AT517476B1 - Cell culture status determination - Google Patents

Abstract

The invention relates to a method for determining the status of a microbial culture by measuring at least one culture parameter and then correlating the measured values of the at least one culture parameter with the status of the culture, characterized in that in a microbial methanation process using a hydrogenotrophic methanogenic microorganism before the beginning of the methanation process the specific amino acid secretion pattern of the particular microorganism used is determined; during the process, the concentration of at least one particular amino acid in the culture medium is determined; and, if appropriate, in addition the total protein content in the culture medium and the proportion of the at least one amino acid in the total protein content are determined; according to which the concentration or, where appropriate, the proportion of the respective amino acid in the total protein content is correlated with the status of the culture so as to estimate the viability of the culture and / or to clarify the question whether the methanogen formation rate is gas-limited or liquid-limited.

Description

Description: The invention relates to the determination of the culture status of microbial cultures by measuring the amino acid content.

STATE OF THE ART

In the course of the steadily growing production of renewable energy, especially biological methanation processes (BMP) are becoming increasingly important. In such processes, C02 is reduced to methane by methanogenic microorganisms (which are primarily bacteria, archees, fungi and algae, which are sometimes referred to as microbes for short):

In the later combustion of methane for energy according to the equation:

Carbon dioxide is again formed, which is a C02-neutral cycle. Particularly advantageous is the coupling of BMP with biomass fermentation, in which arise as the main products CH4 and C02, so that the CO 2 portion of the biogas can be converted into CH4. The methane thus obtained is usually of high purity and can be fed, for example, easily into natural gas networks.

Examples of BMP can be found, for example, in US Pat. No. 4,883,753, where fermentations of Metha-nobacterium thermoautotrophicum and Methanococcus thermolitotrophicus are described, and also WO 2008/094282 A1, in which CO.sub.2 from the biomass fermentation is converted to methane by means of methanogens.

In order to be able to optimally control the methanation reaction, it is necessary to know the physiological state of the respective microbial culture, as found by the group of inventors of the present application in earlier research; see S. Rittmann, A.H. Seifert, C. Herwig, "Quantitative analysis of media dilution rate effects on methanothermobacter marburgensis grown in continuous culture on H2 and C02", Biomass. Bioenerg. 36, 293-301 (2012); AH. Seifert, S. Rittmann, S. Bernacchi, C. Herwig, "Method for assessing the impact of emission gasses on physiology and productivity in biological methanogenesis", Bioresour. Technol. 136, 747-751 (2013); AH. Seifert, S. Rittmann, C. Herwig, "Analysis of process related factors to increase the volumetric productivity and quality of biomethane with Methanothermobacter marburgensis", Appl. Energ. 132, 155-162 (2014); S. Bernacchi, M. Weissgram, W. Wukovits, C. Herwig, "Process efficiency simulation for key process parameters in biological methanogenesis", AIMS Bioeng. 1 (1), 53-71 (2014); as well as WO 2014/128300 A1.

A typical fermentation process thus begins with the loading of the bioreactor with culture medium and an initial culture of the microorganism (s) used for the fermentation, which is referred to as the "setup phase". In the following so-called "initial phase", "biomass" is generated by increasing the number of microorganisms used. By the latter term is meant hereinafter in the mass of methanogenic microorganisms, which is used in the sequence for the above-described reaction of C02 to CH4. For this purpose, the biomass, which is increased in the reactor and continues to multiply, is continuously fed with CO 2, H 2 and liquid nutrient medium.

In the course of this research, in addition to conventional factors such as the gas pressures of injected C02 and H2, the supply rate of the medium as a nutrient source, the concentration of nutrients and metabolites and the volumetric productivity, also as a specific methane formation rate ("methane evolution rate", MER), as well as the specific methane production rate (qCH4) of the respective culture as revealing parameters in the methane production determined. These are related as follows: MER (mmol / lh) = biomass concentration (g / l) x qCH4 (mmol / gh) Thus, the volumetric productivity or methane formation rate MER indicates the molar amount of methane per liter Culture and hour of a microbial culture with a defined concentration (in g / l) is produced. The ability to produce methane from this culture is expressed by the specific methane production rate qcm, which in turn indicates the molar amount of methane produced by one gram of biomass per hour.

