5 Oxygen influence on the Gluconsäurefermentation with Aureobasidium strains

The oxygen supply is similar to Aspergillus niger of great importance to the glucon acid fermentation with the isolated strains.

A direct oxygen dependence could at Schüt telcool and small-fermenter experiments become. An isolate of meadow flowers from Holland reached 54.17 g / l gluconic acid in a shake flask without baffles and 97.8 g / l gluconic acid in a shake flask with Schika NEN. The influence of oxygen illustrates the following Table.

Oxygen influence on glucose consumption ventilation Glucose consumption after 48 h (g / l) 1. Shake flasks without baffles 57 2. Shake flasks with baffles 100 3. N₂ (small fermenter) 0.7 4. 0.3 vvm (atmospheric oxygen) 9.8 5. 1.4 vvm (atmospheric oxygen) 22.5

For fermentations with other isolates in Kleinfer menter (with magnetic stirrer) was an oxygen supply with air not sufficient for optimal Gluconsäu reproduction. In the following experiments, the because of working with pure oxygen to the better To simulate oxygen supply in the stirred tank.

Example 6 Oxygen influence on the continuous gluconic acid fermentation with DSM 7085

The influence of oxygen was measured in a stirred tank (Biostat E von Braun-Diessel) in the continuous process with the Production strain DSM 7085 with residence times between 12 and examined in detail for 13 hours. In the various The experiments were the air / oxygen ratio varied. The composition of the fermentation medium except for one increased to 0.5mM Iron concentration and a copper sulfate concentration of 1 mg / L substantially Example 4.

The dissolved oxygen concentration affects both growth ( Figure 3) and gluconic acid production ( Figure 5) and various quantities such as turnover, space-time yield, biomass-specific productivity ( Figure 4), selectivity and yield.

With DSM 7085, gluconic acid can be between 40 and 360% Dissolved oxygen will be produced (100% equivalent Oxygen saturation with atmospheric oxygen). The bio Mass-specific productivity rose linearly with stei gender dissolved oxygen concentration and was at a residence time of 13.29 hours and dissolved acid substance concentration of 360% 3.2 g / g · h.

The growth increases in a range between 39 and 77% and decreases again at higher dissolved oxygen concentrations. A correlation is observed in the CO₂ of the exhaust air. The optical density does not correlate with the biomass concentration and decreases linearly from 39% to 360% when the oxygen concentration in the fermenter is increased. This is due to pelletization (lower optical density) at higher oxygen concentrations and may have a protective function ( Figure 3).

The space-time yield reached at 122% dissolved oxygen with a residence time of 12.71 hours, their maximum with 16.57 g / l · h ( Figure 4). The selectivity was over 90% in the total dissolved oxygen range. With these short residence times over 200 g / l gluconic acid could be produced continuously. The use of higher glucose concentrations and biomass retention with microfiltration allows maximizing gluconic acid concentration with short residence times at high dissolved oxygen concentrations. An alternative is to cultivate the microorganism in a small fermenter at optimum growth oxygen concentrations and to continuously feed the production medium into a second high-oxygen-content production fermenter. Nitrogen limitation can positively influence the specific parameters.

Example 7 Continuous gluconic acid fermentation with DSM 7085 at different residence times (X / D diagram)

The fermentation medium corresponded substantially Example 4:

fermentation medium glucose 110 g / l and 249-300 g / l NH₄Cl 3 g / l KH₂PO₄ 0.7 g / l MgSO₄ · 7 aq 0.35 g / l CuSO₄ · 5 aq 100 μg / l ZnSO₄ · 7 aq 0.01 g / l CoSO₄ · 7 aq 0.004 g / l H₃BO₃ 0.04 g / l MnSO₄ · 4 aq 0.5 mM FeSO₄ · 7 aq 0.5 mM CaCl₂ 0.1 g / l NaCl 0.1 g / l KJ 0.1 mg / l Na₂MoO₄ · 2aq 200 μg / l citric acid 2.5 g / l temperature 30 ° C PH value 6.5 workload 500 ml correction means 22.5% NaOH

In continuous gluconic acid formation with DSM 7085, upon ingestion of a glucose concentration of 360 g / L with short residence times of about 10-12 hours, over 200 g / L gluconic acid can be produced for a prolonged time ( Figure 6). By using higher glucose concentrations, gluconic acid concentration can be maximized. The gluconic acid concentration correlates with the course of the biomass concentration and decreases with shortening of the residence time. Conversely, the glucose concentration behaves.

