DD283737A7 - PROCESS FOR CULTURING METHANOTROPHIC BACTERIA - Google Patents

Abstract

The invention relates to a method for the cultivation of methanotrophic bacteria. The invention belongs to the field of microbiological protein production. The invention aims to develop for the natural gas process a Prozeszregime that provides the concentrations of the gas mixture outside the explosive range, which ensures maximum growth during startup and pressure fermentation and thus to a safe continuous Prozeszfuehrung and shortening the transition time of discontinuous to continuous operation leads. It has been found that the ratio n1 / n2 of the concentration of the dissolved oxygen in the culture liquid to the concentration of the oxygen in the gas mixture is a suitable process quantity. The process provides a gas supply within the limits of n1 / n2 of 5-30, preferably 8-25, with the upper limit set either by increasing or increasing the O2 concentration in the gas mixture and reaching the lower value after appropriate growth. Bacteria; Pressure fermentation; Oxygen concentration; Eiweiszgewinnung; discontinuous; Natural gas; continuously}

Description

Process for the cultivation of methanotrophic bacteria

Field of application of the invention

The invention belongs to the field of microbiological protein production and can be used in plants in which methane or natural gas are microbiologically converted by means of methanotrophic bacteria.

Characteristic of the known technical solutions

The known methods assume that methanotrophic bacteria are cultured in a nutrient-containing fermentation medium in the presence of a methane- and oxygen-containing gas, working discontinuously and continuously.

Temperature and pH of the fermentation medium are kept in the optimal ranges for each culture

 Methods are known at atmospheric pressure and at elevated system pressure, although no examples are given for extremely high pressures (40 at in GB-PS 1150401, 1500 psig in GB-PS 1320722).

The composition of the gassing mixture varies widely, in DE-CS 2610478 ratio of methane to air of 2: 1 to 1: 6, in GB-PS 1320722 methane 1-97%, Ö2 3-74% or in DE-OS 2241258 methane 10-50 %, preferably 15-25%, O2 10-19%, preferably 16-18%.

The avoidance of explosive gas mixtures is mentioned several times. GB-PS 1150401, however, the method of DE-OS 2417848 expressly works in the explosive area and provides for an energetic use of the exhaust gas.

The composition of the gassing mixture is considered according to various aspects, e.g. economic or from the mass transfer

conditionally chosen, but it never comes to an optimal CH4 or 02 supply for the culture itself.

Although it is spoken of inhibition of growth at too high an offer of O2 in GB-PS 1320722 and a dissolved Konaentration of 1-30 ppm 0?. proposed, but is not guaranteed in all the proposed method, especially when using elevated pressure that limiting or inhibiting concentrations of dissolved gases are avoided if the current growth does not match the supply of gas components, which are usually dosed in constant conditions. Only in DE-OS 2308087, however, places the emphasis on the gas recirculation, it is spoken of supplemented gas to supplement the respective process state.

There are also known methods which are intended to achieve maximum growth by using oxidized derivatives of methane (DE-OS 2460672) or Stoffwecheelprodukten (DE-OS 2610478), but do not use the potential of optimal gas supply, because the claimed in these methods Objectives maximum growth and maximum gas utilization are mutually exclusive

Finally, most processes only require certain stationary conditions of the continuous process, but leave the 'most difficult transient phase of the start-up, in which, for example, at all times limitations and inhibitions, leaching of the culture or explosive gas mixtures must be avoided. Only in DE-OS 2460672 and DE-OS 2535782 the transition from the discontinuous to the continuous phase is described, but only with respect to the supply of liquid nutrient medium, but not with respect to the gas supply. Thus, DE-OS 2460672 at the beginning of the growth medium with D = 0.08 n- ^{1 is} supplied and then increased in two-day intervals D by 0.02 until reaching D = 0.22 h- ¹ . In DE-OS 2535782 beginning of the continuous process after 24 h, every 2 h, D is increased by 0.02 until reaching 0.175 hi. In the described method, the process of starting takes a relatively long time, only low biomass concentrations are achieved. Measures for the design of the

driven and to the leadership of the process under increased pressure are not given at all.

Overall, the described method has the disadvantage that never equilateral optimum gas supply and thus optimal growth and the security that the gas mixture is outside the explosive range, guaranteed.

Object of the invention

The invention aims to develop a process regime for a method of breeding methanotrophic bacteria, which provides gas concentrations outside the explosive range of methane-oxygen mixtures, ensures maximum growth during startup and pressure fermentation and thus safe continuous process control and shortening the transitional period from discontinuous to continuous operation.