By measuring various parameters can thus draw conclusions about the state of the culture, including, however, usually several, repeated at a time interval measurements must be performed. Accordingly, there is a need for a method of simplifying the determination of the state of the culture by means of one-time measurements, i. Measurements at a single time. Thus, the object of the present invention was the development of such a simplified method.

DISCLOSURE OF THE INVENTION

[0012] This object is achieved by providing a method for determining the status of a microbial culture by measuring at least one culture parameter selected from the concentrations of nutrients and metabolites, in particular amino acids, in the culture medium and gas partial pressures, and then correlating the measured values of the at least one culture parameter with the status of the culture, characterized in that: in a microbial methanation process using a hydrogenotrophic methanogenic microorganism a) before the start of the methanation process, the specific amino acid secretion pattern of the particular microorganism used is determined; B) during the process, the concentration of at least one specific amino acid in the culture medium is determined; and c) optionally additionally determining the total protein content in the culture medium and the proportion of the at least one amino acid in the total protein content; according to which d) the concentration or optionally the proportion of the respective amino acid on

Total protein content is correlated with the status of the culture, so as to assess the viability of the culture and / or to clarify the question of whether the methane production rate is limited by the gas exchange of the culture with the environment, i. "gas limited", or limited by the liquid nutrient supply, i. "liquid limited" is.

Namely, the inventors have surprisingly found in their recent research that not only the total protein content (ie, amino acid content of fully lysed cells), which is known to be indicative of the viability of a microbial culture, because at high protein concentrations, the viability of the culture in the Is usually restricted, but also the presence of individual amino acids in the culture allows direct conclusions about the status of culture.

In fact, it has been found that microorganisms, even in the fully viable state, apparently secrete certain amino acids into the culture medium, so that for each microorganism a specific secretion pattern exists in the fully viable state. Once this specific secretion pattern has been determined, it is sufficient to determine the concentration of a particular amino acid during the methanation process in order to be able to draw conclusions about the status of the culture - in many ways.

The determination of the specific secretion pattern in step a) comprises both determining which amino acids are secreted by the respective microorganism in the fully viable state generally in the surrounding medium, and determining whether and how the secreted amount of the respective Amino acid in the transition from the gas-limited to the liquid-limited state or vice versa changes. In addition, in those cases where such a change in amino acid secretion occurs, in step a) a threshold for that particular amino acid (s) transition can be determined by also determining the total protein content and the proportion of total amino acid content of the amino acid gas-limited as well as in the liquid-limited state is calculated.

In addition, the specific secretion pattern may also include information about the course of viability. Thus, thresholds for the total protein content and / or for the proportion of an amino acid which is not part of the specific secretory pattern can also be determined in the total protein content in step c), ie. it is ascertained from which total protein content or from what proportion of the respective amino acid on the total protein content limited viability exists and from when a dying culture is present. Herein, restricted viability means a proportion of lysed cells greater than about 15% and a dying culture greater than about 30%.

As a result, it is sufficient according to the method of the invention to determine at any time the concentration of one of these amino acids and optionally also the total protein content at this time and the proportion of the respective amino acid thereto, conclusive statements regarding the status of the culture to meet. Further measurements at later times are therefore not absolutely necessary, but can of course be performed to track status changes.

In the simplest preferred embodiment of the invention, only in step b) the concentration of an amino acid is determined, which was determined in step a) that it is not part of the specific secretion pattern of the microorganism, wherein a concentration of this amino acid> 0 an indicator of at least limited viability of the microorganism in the culture, since the microorganism in the fully viable state does not secrete this amino acid in the medium yes.

If, in addition, in step c) the total protein content and / or the proportion of the amino acid determined in step b) is determined on the total protein content and before a specific threshold value has been determined in step a) shows a total protein content or a proportion of the amino acid in the total protein content Viability limited under the specific threshold determined in step a), and a total protein content or content of the amino acid in the total protein content above the respective threshold indicates a dying culture.

In a preferred embodiment of the method according to the invention additionally or alternatively in step b) the concentration of an amino acid is determined, which was determined in step a) that it is part of the specific secretion pattern of the microorganism, and in step c) the total protein content in the culture medium and the gravimetric fraction of the amino acid determined in step b) are determined on the total protein content, the proportion of this amino acid in the total protein content, depending on whether this proportion is below or above a threshold previously determined in step a), an indicator represents a gas-limited or a liquid-limited methane production.