The space-time yield only increases linearly when the residence time is reduced to 10.77 hours and then collapses at even shorter residence times. The bio mass-specific productivity increases throughout Be with shortening of the residence time ( Fig. 7). The conversion and the yield (moles of product / mole of glucose used) fall in a similar way to the gluconic acid concentration while shortening the residence time. The selectivity is over 90% in the entire range. In order to maximize gluconic acid concentration, it is better to work at residence times of over 20 hours and at high glucose concentrations.

In the X / D diagram with 110 g / l glucose in the feed increases first the gluconic acid concentration with shortening of the Residence time and decreases again at very short dwell times. For longer residence times, the formed Gluconic acid reacted and fumaric acid as a byproduct educated.

Example 8 Iron and manganese influence on the discontinuous Gluconic Acid

With manganese and iron ions you can grow and control gluconic acid production. With a definier Minimal medium was found to be deficient in iron and manganese with a characteristic pellet-like growth a turnover of almost 100% a selectivity of 98% be achieved.

However, the influence of iron is not as great as in the case of Manganese. Strain DSM 7085 showed a similar glucone Acid production at iron concentrations between 50 and 500 μM, at a NH₄Cl concentration of 0.3 g / l.  

Composition of the medium

Medium to iron influence Where. MnSO₄ · 4 aq 0.5 mM FeSO₄ · 7 aq 50, 100, 250 and 500 μM

Manganese limitation (10 μM iron and 0 μM manganese in the fermentation solution) led similar to the Iron limitation to a selectivity of 98% a turnover of almost 100%. After 54 hours could with the o.a. Medium 147 g / l gluconic acid who produces the. Characteristic here was also a pelletar constant growth. Without manganese and without iron could after Only 32 g / l for 54 hours, with 0 μM iron and 10 μM manganese  118.86 g / L and with 10 μM iron and 0 μM manganese 147.07 g / l of gluconic acid are formed.

When increasing the manganese concentration, the wax could growth and production. With 0.25 mM Manganese and iron were 29.08 g / l after 24 hours, with 0.5 mM 44.27 g / l and with 2 mM 35.67 g / l gluconic acid educated. With 10 μM manganese were only after 24 hours 11 g / l formed. The selectivity was over 90%.

Example 9 Manganese influence on the continuous gluconic acid mentation with DSM 7085

The manganese influence was in continuous fermentation tions in the small fermenter. In these Experiments were a medium as in Example 4 be uses, except that the manganese concentration after each steady state was varied. This manganese concertos were concentrations between 0.25 and 7.5 mM. Until the Reaching the respective steady states were at least 5 residence times required.

media composition Glucose | 360 g / l NH₄Cl 3 g / l KH₂PO₄ 0.7 g / l MgSO₄ · 7 aq 0.35 g / l ZnSO₄ · 6 aq 0.01 g / l CuSO₄ · 5 aq 1 mg / l CoSO₄ · 7 aq 4 mg / l H₃BO₃ 0.04 g / l CaCl₂ 0.1 g / l NaCl 0.1 g / l KJ 0.1 mg / l FeSO₄ · 7 aq 0.1 mM MnSO₄ · 5 aq 0.25-7.5 mM temperature 30 ° C PH value 6.5 workload 500 ml SKE 1300 dwell about 18 h; <20 h (Vitamin addition as in Example 4)

In continuous gluconic acid fermentation with DSM 7085, gluconic acid can be produced at manganese concentrations between 0 and over 7.5 mM manganese. The optimum is in the range of 2.75 to 5 mM. As the manganese concentration increases from 0.25 to 5 mM manganese, the biomass increases and decreases again at a further increase. At residence times over 20 hours, the glucose is largely consumed with 5 mM manganese and formed gluconic acid z. Part implemented ( Fig. 9). At the lower residence times it could be shown that with 5 mM manganese the highest gluconic acid concentration of 260 g / l can be produced continuously (see Fig. 8). Above and below this Mangankonzentra tion, the gluconic acid concentration decreases and is at 0.25 mM Mn only 92.73 g / l ( Figure 8).

Example 10

Iron influence on the continuous Gluconsäurefermen with DSM 7085

fermentation medium as in Example 9, with NH₄Cl | 3 g / l MnSO₄ · 4 aq 5mM FeSO₄ · 7 aq 0.1 to 2 mM

In the continuous gluconic acid production were the differences across a wide spectrum of iron con concentrations (from 0.1 to 2 mM) similar to the discontinuous gluconic acid formation is not so great. One can with 0 to over 2 mM iron continuously Concentration of gluconic acid is preferred between 0.1 and 1 mM iron.

