DD139870A1 - METHOD FOR MICROBIAL CONVERTING OF SUBSTR & T COMBINATIONS - Google Patents

Abstract

The invention relates to the field of microbiological industry and has the goal to increase the yield of biomass or product by increasing the substrate utilization and specific growth rate or product formation rate. Measured by the enthalpy of combustion of the microbial biomass, the substrates for growth and for product formation can be divided into: 1. substrates with excess energy, 2. with a nearly balanced carbon / energy ratio and 3. with energy shortage.

Description

Title ·. ,

Process for the micro-artificial co-transformation of substrate combinations

Field of application of the invention

The invention relates to the field of the microbiological industry, especially to processes for the production of microbial biomass, and products,

Characterization of the known technical solutions

The known technical solutions for biomass production and product synthesis are characterized in that a substrate is used as carbon and energy source, z, B. for biomass: methanol · - 76,26,280 Japan, Cl. C 12 C 11/08 (1976, Glucose - 76.48.489 Japan CI C 12 D 13/06 (1976), n-alkanes - 2,348,753 FRG, C 12 C 11-18 (1973), 1,442,070 Great Britain, C 12 B _1- O (1975), ethanol or acetic acid - 77,105,275 Japan, CI C 12 C 11/08 (1977); for product syntheses: glucose - 1,812,710 Japan and 3,622,455 US (1976) , n-alkanes - Tabuchi et al. (1970) Nippon Nogei Kogaku Kaishi 44, 562..

Through phenotypic optimization of the fermentation processes, yield coefficients (biomass production) are achieved, which are generally around 45% and not more than 50%. When using mixed substrates, the selection and combination of substrates is on an empirical basis.

Sulfite liquors / ethanol - 2506149 BRD, C 12 D 13/06 (1976), glucose / lactose - Baidya et al. (1967) Biotechnol. Bioeng. IX, 195).

563

Aim of the invention

The aim of the invention is to increase the yield of biomass or product by increasing the utilization of the substrate and the specific growth rate or product formation rate 5 and thus to substantially improve the economics of the processes for producing biomass and microbial products.

Explanation of the essence of the invention .

Substrates for microbial growth, which are carbon and energy sources at the same time, differ in carbon / energy ratio. Measured by the energy content of the cell substance produced, the embossed substrates can be evaluated for their balance in carbon / energy ratio. The substrates can be divided into three groups (see Table 1).

1 · Substrates with excess energy (E> C)

2. Substrates with a balanced carbon: energy ratio (E ^ C)

3. Substrates with energy deficiency (E ^ C).

Table 1 % E ai c % E ^ C % 20 E > C 74  glycerin 96.5 sucrose 113 _: methanol 78 arabitol 103 xylose 113 ethanol 80 Manni t 105 ribose 113 pentadecane 80 glucose 114 hexadecane 86 lactic acid 118 25 palmitic acid acetic acid 122 hot maid 137 bensäure Amber 143 acid 30 malic acid 159 fumaric acid 160 lemons 161 acid ants 208 acid oxalic acid 435 - "3 -

563

Numerical values mean: % of the energy consumed with 100% carbon incorporation and a C content of the biomass of 47%

The essence of the invention is to use substrate mixtures for microbial biomass productions as well as for product syntheses, which are to be combined in such a way that a balanced carbon / energy ratio is achieved, so that yield coefficients are achieved which approach the theoretical optimum (in biomass production approx ,

67% based on carbon). This has a significant impact on the economics of the processes by increasing substrate yield and reducing waste heat. Conditions for the successful application of this principle are microorganisms which are able to adsorb on the one hand on each substrate of the chosen combination and on the other hand combine the substrates in combination simultaneously and in that combination one substrate does not inhibit the utilization of another substrate and / or repress-t. The simultaneous utilization of the substrates simultaneously leads to an increase in the specific growth rate, which is advantageous for the dimensioning of the plant and the biological stability in the unprotected fermentation process.