This applies to any amino acid for which it was found in step a) in the determination of the specific secretion pattern that changes in the culture conditions from gas-limited to liquid-limited state or vice versa, a change in the medium secreted amount of this amino acid, wherein a threshold between the two different sets is determinable or determinable, as demonstrated in the later examples.

Of course, knowing the state of the culture, it is possible to respond to undesirable conditions or developments of the particular culture by adapting the fermentation conditions accordingly.

The hydrogenotrophic methanogenic microorganism according to the present invention is not particularly limited as long as it is capable of reducing carbon dioxide to methane. Preferably, however, the methanogenic microorganism of the present invention is a methanogenic bacterium, and more preferably a methanogenic archaee, e.g. Methanobacterium alcaliphilum, bryantii Methanobacterium, congolense Methanobacterium, defluvii Methanobacterium, espanolae Methanobacterium, Methanobacterium formicicum, Methanobacterium ivanovii, Methanobacterium palustre, uliginosum Methanobacterium thermaggregans, Methanobacterium, Methanobrevibacter acididurans, Methanobrevibacter arbor iphilicus, Methanobrevibacter gottschalkii, olleyae Methanobrevibacter, Methanobrevibacter ruminantium, Methanobrevibacter smithii, woesei Methanobrevibacter, Methanobrevibacter wolinii, marburgensis Methanothermobacter, Methanothermobacter thermoautotrophicus, Methanobacterium thermoautotrophicus, Methanothermobacter thermoflexus, Methanothermobacter thermophilics, Methanothermobacter wolfeii, bavaricum Methanothermus sociabilis, Methanocorpusculum, Methanocorpusculum parvum, Methanoculleus chikuoensis, Methanoculleus submarinus, frigidum Methanogenium, Methanogenium liminatans, Methanogenium marinum, Methanosarcina acetivorans, Methanosarcina Methanosarcina mazei, Methanosarcina thermophila, Methanomicrobium mobile, Methanocaldococcus jannaschii, Methanococcus aeoli-cus, Methanococcus maripaludis, Methanococcus vannielii, Methanococcus voltaei, Me-thanothermococcus thermolithotrophicus and Methanopyrus kandleri. Particular preference is given to methanogenic archaea of the species Methanosarcinia barkeri, Methanothermobacter marburgensis, Methanobacterium thermoautotrophicus, Methanocaldococcus jannaschii, Methanothermobacter thermoautotrophicus, Methanococcus maripaludis, which can each be used individually or as a mixture of several thereof. In particular, the methanogenic archae of the species Methanothermobacter marburgensis, e.g. the strain deposited under DSM 2133 in the German strain collection for microorganisms and cell cultures (DSMZ, Braunschweig, Germany), which represented the starting point for the researches of the inventors.

Archaea are both due to the simple structure of their cell structure as well as the fact that it is a large part to extremophiles that are viable under extreme conditions, and the resulting stability and comparatively easy handling of the cultures and the predictability of their Metabolism and reproducibility of the fermentations are particularly preferred.

Using the above-described Methanothermobacter marburgensis strain as a model organism, the inventors have come to the following results.

In terms of viability, it has been found for this particular organism in step of the method of the invention that aspartic acid is not part of its specific secretory pattern. In order to determine the status of a culture of this microorganism, the concentration of aspartic acid is now determined in step b), whereby at least a limited viability of the culture is indicated by a concentration> 0. This is based on the finding that as soon as aspartic acid is found in such a culture, cell lysis must have been used to a greater or lesser extent. With this in mind, the remaining parameters of the fermentation, e.g. Nutrient supply in the medium, dilution, etc., be adjusted accordingly to counteract the death of the cells.

In a preferred embodiment of the method according to the invention using this strain of Methanothermobacter marburgensis additionally determines the total protein content, by a total protein content> 0.5 pg / ml limited viability and by a total protein content> 2.0 pg / ml a dying culture because the inventors have determined these specific thresholds in step a) of the method of the invention. Such an exact determination of the culture status in terms of viability based on the total protein content was previously impossible according to the prior art.