The influence of iron was investigated in chemostat experiments with residence times of about 13 and 18 hours. The medium used was given in Example 10. At a residence time of about 18 hours and at almost complete conversion, no measurable effects were observed ( Figure 10), but only at the shorter residence time (no complete conversion, Figure 11).

Over a concentration range between 0.1 and 2 mM Iron was not compared to manganese big differences in growth and in gluconic acid production observed.

Fig. 11 shows the influence of iron on the continuous gluconic acid fermentation with a residence time of about 18 h: 5.96 g / l of biomass were formed with 0.1 mM iron and 7.4 g / l with 2mN Fe. Nitrogen in the fermenter was measured throughout. The residual nitrogen concentration in the fermenter was 0.1 mg Fe at 197 mg / l and 2 mM at 201 mg / l.

The maximum gluconic acid concentration was reached with 1 mM iron and was 223.25 g / l. With 0.1 mM Fe, 181.95 g / l and with 2 mM 215.66 g / l gluconic acid were formed. The maximum space-time yield and maximum specific productivity were also achieved with 1 mM Fe and were at a residence time of 13.28 h each Weil at 16.81 g / (lh) and 2.46 g / (g · H). With 0.1 mM iron, a space-time yield of 14.47 g / (lh) and a specific productivity of 1.82 g / (gh) were achieved with a residence time of 13.71 h ( Fig. 12).

Significant differences in iron concentration were observed at this short residence time (no full conversion), surprisingly at product selectivity (mole of product / mole of converted glucose). As the iron concentration increased from 0.1 mM to 2 mM, product selectivity increased continuously. The selectivity was 84.26% with 0.1 mM and 98% with 2 mM iron. At iron concentrations of more than 0.5 mM, the selectivity was above 90% ( FIG. 13).

The optimal residence time for each used Glucose concentration is for optimal production Of great importance and should be taken into account at very long residence times, the gluconic acid formed partially implemented.

More than 250 g / l of gluconic acid were formed with all the investigated iron concentrations at a residence time of about 18 h. The highest gluconic acid concentration of 260.94 g / l was measured with 0.1 mM iron. With 2 mM iron, 252.35 g / l gluconic acid was formed ( Figure 11). The selectivity decreased with increasing iron concentration because of the continued Gluconsäurever consumption.

Fig. 12: Iron influence on the specific productivity (BSP) and space-time yield (RZA) with a residence time of about 13 hours.

Fig. 13: Iron influence on the product selectivity with a residence time of about 13 hours.

Example 11 nitrogen influence

Nitrogen limitation is also of great importance interpretation. Gluconic acid production in discontinuous process with the isolated strains is a three- Phase process and is similar to a typical one Growth curve with a lag phase followed by a lag phase accumulating log phase and a stationary product tion phase. The maximum logarithmic gluconic acid pro production was used on all isolates examined immediately after the consumption of nitrogen.

Under Phosphorlimitierung with 0.1 g / l KH₂PO₄ was the Nitrogen influence in small fermenters at 30 ° C and pH 6.5 examined. In this case, 0.5 g / l, 1 g / l, 2 g / l were so as 4 g / l NH₄Cl in four parallel fermentations under otherwise used the same conditions. Interestingly Wise was also despite remaining nitrogen at the higher N concentrations still gluconic acid produ ed. The gluconic acid production with the isolated Microorganisms are growth-linked. The best spe specific productivities were recorded at the lowest Concentration (0.5 g / l NH₄Cl) determined. In the highest N concentration (4 g / l NH₄Cl) was the best Growth and, surprisingly, a better product than at the medium concentrations (1 g / l and  2 g / l).

Example 12

In the fed-batch process could with DSM 7085 within over two days 400 g / l gluconic acid are produced. The selectivity was similar to that of the continuous fermentation over 90%. After the growth phase a 75% glucose solution was periodically added to the Fer supplied.

medium composition

The strain DSM 7085 could also at a Glucosekon concentration of 360 g / l grow. Only after a delay The lag phase started the log phase. So it is possible high glucose concentrations.

It makes sense, however, to start with glucose concentration of about 150 g / l and gives a highly conc. Glu coselösung or glucose in solid form to the fermenter to.

