JPH03133947 | RECOVERY OF AMINO ACID |
WO/2014/013081 | METHOD |
SCHUPP BENJAMIN (AT)
REISCHL BARBARA (AT)
TAUBNER RUTH-SOPHIE (AT)
PALABIKYAN HAYK (AT)
FINK CHRISTIAN (AT)
ERGAL IPEK (AT)
FENNESSY ROSS (AT)
WO2016179545A1 | 2016-11-10 | |||
WO2020252335A1 | 2020-12-17 | |||
WO2012110256A1 | 2012-08-23 | |||
WO2014128300A1 | 2014-08-28 | |||
WO2017070726A1 | 2017-05-04 | |||
WO2016179545A1 | 2016-11-10 | |||
WO2020252335A1 | 2020-12-17 |
US20110281333A1 | 2011-11-17 | |||
US20110281333A1 | 2011-11-17 | |||
US20180179559A1 | 2018-06-28 | |||
EP2192170A1 | 2010-06-02 | |||
US20180163240A1 | 2018-06-14 | |||
US20190194630A1 | 2019-06-27 | |||
US20170130211A1 | 2017-05-11 | |||
US11260039B1 | 2022-03-01 | |||
CN106520651A | 2017-03-22 | |||
US20060057685A1 | 2006-03-16 |
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CLAIMS 1. A method of producing amino acids by fermentation in a bioreactor, wherein the bioreactor comprises methanogenic microorganisms in a fermentation broth, the method comprising at least the step of feeding a gaseous carbon source comprising carbon dioxide and/or carbon monoxide, a nitrogen source comprising nitrogen gas and preferably a sulfur source to the bioreactor under conditions such that the methanogenic microorganisms produce the amino acids, wherein the fermentation broth comprises ammonium at a concentration of 0.1 mmol/L to 200 mmol/L, preferably 2 mmol/L to 100 mmol/L, more preferably 4 to 40 mmol/L. 2. The method of claim 1, wherein hydrogen gas, acetate, a methyl compound preferably selected from methylamines, methyl sulfides and methanol, any other alcohol, preferably a secondary alcohol such as 2-propanol or 2-butanol, a methoxylated aromatic compound and/or formate are fed to the bioreactor; preferably wherein hydrogen gas, acetate, methanol or combinations thereof are fed to the bioreactor. 3. The method of any one of claims 1 to 2, further comprising the step of harvesting at least a portion of the amino acids from the bioreactor. 4. The method of any one of claims 1 to 3, wherein the amino acids further comprise, or wherein said portion further comprises, at least one, preferably at least two or even at least three, more preferably at least four or even at least five, even more preferably at least seven or even at least nine, yet more preferably at least 12 or even at least 15, yet even more preferably at least 17 or even at least 18, especially all of the 20 canonical amino acids. 5. The method of any one of claims 1 to 4, wherein the methanogenic microorganisms comprise archaea selected from any of Methanobacteriales, Methanococcales, Methanomicrobiales, Methanosarcinales, Methanopyrales, Methanocellales, Methanomassiliicoccales, and Methanonatronarchaeales, preferably Methanobacteriales and Methanococcales; more preferably selected from Methanobacteriaceae, Methanocaldococcaceae and Methanococcaceae; in particular selected from Methanothermobacter, Methanothermococcus, Methanocaldococcus and Methanococcus. 6. The method of any one of claims 1 to 5, wherein the methanogenic microorganisms comprise at least two different species. 7. The method of any one of claims 1 to 6, wherein the total amino acid production rate per volume of fermentation broth is at least 0.01 µmol L-1 h-1, preferably at least 0.05 µmol L-1 h-1, more preferably at least 0.1 µmol L-1 h-1, even more preferably at least 0.5 µmol L-1 h-1, yet even more preferably at least 1.0 µmol L-1 h-1, especially at least 5 µmol L-1 h-1 or even at least 10 µmol L-1 h-1. 8. The method of any one of claims 1 to 7, wherein the total amino acid production rate per biomass is at least 0.1 µmol g-1 h- 1, preferably at least 0.5 µmol g-1 h-1, more preferably at least 1.0 µmol g-1 h-1, even more preferably at least 5 µmol g-1 h-1, yet even more preferably at least 10 µmol g-1 h-1, especially at least 50 µmol g-1 h-1 or even at least 100 µmol g-1 h-1. 9. The method of any one of claims 1 to 8, wherein the method is a continuous process, a fed-batch process, a batch process, a closed batch process, a repetitive batch process, a repetitive fed-batch process or a repetitive closed batch process, preferably a continuous process, a fed-batch process or a repetitive fed-batch process, especially a continuous process. 