In other preferred embodiments of the invention, however, it is again determined whether the methane production is limited by the gas exchange of the culture with the environment, i. E. "gas limited", or limited by the supply of nutrients in the liquid medium, i. "liquid limited" is. For this purpose, in one embodiment, the total protein content and the alanine content are determined therefrom, as in the determination of the specific secretion pattern of this strain of Methanothermobacter marburgensis in step a) it was found that alanine - as well as glutamic acid and glycine - are also in a fully viable state by the microorganism are secreted and thus part of this pattern. In addition, in step a) a threshold for the proportion of alanine in the total protein content in the transition from gas-limited to liquid-limited methane production of 0.5 was determined.

Thus, by a measured in step c) gravimetric alanine content <0.5, a gas-limited methane production and by an alanine content> 0.5, a liquid-limited methane production indicated. This embodiment is based on the surprising discovery of the inventors that in a liquid-limited methanation process, the microorganism produces predominantly the amino acid alanine. Once the process is gas limited, i. On the other hand, if the nutrients supplied with the medium can not be completely used up, the proportion of alanine in the total protein content drops below 50%.

Alternatively or additionally, in one embodiment of the present invention in step c) the total protein content and the proportion of glutamic acid are determined there, wherein by a glutamic acid content> 0.5 a gas-limited methane production and by a glutamic acid fraction <0, 5 a liquid-limited methane production is displayed. It is also surprising that the inventors have found that the ratios of glutamic acid are exactly the opposite of alanine: as long as sufficient nutrients are provided to the microorganism with the medium, i. As long as a gas-limited process takes place, mainly glutamic acid is produced. If, on the other hand, nutrients become scarce and the process is therefore limited to liquids, the proportion of glutamic acid in the total protein content drops below 50%.

The concentrations or proportions of other amino acids in the total protein content do not allow such statements regarding the culture status, as evidenced by the later examples, especially since in the fully viable state only a third amino acid, namely glycine, is secreted into the medium, the amount thereof does not change significantly at the transition from gas to liquid-limited state.

In a particularly preferred variant of the latter embodiment of the method of the invention using the above strain Methanothermobacter marburgensiss in step c) in addition to the proportion of glutamic acid, the methane formation rate is determined and based on the amount of biomass supplied to the specific methane production rate qCH4 which is subsequently correlated with the proportion of glutamic acid in the total protein content, whereby a transitional state between gas-limited and liquid-limited methane production becomes detectable.

In knowledge, whether the methanation reaction at the time of measurement gas or liquid is limited, the feed streams can in turn be readjusted accordingly to provide optimum reaction conditions.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be further described hereinafter by way of concrete examples with reference to the accompanying drawings, which show the following.

Fig. 1 shows the specific expression pattern determined in Example 1 of the microorganism used in the examples.

Fig. 2 shows the relationship between the proportion of aspartic acid and the total protein content and the derivable information on the viability of the culture according to Example 2 of the invention.

FIG. 3 shows the relationship between the proportion of aspartic acid and the total protein content as well as the non-derivability of information regarding the process status according to Comparative Example 1.

Fig. 4 shows the relationship between the proportion of glutamic acid and the total protein content and the information derived therefrom with respect to the process status according to Example 3 of the invention.

5 shows the relationship between the proportion of alanine and the total protein content as well as the information derivable therefrom with respect to the process status according to Example 4 of the invention.

6 shows the relationship between the proportion of glycine and the total protein content as well as the non-derivability of information regarding the process status according to Comparative Example 2.

Fig. 7 shows the relationship between the proportion of asparagine and the

Total protein content and the undetrability of information regarding the process status according to Comparative Example 3.

Fig. 8 shows the relationship between the content of glutamic acid and the volumetric productivity and the undetectability of information regarding the process status according to Comparative Example 4.

Fig. 9 shows the relationship between the proportion of glutamic acid and the specific methane production rate and the information derived therefrom with respect to the process status according to Example 5 of the invention.