Example 13 Phosphorus influence on the continuous Gluconsäurefer mentation A. pullulans DSM 7085

In the study on the influence of phosphorus, a Me dium analogous to Example 10, but with the halved Amount of nitrogen, vitamins and trace elements and with 0.25 mM iron. The experiments were carried out at Ver times of about 12.5 and 17 hours. The phosphor effect became clearer at the shorter Dwell time.

FIG. 14 shows the influence of phosphorus on the gluconic acid fermentation with a residence time of about 13 hours. With increasing KH₂PO₄ concentration, the biomass concentration decreases continuously. With 0.35 g / l KH₂PO₄ (this corresponds to the Ausgangskonzen concentration) were 4.47 g / l and formed with 2.1 g / l KH₂PO₄ 1.91 g / l biomass. With increasing KH₂PO₄ con centration of 0.35 g / l up to 1.4 g / l continuously increased the gluconic acid concentration. With 1.4 g / l KH₂PO₄ the highest product concentration of 191.35 g / l was reached and dropped again at higher KH₂PO₄ concentrations. With 0.35 g / l KH₂PO₄ were compared for comparison 158.99 g / l and with 2.1 g / l 138.36 g / l gluconic acid continuously formed ( Fig. 14).

The maximum space-time yield was also achieved with 1.4 g / l KH₂PO₄ and was 15.43 g / (l · h). At 0.35 g / l, a space-time yield of 12.62 g / (lh) was determined. The biomass-specific productivity increased continuously with increasing phosphorus concentration. At 0.35 g / l KH₂PO₄ it was 2.82 g / (gh) and at 2.1 g / l KH₂PO₄ 5.78 g / (g · h) ( Fig. 15). A higher phosphorus concentration is therefore in contrast to the literature, with lower optimal phosphorus concentrations are given as Aspergillus niger (RÖHR & KUBICEK, 1983), in Aureobasidium pullulans advantage for a higher biomass specific productivity, space-time yield and product concentration.

With increasing KH₂PO₄ concentration up to 1.05 g / l rose at a residence time of about 17 h the Gluconic acid concentration and decreased again at higher KH₂PO₄ concentrations. With 0.35 g / l KH₂PO₄ (this corresponds to the initial concentration) 200 g / l gluconic acid were at a residence time of about 17 hours continuously formed. At a rate of 1.05 g / l, approximately 270 g / l gluconic acid was formed under otherwise identical conditions. Great differences in growth were not observed. The highest space-time yield was 1.05 g / l KH₂PO₄ reached and was 15.31 g / l · h.

Fig. 14 Phosphor influence on the continuous Gluconsäurefer mentation at a residence time of about 13 hours

Fig. 15 phosphorus influence on the biomass specific productivity and space-time yield at a residence time of about 13 hours.

Example 14 Magnesium influence on the continuous glucon acid fermentation with DSM 7085

The influence of magnesium was investigated in chemostat experiments with a residence time of about 20 hours. The Medium corresponds to Example 10 (with 1.4 g / l KH₂PO₄, 450 g / l glucose, 5 mM manganese, 1 mM iron, etc.).

Fig. 16 shows the progressions of the biomass, glucose and gluconic acid concentration at various magnesium concentrations. A strong influence of magnesium on the growth of Aureobasidium pullulans was observed in these experiments. At magnesium concentrations of 0.8 to 1 mM, the biomass remained unchanged. However, as the magnesium concentration increased from 1 mM to 3 mM magnesium, a strong continuous increase in biomass was observed to a factor of 2.17. 11.26 g / l were continuously formed with 1 mM magnesium and 24.45 g / l biomass with 3 mM ( FIG. 16). Despite the ventilation with pure oxygen, the dissolved oxygen concentration in the glass fermenter was insufficient for optimal production of gluconic acid in the case of the high biomass.

No significant differences were observed in the gluconic acid concentration. The Gluconic acid concentration increases with decreasing Mg concentration and reaches maximum concentrations of less than 200 g / l. The specific productivity decreases with increasing magnesium concentration. The Magnesium concentration of 1.42 mM, which was used in all other experiments, is optimal Concentration range.

From the gluconic acid and glucose concentrations in Fig. 15, a deterioration in selectivity at magnesium concentration of over 2 mM becomes apparent due to the high biomass. Surprisingly, however, the selectivities are also very poor even at low magnesium concentrations of less than 1 mM. According to references, low magnesium concentrations are of advantage for the synthesis of FAD of the cofactor of glucose oxidase and thus for optimal production of gluconic acid. According to our findings, however, low magnesium concentrations have a negative effect on the product selectivity and thus the economic efficiency of the process.