Example 1 :

The methanol-neutralizing yeast strain Hansenula polymorpha was cultured in the batch process under the following conditions: T = 38 ^° C. pH = 4.5

Composition of the nutrient solution: Component M £ 2S ^e (2 ^ro

H ₃ PO ₄ 1 ml

KCl 0.5 g

NaCl. 0.1 g

209 56 3

MgSO ₄ , 0.182 g

Peso. 30 mg

MnSO ₄ , 5H ₂ O 24 mg ·

ZnSO ₄ 4 mg

CuSO ₄ 1 mg

CoSO ₄ 2 mg

₂₄ 10 mg

Biotin 15 mg

Thiamine 800 mg

-] 0 UH-a ~ N entry continuously via pH control Permentor = 2.51 Laboratory fermentor (LKB, Sweden) The C source was a combination of methanol and xylose (concentration = 50 mM methanol

+12 mM xylose),

The following values for the specific growth rate (/ u) and the yield coefficient (Yc) were calculated from the simultaneous substrate consumption and the biomass yield:.

^Substrate / U (h- ¹ ) Yc (gTS / gC)

Methanol - '

+ Xylose. 0.210 1.2

On the other hand, when methanol and xylose were used separately as C sources (concentration of methanol = 25 mM constant, and experiment, concentration of xylose = 12 mlvl), the yield coefficients and the specific growth rates were only: "

Substrate, u (h ~ ¹ ) Yc (gTS / gC)

Methanol 0.110 0.89

Xylose 0.070 1.0 ·

Under the condition of the combined substrate addition, an increase in the specific growth rate by almost 100% and a significant increase in the yield coefficient can be observed.

- 5 -

Example 2 :

The methanolutilizing strain Hansenula polymorpha sp. was cultured under the conditions described in Example 1. »The fermentation was carried out continuously. The substrate used was the combination of methanol with xylose (mixing ratio as in Example 1, concentration of methanol <12 mM).

By using the substrate combination could be a stable

_1 process with D. , = 0.138 h, the earnings

1.0 coefficient was Yc = 1.5 gTS / gC.

On the other hand, when methanol was used as sole C source and energy source (concentration <12 mM), a maximum flow rate of only D.I. = 0.07 h could be achieved; the yield coefficient was Yc = 1.18 gTS / gC.

By using the substrate combination, the critical flow rate increased by approximately 100% and the yield coefficient by 27% compared to the sole substrate methanol.

Example 3 :

The yeast Candida utiis would be cultured with a mixture of ethanol and sucrose in a weight ratio of 3: 1 in a stirred fermentor with 5 kg working contents under the following conditions:

Fermentation temperature: 35 ^° C.

pH value: 4.0

Residence time: 3.0 h.

Dissolved Op Concentration: ^ 2 ppm 0

Composition of the nutrient solution:.

Ethanol 12.0 g / l

Sucrose 4.0 g / l.

H ₃ PO ₄ (85%) 0.8 ml / 1

0.76 g / l 0.28 g / l 26.0 mg / l 8.8 mg / l 8.8 mg / l

1.6 mg / 1

    - 6 -

H ₂ SO ₄ , 7 H ₂ O _MgSO4 7 H ₂ O PeSO ₄ , 7 H ₂ O ZnSO , 5 H ₂ O MnSO ₄ , 5 H ₂ O CuSO ₄

9

The nitrogen supply of the yeast was carried out with ammonia water via the pH control. Both substrates were fully utilized. A yeast concentration of 11.0 g HTS / 1 was obtained. This corresponds to a yield coefficient of 0.68 g HTS / g substrate or 1.38 g HTS / g C.

In contrast, ethanol and sucrose were among the o.g. Conditions used individually as a C source, the following yield coefficients were obtained:

Substrate Ys (gHTS / g substrate) Yc (gHTS / g carbon

Ethanol 1.5 1, 28

Sucrose 2.0 1.19

For the used ethanol / sucrose mixture with the weight ratio 3: 1, it is possible to calculate therefrom an Ys yield coefficient of 0.63 gHTS / g substrate or Yc of 1.26 gHTS / g carbon.