Explanation of the essence of the invention

The invention is therefore based on the object, certain process parameters that are suitable as a measure of the Proseßführung to select and set their limits.

According to the invention, the object is achieved by using the ratio nl / n2 of the dissolved oxygen concentration (in ppm) in the fermentation medium to the concentration of oxygen in the fermentation waste gas (in volume fractions) including the limits of this ratio as a parameter for the sample guide.

The limits of this ratio nl / n2 are 5 and 30, preferably 8 and 25. The process control provides for a gas supply within these limits, the upper value set either by pressure increase or by increasing the O2 concentration in the gas mixture and the lower value is achieved after appropriate growth.

If the ratio nl / n2 exceeds a value of 30, 2.B. increases, 02-inhibition is observed, the growth rate decreases considerably and the transition period of discontinuous to continuous operation is extended, or in the continuous process takes place a flushing of the culture.

If the ratio falls below 5, either an O2 limitation is observed or the gas mixture is in the explosive range.

The following examples illustrate the invention in more detail.

example 1

In a stirred fermentor 5 1 liquid are added, which was brought to a initial concentration of 1.5 g / l with a suspension of the methane-oxidizing 'bacterial strain ZIMET B 502, and a liquid nutrient medium, which was calculated for a biomass increase of 1-2 g / l, contains.

The composition of the nutrient solution was as follows per liter of water (for Ig biomass):

H3PO4 (80%) 0.028 ml

KK2PO4 35 rag

MgSO4 * 7 H2O 25 mg

CuSC) 4 * 5 H2O 0.785 mg

M11SO4 * 4 H2O 1.825 mg

FeCl2 * 4 HzO 1,200 mg

Z11CI2 0.322 mg

C0SO4 * 7 H2O 0.036 mg

NiSO4 * 7 H2O 0.109 mg

Al2 (SO4) 3 * 18 HzO 0.186 mg

Ca (NO3) 2 * 4 H2O 0.883 mg

Na2 Mo04 * 2 H2O 0.041 mg

H3BO3 1.286 mg

CrCl3 * 6 HzO 0.077 mg

An excess of nutrient and trace salts is constantly created as a function of biomass growth. It will set a temperature of 38 ° C and a pH of 5.7

 Then you start to introduce gases into the fermenter. First, N2 is introduced as an inert gas (100 l / h) to drive off remaining oxygen. Then you start with the introduction of methane (35 l / h), during which you pay attention to the fact that always methane in excess (no methane limitation!) Is present. Finally, air is introduced in an amount of 69 l / h.

This gas mixture (7.5% O2 at the inlet) achieves 5.8% O2 in the exhaust gas and a dissolved O2 concentration of 1.46 ppm, which corresponds to a ratio nl / n2 of 25.

Then one waits until growth has reduced the dissolved O 2 concentration to 0.38 ppm and the exhaust gas concentration to 4.75%. This corresponds to a ratio nl / n2 of 8.

Now you increase the air flow to 123 l / h (equivalent to 10% O2 at the entrance). The 02 concentration in the exhaust gas rises to 8.5% (safety limit of the explosive range) and the dissolved O2 concentration to 2.03 ppm. These values correspond to a ratio nl / n2 of 24.

Again, wait until the dissolved O 2 concentration (CL) and the exhaust gas concentration have decreased so that the ratio n 1 / n 2 becomes 8. This is the case at 7.28% O2 and Ci = 0.6 ppm.

Then you start to increase the system pressure. First to 0.178 MPa (here nl / n2 = 24.8 at Ci = 1.81 ppm and 7.3 % O2 is reached), in a second step to 0.233 MPa, in both cases waiting until the lower value of the ratio nl / n2 of 8 sets.

Because the exhaust gas concentration would fall below 5% after a further increase in pressure (currently it is 5.5%), the air volume is increased again. It is increased to 158 l / h and then the pressure in two further steps to 0.284 and 0.336 MPa, always up to an initial value nl / n2 = 25 and with a waiting time to

vi = 44 and vi = 27 and vi = 17 and vi = O and

sum decrease to nl / n2 = 8.

Before the last pressure increase in this test (to 0.391 MPa), the air volume is increased again (to 180 l / h) and then to 199 l / h, always in compliance with the limit values for nl / n2 between 8 and 25.