Fig. 16 Magnesium influence on the continuous gluconic fermenation.

Example 15 Temperature influence on the continuous glucon acid fermentation with DSM 7085

The influence of temperature on the continuous gluconic acid formation with Aureobasidium pullulans DSM 7085 was in a chemostat experiments at a residence time of about 12-13 h with a defined medium analog Example 10, but with 1 mM iron, 1.4 g / l KH₂PO₄ and 360 g / l glucose examined.

All the above experiments were carried out largely at 30 ° C. However, the organisms are also in the Able to grow and produce at lower temperatures. Different temperature optima could be identified for growth and production.

FIG. 17 shows the courses of biomass, glucose and gluconic acid concentration as a function of the temperature. A continuous increase in biomass concentration was observed with decreasing temperature. The biomass was 12.45 g / l at 31 ° C and 30 ° C and 14.8 g / l at 27 ° C. A temperature rise of just one ° C (from 31 ° C to 32 ° C) caused a drastic decline in the biomass concentration. The highest product concentrations were achieved at temperatures between 29 ° C and 31 ° C. 234.53 g / l gluconic acid were continuously formed at 29 ° C, 232.19 g / l at 30 ° C and 223.5 g / l at 31 ° C. At 32 ° C, the product concentration dropped drastically and at steady state was only 78.09 g / l. A continuous decrease in product concentration was observed at temperatures below 29 ° C ( Figure 17).

The glucose concentration in the fermenter was between 66.21 g / l at 30 ° C and 290.13 g / l at 32 ° C. The maximal Space-time yield and biomass-specific productivity was achieved at 29-30 ° C.

Fig. 17 Temperature influence on the continuous gluconic fermentation of a residence time of about 12 h

Fed batch fermentation

In a fed batch experiment, a maximum gluconic acid concentration of 504 g / lg / l was achieved. The glucose was largely reacted ( Figure 18). The delay in the increase in gluconic acid concentration above 450 g / l is due to technical problems (eg dilution effects, substrate addition).

Fig. 18 Fed batch Gluconsäurefermentation with A. pullulans.

Example 16 Process Optimization of Continuous Gluconic Acid fermentation 16.1. Continuous gluconic acid fermentations under optimized conditions

With the optimized medium (analogous to Example 10, however with 1 mM iron, 1.4 g / l KH₂PO₄ and 450 g / l glucose) could at pH 6.5, oxygen saturation of about 155% and 30 ° C at a residence time of 21 hours 315 g / l and from 25 hours 330 g / l gluconic acid continuously be formed in a stirred tank (3 l). The biomass was at steady state at 6.8 g / l.

By extending the residence time and with higher Glucose concentrations may be the gluconic acid concentration be maximized. At gluconic acid concentrations above 300 g / l, however, is a strong product ingredient to register.

16.2 Continuous gluconic acid fermentation with Bio Mass retention by microfiltration

A good alternative for accelerating the maxi The product concentration offers the partial Biomass retention using microfiltration (cross over filtration). The biomass retention using microfil tration makes it possible to decouple the growth of Ver because time. It also allows the implementation of the Pro at higher, toxic for growth, acid substance concentrations, with the highest productivity. In a Ver time of about 23 hours, 290% oxygen saturation and with 23 g / l accumulated biomass (about 80% microfil trypsin content), 375 g / l gluconic acid were completely according to sales are formed continuously. At 19 hours Residence time and with 25 g / l biomass were 370 g / l Gluconic acid also at complete conversion (glucose not measurable) produced continuously. A shortening The residence time seems to be the same after the o.a. results possible.

16.3 Cascading of two fermenters

Another alternative process control is the cascading of two or more than two fermenters. Thereby a distribution of the residence time is possible, with different optima, z. For growth and production can be adjusted independently of each other. In this series of experiments, two stirred tank fermenters were used used with a total working volume of about 4.5 1. The medium used corresponded to Example 9, but with 450 and 600 g / l glucose, 1 mM Fe, 5 mM Mn, 1.4 g / l KH₂PO₄.

The first fermenter was at an oxygen saturation between 120 to 180% and the second of more than 300% operated.