EXAMPLES

The microorganism used in all model experiments was deposited in the German strain collection for microorganisms and cell cultures (DSMZ, Braunschweig, Germany) strain Methanothermobacter marburgensis DSM 2133 and thus a hydrogenot-rophe, thermophilic, methanogenic Archaee. The cultivation was carried out in a 10-i Biostat C + laboratory reactor (Sartorius Stedim Biotech AG, Göttingen, Germany) with the parameters described in earlier publications of the group of inventors (AH Seifert, S. Rittmann, S. Bernacchi, C. Herwig , "Process efficiency simulation for key process parameters in biological methanogenesis", AIMS Bioeng. 1 (1), 53-71 (2014), S. Rittmann, AH Seifert, C. Herwig, "Quantitative analysis of media dilution rate effects on Methanobacter marburgensis grown in continuous culture on H2 and C02 ", Biomass Bioenergy 36, 293-301 (2012), S. Bernacchi, S. Rittmann, AH Seifert, A. Krajete, C. Herwig," Experimental methods for screening parameters influencing the growth to product yield (Y (x / CH4)) of a biological methane production (BMP) process performed with Methanobacter marburgensis ", AIMS Bioeng. 1 (2), 72-86 (2014)).

The 10-L bioreactor was gravimetrically controlled and the harvest collected within the reaction vessel. All experiments were carried out at a pressure of 2 bar. The dilution rate of the starting medium was controlled by a gravimetric flow controller to a constant value of 0.05 h'1, with the exception of the targeted production of impaired viability and non-viability of the cells, for which purpose the dilution rate was 0.01 or 0, The feed of gas was adjusted to 0.5 volumes per reaction volume per minute (l / l.min) with a ratio H 2 / CO 2 of 4: 1. The culture was stirred at 1500 rpm, a pH of 7 and a temperature of 65 ° C were set, and the reaction volume was kept constant at 5 ℓ (unless otherwise specified).

The trace elements were kept constant at 6x and therefore, as known from the literature, were not limiting for the growth rate. The only difference with the conditions known from the literature was the exchange of the base for a 1.5 M NH 3 solution, which was used both for adjusting the pH and as a nitrogen source. The starting medium therefore further contained KH2PQ4, NaCl and trace elements in a known composition. The process control was carried out using the software Lucullus (available from Securecell, Schlieren, Switzerland).

The determination of the specific secretion pattern and the amino acid concentrations in the culture medium by means of HPLC analysis (Thermo Ultimate 3000 with an Eclipse Plus C18 column and an Agilent 1200 fluorescence detector, all available from Agilent Technologies, Santa Clara, USA). The total protein content was determined by means of the multi-plate reader Infinite M200 Pro (Tecan, Männedorf, Switzerland) with 96-well microplates (Greiner Bio-one, Frickenhausen, Germany) and Bradford reagent from Sigma Aldrich and one using bovine serum albumin (Carl Roth, Karlsruhe, Germany) created calibration curve.

EXAMPLE 1 - Determination of the specific secretory pattern according to step a) of the method according to the invention. Methanothermobacter marburgensis DSM 2133 was cultured until stationary, after which samples were taken and analyzed by HPLC. The result of a triplicate determination is shown in Fig. 1, which shows that this microorganism, in the fully viable state, secretes four different amino acids into the culture medium, namely (in decreasing amounts) alanine, glutamic acid, glycine and traces of asparagine.

As long as no other amino acids are found in the culture medium, such a culture is therefore considered to be fully viable.

Example 2 - Determination of aspartic acid according to step b) and the total protein content according to step c) of the method according to the invention As an illustrative example of an amino acid which is not part of the specific secretion pattern of this microorganism, in step b) of the method In the fully viable state (dilution rate 0.05 h "1), in the restricted viable state (dilution rate 0.01 h" 1) and in the dying state (dilution rate 0.00 h "1) of aspartic acid Culture In parallel, step c) of the method was used to determine the total protein content in all cases.

The results are shown in Fig. 2, wherein the gravimetric fraction of aspartic acid is plotted on the total protein content over the total protein content.

As expected, no aspartic acid was secreted into the medium in the fully viable state. On the other hand, once the viability of culture decreased, a varying amount of aspartic acid was found in the medium, but no conclusions could be drawn about the exact state of the culture (viable or dying). However, viability thresholds could be determined from the total protein content, a threshold of about 0.5 pg / ml for the transition from unrestricted to impaired viability and a threshold of about 2.0 pg / ml for the transition to impaired viability a dying culture.