10. The method of any one of claims 1 to 9, wherein at least 50%, preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, yet even more preferably at least 90% or even more preferably at least 95%, especially at least 99% or even at least 99.9% of all nitrogen atoms of all nitrogen sources fed to the bioreactor are fed to the bioreactor in the form of nitrogen gas. 11. The method of any one of claims 1 to 10, wherein at least 50%, preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, yet even more preferably at least 90% or even more preferably at least 95%, especially at least 99% or even at least 99.9% of all carbon atoms of all carbon sources fed to the bioreactor are fed to the bioreactor in the form of carbon dioxide gas and/or carbon monoxide gas. 12. The method of any one of claims 1 to 11, wherein the sulfur source is fed to the bioreactor, preferably wherein the sulfur source comprises cysteine and/or sulfide, especially wherein the fermentation broth comprises sulfide at a concentration of 0.001 – 150 mg/L, preferably 0.01 – 100 mg/L, especially 1 to 80 mg/L and/or wherein the sulfide feed rate or cysteine feed rate is 0.0001 – 0.2 mol L-1 h-1, preferably 0.001 – 0.05 mol L-1 h-1. 13. The method of any one of claims 1 to 12, wherein methane is harvested from the bioreactor. 14. The method of any one of claims 1 to 13, wherein the fermentation is started with the methanogenic microorganisms in chemically defined fermentation medium. 15. Use of methanogenic microorganisms for producing amino acids from an electron donor compound, a gaseous carbon source comprising carbon dioxide and/or carbon monoxide, a nitrogen source comprising nitrogen gas and preferably a sulfur source in a fermentation broth, wherein the fermentation broth comprises ammonium at a concentration of 0.1 mmol/L to 200 mmol/L, preferably 2 mmol/L to 100 mmol/L, more preferably 4 to 40 mmol/L; preferably wherein the electron donor compound is selected from hydrogen gas, acetate, a methyl compound preferably selected from methanol, methylamines and methyl sulfides, any other alcohol, preferably a secondary alcohol such as 2-propanol or 2-butanol, a methoxylated aromatic compound, formate, and mixtures thereof. |
Examples Example 1: Amino acid production and active secretion by Methanothermobacter marburgensis under N2-fixing conditions The aim of our research was to examine the physiological and biotechnological characteristics of biological N 2 fixation in connection to H 2 /CO 2 utilization of different methanogens. Among several methanogens analyzed, Methanothermobacter marburgenis was prioritized to investigate N 2 fixation, CH 4 production, and amino acid excretion characteristics in closed batch and fed-batch cultivation modes and at different NH 4 + concentrations. In brief, M. marburgensis was grown on chemically defined minimal medium with different concentrations of ammonium chloride (NH 4 Cl) in a N 2 /H 2 /CO 2 atmosphere. This enabled the quantification of ammonia uptake, N 2 fixation, amino acid excretion and the conversion of H 2 /CO 2 to CH 4 . N 2 fixation by M. marburgensis was be confirmed in all experiments with H 2 /N 2 /CO 2 in the gas phase. Furthermore, the active excretion of proteinogenic amino acids was found, with highest detected values of glutamic acid, alanine, glycine and asparagin. The highest general production of 7.5 µmol L -1 h -1 was detected under “100%” NH4 + concentration (see Table 1 below) in closed batch after 40 h. Hence, the concomitant production of amino acids and CH 4 from CO 2 turned out to be of biotechnological relevance in an integrated approach coupling biomethanation and N 2 fixation in a biorefinery concept. Materials and methods Strains The following strains were selected for experiments: Methanothermobacter marburgensis (Schönheit et al. 1980; Wasserfallen et al. 2000), Methanobacterium thermaggregans (Blotevogel and Fischer 1985), Methanococcus maripaludis (Jones et al. 1983), Methanocaldococcus villosus (Bellack et al. 2011) and Methanothermococcus okinawensis (Takai et al. 2000). These strains may e.g. be obtained from the DSMZ, Braunschweig, Germany. Media Pre-cultures of M. villosus and M. okinawensis, were grown on chemically defined medium according to Taubner & Rittmann, 2016. M. maripaludis was grown in McN medium (cf. Mauerhofer et al. 2021). Methanobacterium thermaggregans and Methanothermobacter marburgensis were cultivated on MM medium. MM Medium (see also Table 1 below): NH 4 Cl 2.1 g/L and KH 2 PO 4 6.8 g/L in ddH 2 O. 200x trace element (TE) solution to be added (e.g. up to a concentration of 5 mL per L of MM medium): Titriplex I 9 g/L, add 800 mL H 2 O and adjust the pH to 6.5 with 5 mol/L NaOH solution, then add (up to target concentration): MgCl 2 ·6H 2 O 8 g/L, FeCl 2 ·4H 2 O 2 g/L, CoCl 2 ·6H 2 O 40 mg/L, NiCl 2 ·6H 2 O 240 mg/L, NaMoO 4 ·2H 2 O 40 mg, then adjust to pH 7.0 and volume of 1 L with 1 mol/L NaOH and ddH 2 O. The medium may further contain e.g. 3.6 g/L NaHCO 3 as a carbon source. As a sulfur source, e.g. 2 mL of 0.5 mol/L Na 2 S·9H 2 O per liter may be added