Combined addition of substrate therefore shows an increase in the yield coefficient of 9.5%.

Example 4 :

The paraffinutilizing end yeast strain Candida guilliermondii D was cultured in a batch process in a 40 L laboratory stirred fermentor under the following conditions:

Temperature: 33 ⁰ C

pH: 4.2

pH control: with 10% NaOH

Fumigation: 100 1 air / kg.h

Composition of the nutrient solution:

1 1 contains: 13.4 g (NHj ₀ SO,

3.5 G KH ₂ PO ₄ 7H ₂ O 0.84 G H ₃ PO ₄ ¹ V XJ Γ} ( -Tl ₂ VJ 0.53 G MgSO _4. f "7 TT r \ I -HpVJ 42.0 mg PeSO ₄ . 5H ₂ O 33.0 mg MnSO ₄ . 21.0 mg CuSO ₄ . 20.0 mg ZnCl ₂

- ⁷ - 20 9 563

C-source: mixture of 140 g petroleum distillate / kg broth (boiling range 240-360 ⁰ C at 18.5% by mass of n-alkanes.) And 20 g of glucose / kg broth

Under these culture conditions, a specific -1 growth rate Ai of 0.41 h was achieved.

Separate cultivation on petroleum distillate (140 g / kg nutrient solution) and glucose (20 g / kg nutrient solution). an inert oil content of 140 g / kg nutrient solution, on the other hand, was only limited to specific growth rates of

0.28 h " ¹ for ED and

Reached 0.24 h ^-1 for glucose.

, Under the conditions of the combined substrate supply, there was an increase in the specific growth rate compared to petroleum distillate as the sole carbon source of 45% and to glucose as the only carbon source of 70%.

In the continuous fermentation under the above conditions, with a residence time of 5 hours by the substrate combination of petroleum distillate (200 g / kg nutrient solution) and glucose (10 g / kg nutrient solution) compared to a fermentation with petroleum distillate as sole carbon source, an increase in productivity χ of 2 , 94 to 3.99 (gTS / kgh) and a reduction in oxygen consumption from 2.71 to 1.86 g 0 _? / g TS

2.5 can be achieved.

Example 5 :

Based on the following fermentation conditions

- Temperature: 32 ⁰ C. - pH: 4.2

- Ventilation rate: 15% dissolved O ₂

Stirrer speed: 2400 rpm

- Reactor: 12 ILaboratory fermentor with ground power

- Cell concentration: 20 g HTSAg

Residence time: 50 h (one-step process control) -nutrient composition.

per 1 (20 g HTS)

KH ₂ PO ₄ MoSO ₄ 4 7 H ₂ O CuSO ₄ , ZnCl ₂ 5 H ₂ O CoSO ₄ V 7 H ₂ O MnSO ₄ ♦ 4 H ₂ O H ₃ BO ₃ CaCl ₂ ♦ 6 H ₂ O

0.042 g

0.114 g

0.007 g FeCl ₂ 50 g glucose · 10 ml 0.01% thiamine solution

is prepared using the yeast strain IMET H 134 (deposited in the IMET collection of the ZIMET Jena) with a supply of 1.5 ml / min of the mentioned nutrient solution and 0.25 ml / rnin a paraffin mixture of the chain length range C _1? - C ₁ □ a productivity of 1.6 g of citric acid / kg.h, or a specific

If only paraffins are used as the carbon source, a maximum productivity of 1.2 g of citric acid / kg is achieved at the same feed rates after reaching a stable stationary state (continuous fermentation time about 150 hours) .h, corresponding to a specific product formation rate of 0.06 h.

Claims (1)

claim A process for the microbial conversion of substrate combinations, characterized in that simultaneously utilizable substrates, according to their carbon / energy ratio, are combined so as to produce balance and thus the substrate mixture of the employed microorganism strain with increased speed with low loss, i. be converted into cell substance or microbial products with both the material and energy-optimal efficiency.