Thus, according to the present invention, impaired viability of a culture of this microorganism can be detected by detecting the presence of aspartic acid in the culture medium and estimating the extent of the restriction by the total protein content.

COMPARATIVE EXAMPLE 1 - Correlation of aspartic acid portion with gas-limited or liquid-limited state of culture.

By varying the ratio between the feed streams of nutrient medium and gas supply, in each case a gas- and a liquid-limited state of the unrestricted viable culture was brought about and again the aspartic acid concentration and the total protein content were determined. The results are shown in FIG. As expected, the amount of aspartic acid was zero, while the total protein content varied widely and therefore did not allow statements about the state of the culture.

Example 3 - Determination of glutamic acid according to step b) and the total protein content according to step c) of the method according to the invention Comparative Example 1 was repeated, but instead of aspartic acid, which is not part of the specific secretion pattern, glutamic acid was determined by the Microorganism is secreted in an unrestrictedly viable state. The results are shown in FIG.

It can be seen that the microorganism secretes glutamic acid in the gas-limited state, as can be seen from the proportion of glutamic acid in the total protein content of nearly 1. In the liquid-limited state, however, little glutamic acid is secreted, which can be concluded from a proportion of the total protein content below 0.2. Thus, for cultures of this microorganism, a threshold for the proportion of glutamic acid in the total protein content of about 0.5 can be established and the latter used as an indicator of the state of the culture, i. If the proportion of glutamic acid in the total protein content is greater than 0.5, the culture is in the gas-limited state; if it is below that, it is in the state limited to the liquid.

Example 4 - Determination of alanine according to step b) and the total protein content according to step c) of the method according to the invention.

Example 3 was repeated, but alanine was now determined instead of glutamic acid, which is also secreted by the microorganism in the fully viable state. The results are shown in FIG.

In the gas-limited state, the microorganism secretes almost no alanine, in the liquid-limited state, however, predominantly alanine, which can be concluded from proportions of the total protein content between 0.7 and 0.95. However, as the total protein content increases, alanine secretion decreases in the liquid-limited state and increases slightly in the gas-limited state. Thus, for cultures of this microorganism, a threshold for the alanine content in the total protein content of, for example, about 0.5 may be re-established and the latter used as an indicator of the state of the culture, i. If the proportion of alanine in the total protein content is less than 0.5, the culture is in the gas-limited state; if it is above it, it is in the liquid-limited state.

COMPARATIVE EXAMPLE 2 - Correlation of glycine content with gas-limited or liquid-limited state of the culture To verify the ratios in the case of glycine, the third amino acid from which the microorganism in the fully viable state secretes appreciable amounts into the medium, the examples 3 and 4 repeated to determine glycine. The results are shown in FIG.

It can be seen that on the basis of glycine no clear conclusions can be drawn on the culture status, since the amounts vary relatively widely. Although, with increasing total protein content, tendencies towards increased glycine secretion in the liquid-limited state or scarcely any secretion in the gas-limited state are again discernable. However, given the onset of decline in viability of the culture at a total protein content of about 0.5 pg / ml, the glycine content is still not available for reliable conclusions.

COMPARATIVE EXAMPLE 3 - Correlation of the asparagine portion with gas-limited or liquid-limited state of the culture Similar to Comparative Example 2, the proportions of asparagine, the fourth of the fully viable microorganism, are secreted into the medium as shown in FIG , However, asparagine is completely unsuitable for such correlations due to the extremely low levels secreted.

COMPARATIVE EXAMPLE 4 Correlation of glutamic acid content with volumetric productivity In order to examine whether the proportion of glutamic acid in the total protein content permits further conclusions, this was repeated with Example 3 with the volumetric productivity or specific methanation rate MER (in mmol / lh), ie the molar amount of methane formed per liter of culture and hour. FIG. 8 shows that no conclusive statements can be made by means of this correlation.

EXAMPLE 5 - Correlation of glutamic acid moiety with specific methane production rate However, the ratios are different when the glutamic acid moiety is linked to the total protein content with the specific methane production rate qCH4, i. E. the molar amount of methane produced by one gram of biomass per hour. As can be seen from FIG. 9, when the glutamic acid moiety is applied via qcH4, a transition state between the gas-limited and the liquid-limited state can be recognized, in which practically no glutamic acid is secreted into the medium.