after anaerobization and autoclaving. Surprisingly, it turned out that it was advantageous that the TE solution (and consequently, the medium) did not contain tungstate (or, at least, only below 0.1 µmol/L, preferably below 0.01 µmol/L, especially below 0.001 µmol/L), as amino acid yield under nitrogen fixing conditions was lower otherwise. Medium was aliquoted into 117 mL serum bottles (VWR, Austria) to a total working volume of 50 mL, closed with blue rubber stoppers (pre-boiled for ten times 30 min, 20 mm, butyl rubber, Chemglass Life Sciences) and aluminum crimp caps (Ochs Laborbedarf, Bovenden, Germany). Sterile L-Cysteine-HCl·H 2 O, sterile NaHCO 3 solution and Na 2 S·9H 2 O according to the media composition were added after autoclaving in an anaerobic glove box (Coy Laboratory Products, Grass Lake, USA). To ensure conditions are anaerobic the atmosphere in the headspace was changed by vacuuming and gassing with the respective gas (H 2 /CO 2 or H 2 /CO 2 /N 2 ) mixture up to 2 bar rel. (3 bar abs.) repeating the procedure five times (Taubner & Rittmann 2016). For gassing, sterile syringe filters (w/0.2c µm cellulose, VWR International, USA) and sterile needles (disposal hypodermic needle, Gr 14, 0.60 × 30 mm, 23 G × 11/4′′, Braun, Germany) were used. Nitrogen-free (N-free) media were prepared by omitting NH 4 Cl (2.1 g L -1 ) and replace it with a chemically equal molar amount of NaCl (2.3 g L -1 ) to ensure the correct salt concentration in the medium. To replace L-cysteine monohydrate, a diluted HCl solution was used to retrieve the pH value. MM medium with varying NH 4 + concentrations, was made as shown in Table 1. To ensure that CO 2 was the only carbon source, Na 2 CO 3 was replaced by equal molarities of NaCl. Medium without Na 2 CO 3 was manually adjusted to pH 6.8 by titrating 10 mol L -1 NaOH. Table 1: Composition of MM medium with varying NH4 + concentration * g L -1 100% + 100% - 50% - 25% - 10% - 5% - 1% - 0% - KH 2 PO 4 6.8 6.8 6.8 6.8 6.8 6.8 6.8 Na 2 CO 3 0 0 0 0 0 0 0 NH 4 Cl 2.1 1.05 0.525 0.21 0.105 0.021 0 NaCl 0 2 2.15 2.725 3.07 3.185 3.277 3.3 mL L -1 TE 200x 5 5 5 5 5 5 5 5 * in relation to original MM medium in g L -1 . Percentage of NH 4 Cl in relation to original MM medium (which already contains 2.1 g L -1 NH4Cl). -/+ presents the absence and presence of Na2CO3. TE: trace element solution Chemicals H 2 (99.999%), CO 2 (99.999%), N 2 (99.999%), H 2 /CO 2 (80%/20%), H 2 /CO 2 /N 2 (77.74%/11.13%/11.13%) were used for closed batch and fed-batch experiments. For gas chromatography (GC), N 2 /CO 2 (80%/20%), CH 4 (99.995%) and the standard test gas (Messer GmbH, Wien, Austria) (containing 0.01 Vol.-% CH 4 , 0.08 Vol.-% CO 2 in N 2 ) was additionally used. All gases, except the standard test gas, were purchased from Air Liquide (Air Liquide GmbH, Schwechat, Austria). All other chemicals were of highest grade available. Closed batch experiments Cultures were incubated in a water bath (Burgwedel, Germany) at 65°C (M. marburgensis, M. thermaggregans and M. okinawensis) or in a shaking air incubator at 37°C (M. maripaludis) (Burgwedel, Germany) and 80°C (M. villosus) (LABWIT Scientific Pty Ltd, Australia). For the purpose of N2 fixation, all closed batch experiments were performed in a H 2 /N 2 /CO 2 atmosphere. For selecting the strain for prioritization, M. marburgensis, M. maripaludis, M. thermaggregans, M. villosus and M. okinawensis were grown in triplicates (n = 3) with one zero control to an OD 578 of approximately 0.7. As M. marburgensis had one of the highest NH 4 + concentrations in the media, it was additionally examined if growth was affected by reducing the amount of NH 4 + to one tenth of the original media concentration. To remove residual nitrogenous compounds from the media, pre-culture cells were washed before inoculation. For a complete N-free media all cultures were washed three times, for all other experiments one or no time. Experiments of only M. marburgensis were performed in quadruplicates (n = 4) or octuplicates (n = 8) at different NH 4 + concentrations (“0%”, “1%”, “10%”, “25%”, “50%” and “100%”, cf. Table 1 above) in relation to original media composition of 2.1 g L -1 . A pre-culture with one tenth of NH 4 + served as inoculum. The batch with 0% served as negative control and 100% gassed with H 2 /CO 2 in the ratio 4:1 as positive control. After every incubation time the serum bottles were left at room temperature for 45 min to cool down. Pressure was measured with a digital manometer (Keller GmbH, Winterthur, Switzerland). Growth was measured spectrophotometrically via OD (λ = 578 nm, blanked with Milli-Q water) (Beckman Coulter, California, USA). Liquid