The last measures in this experiment relate to the gradual substitution of inert gas by air or methane within the specified limits for nl / n2. The individual steps are (Vi = inert gas, Vm = methane, Vl = air, all in l / h): Vi = 85 and Vl = 214, Vi = 69 and Vl = 230,

Vm = 60

V1 = 247,

Vm = 70

V1 = 260.

By this procedure, after 9 h a Biomaesekonaentration of 10 g / l achieved and maintained until the first phase continuously a growth rate of 0.25 h " ¹

After reaching 10 g / l biomass concentration of the continuous process with D = 0.2 ^h-l is started at a productivity of 2.5 g / l * h.

Example 2

In a fermentor, 0.1 m ^{3 of} liquid, the nutrient medium for a biomass increase of 1-2 g / l and a suspension of the bacterial strain -ZIMET B 502 of 1 g / l contains, and under the same conditions (temperature, pH, regular supplementation of the nutrient medium) as cultivated in Example 1. Begin the fumigation with inert gas (6 m ³ / h), methane (0.6 m3 / h) and air (2 m3 / h). The first value for nl / n2 is 30 (from Ci = 1.38 ppm and 4.6% O2). In the starting phase one waits until the lower value of the ratio nl / n2 has been set to 5 (Ci = 0.2 ppm, 4% O2), but the limits 8 and 24 are always observed

 In this example, from the beginning, when the air

2 6 3 7 3 7

the amount of inert gas is reduced by the amount of increase in the amount of air. The increase in the amount of air takes place when, if the ratio nl / n2 = 24 is maintained, the oxygen concentration does not exceed 8.5%. Therefore, after the start phase after increasing the amount of air in each case two incremental increases in the system pressure are inserted. This process is repeated a total of 3 times via the intermediates Vl = 3.88 m3 / h, P = O.178, P = 0.23 MPa, Vl = 4.7, p = 0.281, p = 0.332, Vl = 5.2, p = 0.383, P = O.435.

If the exhaust gas concentration CH4 falls below 5 % , the amount of methane (with substitution of inert gas) is increased to 1.3 mVh. This is the case after the pressure increase and the subsequent growth. Meanwhile, the biomass concentration has risen to over 9 g / l and the O2 consumption so high that Luftmengen- and pressure increases can be done alternately without the exhaust gas concentrations are in the explosive range in the increase in air volume. This change takes place 7 times (via the intermediates Vl = 5.54, p = 0.486, Vl = 5.84, P = O.537, Vl = 6.1, p = 0.588, Vl = 6.34, p = 0.639, Vl = 6.54, p = 0.689 , VI = 6.72, P = O.739, Vr, = 6.9, p = 0.79).

By increasing the amount of methane to 1.7 mVh (with substitution of the remaining inert gas) is finally achieved that only gassed with methane and air (6.06 % O2, 13.72% CH4 in the exhaust gas).

In a final test phase of the transition phase after reaching the desired system pressure of 0.79 MPa, the air quantity is further increased to 10.32 mVh in 11 steps, to obtain 8.08 for the oxygen exhaust gas concentration as favorable as possible for mass transfer below the explosive range.

After 13 h, the biomass concentration is 21.5 g / l. During the transition phase, a growth rate of / u = 0.25 h- ¹ was constantly ensured.

Subsequently, the continuous process was started with D = 0.2 h-i at a productivity of 5.37 g / l * h.

Example 3

The cultivation conditions are similar to those of Example 1

If the pressure is not increased incrementally but suddenly to the value of 0.39 MPa, the ratio nl / n2 is 100 (at a dissolved ozone concentration of 8 ppm, exhaust gas concentration of 8%), and the growth rate drops to 50 % .

Example 4

The cultivation conditions are similar to those of Example 1.

An even greater decrease in the rate of growth (to 0.11 h- ¹ ) by limitation is observed when no action is taken after reaching the lower value nl / n2 = 5. Then the ratio drops to the value 2 (Ci = 0.16 ppm, 02 = 8%).

However, the value nl / n2 = 2 is also obtained when the exhaust gas concentration is in the explosive range (for example, Ci = 0.3, O2 = 15%).

Claims (2)

claim 1. A method for culturing methanotrophic bacteria »characterized in that the eye-guided amount of oxygen and the pressure are gradually increased while maintaining certain ratios of dissolved oxygen concentration in the culture liquid sum volume fraction of oxygen in the exhaust gas mixture. 2. The method according to claim 1, characterized in that the ratio to be set dissolved oxygen concentration in the culture liquid au volume fraction of oxygen in the exhaust gas mixture in the limits of 5 to 00, preferably 8 to 25, is located.