This is possibly the optimal state for the process development of the fermentation of this microorganism, in which the cell reproduction and methane production balance, so no amino acid is present in excess and therefore no secretion occurs.

In sum, in any case, it can be clearly seen that the present invention provides a new method for determining the state of a culture of methane-producing microorganisms, by means of which - after the initial determination of the specific secretion pattern of the respective microorganism - a variety of states of a corresponding culture are determined with high reliability which, in the simplest case, only requires the determination of the concentration of a single amino acid during the fermentation process.

Claims (12)

claims 1. A method for determining the status of a microbial culture by measuring at least one culture parameter selected from the concentrations of nutrients and metabolites, in particular amino acids, in the culture medium and gas partial pressures, and then correlating the measured values of the at least one culture parameter with the status of the culture, characterized that in a microbial methanation process using a hydrogenotrophic methanogenic microorganism a) before the start of the methanation process, the specific amino acid secretion pattern of the particular microorganism used is determined; b) during the process, the concentration of at least one specific amino acid in the culture medium is determined; and c) optionally additionally determining the total protein content in the culture medium and the proportion of the at least one amino acid in the total protein content; then d) the concentration or, when step c) is performed, the proportion of the respective amino acid in the total protein content is correlated with the status of the culture so as to determine the viability of the culture and / or determine whether the rate of methane formation by the gas exchange of the culture limited with the environment or limited by the liquid nutrient supply. 2. The method according to claim 1, characterized in that in step b) the concentration of an amino acid is determined, which was determined in step a) that it is not part of the specific secretion pattern of the microorganism, wherein a concentration of this amino acid> 0 a Indicator of at least limited viability of the microorganism in the culture. 3. The method according to claim 2, characterized in that additionally determined in step c) the total protein content and / or the proportion of determined in step b) amino acid in the total protein content, wherein a total protein content and / or a proportion of this amino acid in the total protein content under a respective , specific threshold determined in step a) is an indicator of impaired viability and a total protein content and / or a proportion of this amino acid in the total protein content above the respective threshold represents an indicator of a dying culture. 4. The method according to any one of claims 1 to 3, characterized in that in step b) the concentration of an amino acid is determined, which was determined in step a) that it is part of the specific secretion pattern of the microorganism, and in step c) the total protein content in the culture medium and the proportion of the amino acid determined in step b) in the total protein content, the proportion of this amino acid in the total protein content, depending on whether this proportion is below or above a threshold previously determined in step a), an indicator of a gas-limited o-which represents a liquid-limited methane production. 5. The method according to any one of claims 1 to 4, characterized in that a strain of a methanogenic Archaee is used as the hydro-genotropic methanogenic microorganism. 6. The method according to claim 5, characterized in that a strain of Methanothermobacter marburgensis is used as the microorganism. 7. The method according to claim 6, characterized in that in step b) the concentration of at least one amino acid selected from alanine, aspartic acid and glutamic acid, is determined in the culture medium. 8. The method according to claim 7, characterized in that the concentration of aspartic acid is determined, wherein at least a limited viability of the culture is indicated by an aspartic acid concentration> 0. 9. The method according to claim 8, characterized in that additionally in step c) the total protein content is determined, which is indicated by a total protein content> 0.5 pg / ml limited viability and by a total protein content> 2.0 pg / ml a dying culture is shown. 10. The method according to any one of claims 7 to 9, characterized in that in step b) the concentration of alanine and in step c) the total protein content and the proportion of alanine are determined there, wherein by an alanine content <0.5 a gas-limited methane production is indicated and by an alanine content> 0.5, a liquid-limited methane production is displayed. 11. The method according to any one of claims 7 to 10, characterized in that in step b) the concentration of glutamic acid and in step c) the total protein content and the proportion of glutamic acid are determined there, wherein by a glutamic acid content> 0.5 a gas-limited methane production is indicated and is indicated by a glutamic acid content <0.5, a liquid-limited methane production. 12. The method according to claim 11, characterized in that in step c) additionally determines the methane formation rate and based on the amount of supplied biomass to calculate the specific methane production rate qCH4, which is correlated in the sequence with the proportion of glutamic acid in the total protein content whereby a transition state between gas-limited and liquid-limited methane production becomes detectable.