samples of 1 mL were taken and centrifuged at full speed (13200 rpm) for 30 min. Cell pellets and supernatant of each experiment were stored in sterile Eppendorf tubes until further analysis at -20°C. Fed-batch experiments All fed-batch experiments were performed with M. marburgensis in triplicates with H 2 /CO 2 /N 2 gassing at a ratio of 7:1:1 in a DASGIP® 2.2 L bioreactor system (SR1500ODLS, Eppendorf AG, Hamburg, Germany) with 1.5 L working volume of MM medium including 100 µl L -1 of antifoam (Struktol SB2023, Schill und Seilacher, Hamburg, Germany). Best growth conditions are pH of 7 and a temperature of 65°C. Gassing of N 2 and CO 2 was controlled via the MX4/4 unit (Eppendorf AG, Hamburg, Germany). H 2 gas flow was controlled via the C100L Unit (Sierra Instruments, Monterey, USA). Gassing was performed with the same ratio as closed batch experiments. Redox potentials and pH values were monitored by individual redox- and pH-probes (Mettler Toledo GmbH, Wien, Austria). Every fed-batch cultivation was performed with the inoculum of a stock culture of M. marburgensis, adapted to fed-batch cultivation. Before inoculation, the bioreactor was gassed with H 2 /N 2 /CO 2 to ensure conditions are anaerobic and 5 mL of 0.5 mol L −1 Na 2 S·9H 2 O were added. Immediately after inoculation of 30 mL, feeding of 0.2 mL h -1 0.5M Na 2 S·9H 2 O was started and the agitation speed was set to 1600 rpm. Gaseous samples were taken after approximately 0, 13, 16, 19, 22 and 25 h. Batches with and without Na 2 CO 3 in the media, performed under H 2 /CO 2 atmosphere (ratio 4:1) served as reference. For analyzing growth and amino acid excretion, liquid samples were taken and treated as described above. Ammonium determination NH 4 + determination was performed using a modified procedure according to the method described before (Kandeler 1988). The oxidation solution, the colour reagent and the NH 4 Cl stock solution were prepared freshly before the measurement. As standards, nine different concentrations, ranging from 100 µmol L -1 to 1000 µmol L -1 of NH 4 Cl, were prepared. Samples were diluted with MilliQ to an end concentration between the standard ranges. Before the measurement, 300 µL of colour reagent and 120 µL oxidation solution were added immediately to the standards and samples and shortly mixed. After 30 min in the dark, measurement (λ = 660nm) was performed using a 96 well plate (Microtest Plate 96 Well, F, Sarstedt AG & C0, Nümbrecht, Germany) with a plate photometer (Sunrise plate reader, Tecan Group AG, Männedorf, Switzerland). Regression curve R 2 was always higher than 0.999. Gas chromatography Closed batch experiments and the off-gas composition (H2, CO 2 , CH 4 and N 2 ) of the collected gas samples from fed-batch experiments were analyzed by using the Agilent Gas Chromatograph (Agilent 7890A GC, Agilent Technologies, Santa Clara, CA, USA) equipped with a thermal conductivity detector (TDC) and a 19808 Shin Carbon ST Micropacked Column (Restek GmbH, Bad Homburg, Germany). Amino acid analysis For amino acid analyses, supernatant of samples (obtained as described above) were diluted with Mill-Q water at the ratio of 1:4. Measurements were performed on Agilent 1260 Infinity Bioinert HPLC system containing a fluorescence detector, a column oven, an autosampler and a quaternary pump. 1 mL of sample was mixed with 75 µL borate buffer (0.4 N in water, pH = 10.2; Agilent Technologies) followed by 5 µL OPA reagent, (3- mercaptopropionic acid in 0.4 mol L -1 borate buffer and 10 mg mL -1 of o-phthalaldehyde (OPA); Agilent Technologies). 100 µL of the mixture was injected to HPLC system after 2 min at 27 °C. Fluorescent derivates (primary dissolved free amino acids) were separated at 25 °C and a flow rate of 0.8 mL min -1 on a Zorbax ECLIPSE AAA column (4.6 x 150 mm, 3.5 µm particle size, Agilent Technologies) with a Zorbax ECLIPSE AAA guard cartridge (4.6 x 150 mm, 5 µm particle size, Agilent Technologies). Excitation wavelength was 340 nm and emission 450 nm. The use of a gain factors 9 or 10 was depended on the expected concentration and pre-tested before. For identification and quantification of peaks a primary amino acid standard mix (AAS18, Sigma Aldrich) in different concentrations was prepared for each run according to the concentration range of the samples (100 nmol L -1 to 15 µmol L -1 ). AAS18 standard mix lacks five amino acids (asparagine (Asn), glutamic acid (Glu), gamma-aminobutyric acid (GABA), taurine (Tau), tryptophane (Trp); Sigma Aldrich) which were added. In total 20 different AA could be measured with this method. Valine and Methionine were excluded from evaluation as they are located within a signal