The residence time was about 22 hours in the first stage and 37 hours in the second. In the first stage was the maximum space-time yield at a conversion of over 60%, as well as a fast Growth, and in the second, the maximum gluconic acid concentration sought. 366 g / l gluconic acid and a Biomass of 5.3 g / l were in the first fermenter and 433 g / l gluconic acid and a biomass of 7.8 g / l im second temporarily in the experiment with 600 g / l glucose.

In the experiment with 450 g / l glucose 305 g / l gluconic acid were in the first fermenter with a residence time of 22 h, 5.9 g / l biomass and 70 g / l glucose and in the second with a total residence time of 38 h and complete Sales 350 g / l gluconic acid and 8.1 g / l biomass measured.

In a further shortening of the residence time was at 19.5 h residence time in the 1st stage 272 g / l and at a total residence time of 30.8 h in the second stage 370 g / l gluconic acid are continuously formed.

Example 17

In another experiment, using a fermentation medium according to Example 4, but half-concentrated and with 360 g / l glucose; 1.5 g / l NH₄Cl; 0.5 mg / l ZnSO₄ · 6 aq; 0.5 mg / l CuSO₄ · 5aq; 2.5mM Mn; 1mM Fe; 1 mM Mg and 1.4 g / l KH₂ PO₄ in continuous Fermentation without biomass retention (with 3.9 g / l dry biomass in steady state) at 29-30 ° C and pH 6.5 and 21 h residence time a gluconic acid concentration of 305 g / l with a molar Selectivity <98% at a glucose conversion of <95%.

Claims (14)

1. A process for the production of gluconic acid by fermentation in sugar-containing aqueous liquid speed, characterized in that

• isolatable Aureobasidium pullulans (de bary) Arnaud strains isolated from incubation approaches of crushed flowers and stems of Central European forest and meadow flowers collected during the main flowering period
• - form in continuous culture of glucose 200 g / l gluconic acid at 90% conversion and 90% molar selectivity,
• in an Fe and Mn-optimized medium with an N-dependent iron and manganese ion content in the feed, which is at an N content corresponding to 3 g / l NH₄Cl at 0.5 mM Fe and 0.5 mM Mn;
• - Cultured at pH values of 4.5 to 8 and at temperatures of 24-32 ° C.

2. The method according to claim 1, characterized, that one of the Aureobasidium pullulans (de bary) Arnaud tribes with accession numbers DSM 7085, DSM 7086, DSM 7087 or DSM 7088 used.   3. The method according to claim 1, characterized, that at a pH between 6 and 7 and at a temperature of 29-31 ° C is worked. 4. The method according to claim 1, characterized
that working with N-dependent iron, manganese and Magne siumionengehalten in the feed, which at an N content corresponding to 3 g / l NH₄Cl at 0.5-3 mM Fe; 2.5-5 mM Mn or 1-2 mM Mg. 5. The method according to claim 1, characterized, that ensures in the feed for 0.7 g / l KH₂PO₄. 6. The method according to claim 1, characterized, that the fermentation continuously with dwell times of 10-40 h is performed. 7. The method according to claim 6, characterized, that increases with one with increasing residence time with oxygen saturation being worked at 13 h residence time values between 80 and 150%, chosen in particular by 120% oxygen saturation become. 8. The method according to claim 6, characterized, that with unsupported microorganisms and bio water retention is worked.   9. The method according to claim 6, characterized that in at least two cascaded angeord Neten fermenters works. 10. The method according to claim 9, characterized, that in a first stage with 120-170% acid saturation and 10-25 h, especially 12-18 h Ver while and in the second stage with <360% Oxygen saturation and a total residence time  15 h, esb. 18-30 h is worked. 11. The method according to claim 6, characterized, that the fermentation with glucose Zulaufkon concentrations of 300-600 g / l. 12. The method according to any one of claims 1 to 7 and 9 to 11, characterized, that the fermentation in a fluidized bed reactor carried out with supported biomass. 13. Aureobasidium pullulans (de bary) Arnaud strains, which in continuous culture at 90% sales and  90% molar selectivity from glucose 200 g / l Gluconic acid, isolated from incubation Batches of crushed flowers and stems of in the spring to summer collected Central Europe forest and meadow flowers in citric acid Solution at pH 2.5 with 100 g / l glucose under Wetting agent additive and ample Sauerstoffver Care by shaking. 14. Aureobasidium pullulans (de bary) Arnaud strains DSM 7085, DSN 7086, DSM 7087 and DSM 7088.