noise “ammonium peak” and therefore hard to measure within experiments with high NH+ 4 concentrations. The details of this method were as published before (Taubner et al. 2019). GC analysis The relative pressure in bar within the serum bottle was measured with a digital manometer (Keller GmbH, Winterthur, Switzerland). Gaseous substance (n /mol) in the serum bottles headspace was calculated via the ideal gas law. Headspace volume was determined in earlier experiments and adjusted after every OD measurement by the extracted sample volume of 0.75 mL. All measurements were performed at room temperature (25°C). To obtain the actual amount of N 2 the pressure inside the serum bottles was multiplied by 0.11392 based on the exact percentage of N2(11.392 Vol.-%) in the gas mixture, then multiplied with the normalized gas composition gained from GC measurement. The values of the zero-control served as N 2 baseline. Molecular N 2 uptake rate (NUR / mmol L -1 h -1 ) was calculated by dividing the deviation of N 2 before and after incubation ( ^N 2 ) by volume of the liquid medium and the time since last incubation ( ^t): The quantitative/specific nitrogen uptake (qN 2 / mmol h -1 g- 1 ) was determined by dividing the NUR by the biomass concentration (x / g L -1 ) calculated with an experimentally determined coefficient: Carbon dioxide uptake rate (CUR / mmol L -1 h -1 ), molecular hydrogen uptake rate (HUR / mmol L -1 h -1 ), CH4 evolution rate (MER / mmol L -1 h -1 ), carbon balance (C-balance), yields (Y (CH4/CO2) and Y (x/CO2) ) and biomass productivity (r x / c-mmol L -1 h -1 ) was calculated as described elsewhere (Taubner et al. 2016; Rittmann et al. 2012, Bernacchi et al. 2014). The concentrations of H 2 , CO 2 , N 2 and CH 4 after GC measurements were obtained. Results Prioritization of strains Growth of M. marburgensis, M. maripaludis S0001, M. thermaggregans, M. villosus and M. okinawensis was analyzed in a H 2 /CO 2 /N 2 atmosphere in defined, but NH 4 + containing medium, and a N-free medium. This allowed us to screen for NH4 + uptake, N2 fixation, amino acid excretion and the conversion of H 2 /CO 2 to CH 4 in parallel. All methanogens but M. thermaggregans could be grown to an OD 578 of 0.7 in NH 4 + containing medium. Further experiments showed that a certain amount of NH 4 + was necessary for growth under these conditions. Due to most favorable growth characteristics in these experiments, M. marburgensis was selected for further experiments. Growth at specific NH 4 + concentrations with and without bicarbonate (Na 2 CO 3 ) was already performed and showed no or nitrogen-limited growth in 0% and 1 % and similar growth in all other concentrations. In experiments with Na 2 CO 3 in the media, a higher growth rate was shown. NH 4 + uptake kinetics of M. marburgensis Closed batch experiments of M. marburgensis, were then performed with 0%, 5 %, 7.5%, 10 % and 100% NH 4 + in relation to original media composition of 2.1 g L -1 , gassed with H2/CO2/N2 in the ratio 7:1:1. To reduce the possibility of a NH 4 + carryover the experiments were performed with one washing step in octuplicates (n = 8) with additional zero controls. Due to the biomass washing step a slower growth compared to non-washed biomass experiments was observed. After 77.17 h the OD 578 located between 0.17 and 0.20. The 100% 4:1 positive control as expected showed the best OD 578 value around 0.25. Gas samples were taken after approximately 40, 59 and 77 h (Figure 1). Additional fed-batch experiments (n = 3) at NH 4 + concentrations of 0%, 1%, 5%, 10% and 100% were performed. Reference runs showed similar growth up to an OD 578 of 7.0 and 8.1 (Figure 1). Comparing the runs at 100% NH 4 + , a 2.2-fold higher OD 578 was obtained with Na 2 CO 3 in the media. This effect was also visible in closed batch experiments. NH 4 + concentration of 10% showed a stagnation after 20 h reaching a final OD 578 of 1.6, 5% displays stagnation after 15 h reaching an OD 578 of 0.9. A detailed description of H 2 and CO 2 uptake rates, HUR and CUR, respectively, and MER is shown in Table 2. Table 2: Hydrogen Uptake Rate (HUR), Carbon dioxide Uptake Rate (CUR), Nitrogen Uptake Rate (NUR) and Methane Evolution Rate (MER) and C-Balance of closed batch experiments of M. marburgensis with different NH 4 + concentrations. The interplay of simultaneous uptake of NH 4 + and N 2 An NH 4 + uptake during N 2 fixation is evident in closed batch and fed-batch experiments. Comparing Figure 2 (chart a) to Figure 2 (chart b) it is visible that during in fed-batch cultivation an NH 4 + limitation was observed at 0-10% of NH 4 + , whereas in closed batch cultivation NH 4 + was never completely consumed. Further, it is noticeable that within the closed batch experiments the NH 4 + concentration does not differ much between time points, in contrast to fed-batch experiments where NH 4 + decreases over time (Figure 2). The highest consumption was achieved in the positive control experiments. In case of closed batch experiments the highest ammonia uptake rate (AUR) was achieved within the 100% 4:1 run with 243.8 µmol L -1 h -1 at a qN 2 of 4.6 µmol h -1 g- 1 , and in case of fed-batch experiments the highest AUR of 577.3 µmol L -1 h -1 and qN 2 of 10.8 µmol h -1 g- 1 in 100% was obtained with bicarbonate in the media. Highest NUR was calculated from 7.5% and 100% closed batch experiments after 40 h with 0.91 or 0.83 mmol L -1 h -1 respectively and from 10% after 59 h with 0.88 mmol L -1 h -1 . The qN 2 shows the same pattern (Table 3). Earlier sampling time points showed higher NUR, later time points lower, but more balanced NUR values. Table 3: NUR [mmol L -1 h -1 ] and qN2 [mmol h -1 g -1 ] of closed batch experiments in 5%, 7.5%, 10% and 100% NH4Cl. Amino acid excretion by M. marburgensis Active amino acid excretion by M. marburgenis was investigated in closed batch (Figure 3) and fed-batch (Figure 4) experiments. Independent of the cultivation mode almost all detectable amino acids were found. The highest excreted amino acids were glutamic acid (Glu), alanine (Ala), glycine (Gly) and asparagin (Asn). The concentrations of Glu, Gly and Asn were constantly increasing during the course of the cultivation, yet Ala was consumed after a certain timepoint. All cultivation experiments showed a clear NH 4 + dependendence where amino acid excretion in 5%, 7.5% and 10% varies from 100%. (Figure 3 and 4). Taking a closer look to volumetric values, in closed batch the highest value was obtained with glutamic acid with up to 4.59 µmol L -1 h -1 in 5% after 40 h and a smaller amout of alanine with up to 1.36 µmol L -1 h -1 . Highest value of Gly was achieved in 100% after 40 h with 0.99 µmol L -1 h -1 . Fed-batch cultivation lead to Ala as the most excreted amino acid, highest in 5% with up to 2.67 µmol L -1 h -1 . It was also noticable that in 100% Asn was produced 10-fold higher in comparison with the other NH 4 + concentrations with up to 0.79 µmol L -1 h -1 . Examining the total amount of excreted amino acids, closed batch shows an increasing excretion of amino acids over time with the highest in later timepoints with 14.67 to 18.44 µmol L- 1 . Fed-batch shows in general a higher total amount of AA excretion, with highest value of 156.08 µmol L -1 (Figure 5). On the contrary it is to note, that during closed batch experiments showed a higher production rate [µmol L -1 h -1 ] than fed-batch experiments. The presence of too much NH 4 + in the media seems to serve as an inhibitor for amino acid production, as 100% experiments showed slightly lower values (Figure 5). A comparison of the total uptake of NH 4 + with the total amino acids excretion rate indicated that amino acid excretion rate increased with increasing AUR. Furthermore, during depletion of NH 4 + , the concentration of AA did not increase (Figure 5). Conclusions In the context of „power to gas” technology, biological methanation with CO 2 originating from renewable sources and in combination with N 2 fixation, the production of amino acids by methanogens is of high economic interest. To our knowledge there is no study yet that examined a combined CO 2 /N 2 fixation bioprocess. A switch of cultivation mode, closed or fed-batch, could alter amino acid excretion rates and concentration, e.g. in the case of Glu from the highest fed-batch value of 2.74 mg L -1 to 44.83 mg L -1 in closed batch (Table 3). Surprisingly, a variety of amino acids turned out to be actively excreted, with the highest total amount of up to 7.5 µmol L -1 h -1 in early time points (Figure 5). These results underscore that a methanogenic microorganism is well suited for production of amino acids in a biotechnological context, even under N 2 -fixing conditions. Example 2: Amino acid production and active secretion by Methanothermobacter marburgensis in continuous culture Continuous culture of M. marburgensis for amino acid production was successfully established. Experiments were carried out with M. marburgensis in 2 L bioreactors (Eppendorf AG, Hamburg, Germany) and in a 15 L bioreactor (Biostat C+ ,Sartorius Stedim Biotech AG, Göttingen, Germany). For fermentation, original MM medium as described in Example 1 above was used. The same medium was used as feed medium for continuous cultivation mode. To ensure anaerobic conditions inside the reaction vessel, the whole system was flushed with an H 2 /CO 2 , N 2 or H 2 /CO 2 /N 2 mixture for 10 minutes prior to inoculation. Cultivation was performed at 65°C and a stirrer speed of 100 to 1200 rpm (DASGIP parallel bioreactor system, Eppendorf AG, Hamburg, Germany) and from 100 to 1500 rpm (Biostat C+, Sartorius Stedim Biotech AG, Göttingen, Germany). The pH was measured by a pH probe (Mettler Toledo GmbH, Vienna, Austria or Hamilton Bonaduz AG, Bonaduz, Switzerland) and kept constant at a value of 7. The oxidation reduction potential (ORP) was measured by a redox probe (Mettler Toledo GmbH, Vienna, Austria). A 0.5 mol/L Na 2 S·9H 2 O solution was used as sulphur source and fed constantly to the bioreactor at e.g. 0.2 mL/h to 1.32 mL/h. The MM medium was applied using an analog peristaltic pump. The MM medium feed flow rate, the sodium Na 2 S·9H 2 O feed rate and the titration was recorded gravimetrically or adjusted by pump speed. The bioreactor volume was kept constant by withdrawing culture suspension over an immersion pipe using a peristaltic pump controlled on a fixed bioreactor weight, or by using a pipe at a fixed height as a level control system. The withdrawn suspension was collected in a harvest bottle and its volume recorded gravimetrically. All solutions were made anaerobic by flushing with N 2 , H 2 /CO 2 , or H 2 /CO 2 /N 2 . To maintain anaerobic conditions, all bottles were pressurized with N 2 . Pure H 2 /CO 2 (4:1) was used as substrate for M. marburgensis. CO 2 gas flow was controlled via the MX4/4 unit (Eppendorf AG, Hamburg, Germany). H 2 gas flow was controlled via the C100L Unit (Sierra Instruments, Monterey, USA). About thirty different runs of continuous cultures of M. marburgensis were performed under anaerobic conditions. The volume of a run ranged from 1.6 L to 10.29 L. Dilution rate D was varied between runs, in particular with values for D from 0.0125 h -1 to 0.05 h -1 . Volume gas per volume liquid per minute (vvm) was also varied between runs, e.g. from 0.125 to 0.5. Agitation (rpm) was also varied between runs, for instance from 375 to 1500. As a sulfur source, 0.5 mol/L Na 2 S was supplied from e.g. 0.2 mL/h to 1.32 mL/h. Typically, the ammonium concentration was kept between 15 mmol/L and 35 mmol/L. Importantly, volumetric amino acid production rate and specific amino acid production rate ranged from about 25 to about 75 µmol L -1 h -1 and from about 50 to about 2000 µmol h -1 g -1 , respectively (total over all amino acids). The production and secretion into the culture supernatant of the following amino acids (in combination) was typically observed: Asp, Glu, Asn, Ser, His, Gln, Gly, Thr, Arg, Ala, Tyr, Val, Met, norvaline (Nva), Trp, Ile, Phe, Leu, Lys. Individual amino acid production rates were observed up to about 40 µmol L -1 h -1 (volumetric) and up to about 900 µmol h -1 g -1 (specific per biomass). Cys and Pro were not detected due to analytic constraints, but are expected to be produced and secreted as well. In conclusion, reliable production of amino acids was observed in continuous culture. The secretion of these amino acids to the supernatant is especially remarkable, as it simplifies downstream steps (e.g. no cell lysis required for harvesting the product). Also surprising was the observed production of Nva, which has not been observed before in methanogenic archaea (let alone in Methanobacteriales). Example 3: Amino acid production and active secretion in further methanogenic archaea Production of amino acids, including canonical amino acids and Nva, and their active secretion were observed in methanogenic archaea other than M. marburgensis, namely in Methanocaldococcus jannaschii, Methanococcus igneus and Methanocaldococcus villosus. These methanogenic microorganisms were incubated under closed batch conditions similar to the conditions disclosed in Example 1, but at their respective preferred temperatures in 282-based medium (see also Mauerhofer et al, 2021). While for instance Glu production was more pronounced under these conditions, Nva production was also clearly observed for each of Methanocaldococcus jannaschii, Methanococcus igneus and Methanocaldococcus villosus (volumetric Nva production rates reached beyond 1.0 µmol L -1 h -1 , specific Nva production rates beyond 10 µmol g -1 h -1 ). Production of Nva has not been observed before in methanogenic archaea (let alone in Methanococcales). In summary, the production and active secretion into the culture supernatant of the following amino acids (in combination) was observed for methanogenic microorganisms: Asp, Glu, Asn, Ser, His, Gln, Gly, Thr, Arg, Ala, Tyr, Val, Met, Nva, Trp, Ile, Phe, Leu and Lys. Example 4: Amino acid production and active secretion by Methanothermobacter marburgensis in continuous culture (further experiments) Experiments were carried out with M. marburgensis in 2.2 L bioreactors (Eppendorf AG, Hamburg, Germany) and in a 15 L bioreactor (Biostat C+ ,Sartorius Stedim Biotech AG, Göttingen, Germany). For fermentation, original MM medium as described in Example 1 above was used. The same medium was used as feed medium for continuous cultivation mode. To ensure anaerobic conditions inside the reaction vessel, the whole system was flushed with an H 2 /CO 2 , N 2 or H 2 /CO 2 /N 2 mixture for 10 minutes prior to inoculation. Cultivation was performed at 65°C and a stirrer speed of 375 to 1500 rpm (DASGIP parallel bioreactor system, Eppendorf AG, Hamburg, Germany) and from 375 to 1500 rpm (Biostat C+, Sartorius Stedim Biotech AG, Göttingen, Germany). The pH was measured by a pH probe (Mettler Toledo GmbH, Vienna, Austria or Hamilton Bonaduz AG, Bonaduz, Switzerland) and kept constant at a value of 7. The oxidation reduction potential (ORP) was measured by a redox probe (Mettler Toledo GmbH, Vienna, Austria). A 0.5 mol/L Na 2 S·9H 2 O solution was used as sulphur source and fed constantly to the bioreactor at e.g. 0.05 mL/h to 1.32 mL/h. The MM medium was applied using an analog peristaltic pump. The MM medium feed flow rate, the sodium Na 2 S·9H 2 O feed rate and the titration was adjusted by pump speed. The bioreactor volume was kept constant. The withdrawn suspension was collected in a harvest bottle. All solutions were made anaerobic by flushing with N 2 , H 2 /CO 2 , or H 2 /CO 2 /N 2 . To maintain anaerobic conditions, all bottles were pressurized with N 2 . Pure H 2 /CO 2 (4:1) was used as substrate for M. marburgensis. CO 2 gas flow was controlled via the MX4/4 unit (Eppendorf AG, Hamburg, Germany). H 2 gas flow was controlled via the C100L Unit (Sierra Instruments, Monterey, USA). More than 100 different runs of continuous cultures of M. marburgensis were performed under anaerobic conditions. The volume of a run ranged from 1.6 L to 10.29 L. Dilution rate D was varied between runs, in particular with values for D from 0.00625 h -1 to 0.05 h -1 , such as 0.025 h -1 . Volume gas per volume liquid per minute (vvm) was also varied between runs, e.g. from 0.125 to 0.5. Agitation (rpm) was also varied between runs, for instance from 375 to 1500. As a sulfur source, 0.5 mol/L Na 2 S was supplied from e.g. 0.05 mL/h to 1.32 mL/h. Typically, the ammonium concentration was kept between 0.21 mmol/L and 41.63 mmol/L. Importantly, volumetric amino acid production rate and specific amino acid production rate ranged from about 5 to about 250 µmol L -1 h -1 and from about 10 to about 150 µmol h -1 g -1 , respectively (total over all amino acids). The production and secretion into the culture supernatant of the following amino acids (in combination) was typically observed: Asp, Glu, Ser, His, Gly, Thr, Arg, Ala, Tyr, Val, Met, norvaline (Nva), Trp, Ile, Phe, Leu, norleucine (Nle), Lys. Individual amino acid production rates were observed up to about 130 µmol L -1 h -1 (volumetric) and up to about 70 µmol h -1 g -1 (specific per biomass). Cys and Pro were not detected due to analytic constraints, but are expected to be produced and secreted as well. In conclusion, reliable production of amino acids was observed in continuous culture. The secretion of these amino acids to the supernatant is especially remarkable, as it simplifies downstream steps (e.g. no cell lysis required for harvesting the product). Also surprising was the observed production of Nva and Nle, which has not been observed before in methanogenic archaea (let alone in Methanobacteriales). Example 5: Amino acid production and active secretion in further methanogenic archaea Production of amino acids, including canonical amino acids, norvaline, ornithine and homoserine, and their active secretion into the culture supernatant were observed in methanogenic archaea other than M. marburgensis, namely in Methanothermobacter thermautotrophicus and a Methanothermococcus sp. These methanogenic microorganisms were incubated under closed batch conditions similar to the conditions disclosed in Example 1, but at their respective preferred temperatures in MM medium (see Example 1; for Methanothermococcus culture, 30 g/L NaCl was added to the medium)and at various ammonium concentrations. While for instance Ala and Glu production was more pronounced under these conditions, Nva production was also clearly observed for each. Production of Nva has not been observed before in methanogenic archaea (let alone in Methanobacteriales or Methanococcales). 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