Isolation and pure culture of an archaebacterium of order <i>Methanomassiliicoccales</i>

Abstract

The present invention concerns a process for isolating an archaeon of the order of commensal clade Methanomassiliicoccales, the use of a culture and/or isolation medium comprising a sterile extract of a bacterial culture of a bacterium of genus Eggerthella, for the isolation and/or culture of an archaeon of the order of commensal clade Methanomassiliicoccales, and a pure, isolated archaeon Methanomethylophilus alvus Mx-05. It also concerns a culture method of an archaeon of the order of commensal clade Methanomassiliicoccales.

Claims

1. An isolation process for isolating an archaeon of the order of commensal clade Methanomassiliicoccales, said isolation process comprising: a) inoculating a biological sample comprising an archaeon of the order of commensal clade Methanomassiliicoccales under anaerobic conditions, in a gaseous atmosphere containing dihydrogen (H.sub.2) under mesothermal conditions, into a culture medium comprising (i) a base medium comprising KH.sub.2PO.sub.4, NaCl, MgSO.sub.4.7H.sub.2O, NH.sub.4Cl, CaCl.sub.2.2H.sub.2O, sodium acetate, Cysteine-HCl, resazurin, a Widdel trace element solution and a selenite-tungstate solution, (ii) 2% Na.sub.2S, 10% NaHCO.sub.3, and a vitamin mixture, to which is added (iii) one or more compounds providing amino acids, and -(iv) methylated compounds; b) replacing the culture medium of a) by an identical culture medium but depleted of compounds providing amino acids and to which vitamins have been added; c) detecting enrichment with an archaeon of the order of commensal clade Methanomassiliicoccales in said inoculated sample via microscopy, methane production and H.sub.2 consumption, PCR, qPCR, or sequencing; and d) performing serial liquid dilutions or the roll-tube technique of said enrichment obtained in c) using a medium as defined in a) or b) to which a sterile extract is added of a bacterial culture a bacterium of genus- Eggerthella, until a clone is obtained of an archaeon of the order of commensal clade Methanomassiliicoccales.

2. The isolation process according to claim 1, further comprising c′) between c) and d), wherein the culture medium comprising the biological sample is filtered on a filter, the filtrate obtained after c′) being subsequently used at d) to perform the serial dilutions.

3. The isolation process according to claim 2, wherein the filter has a pore size of at least 0.45 μm.

4. The isolation process according to claim 1, wherein determination of the obtaining of a clone of an archaeon of the order of commensal clade Methanomassiliicoccales is performed via microscopy, methane production and H.sub.2 consumption, PCR, qPCR, or sequencing.

5. The isolation process according to claim 1, wherein the sterile extract of a bacterial culture of a bacterium of genus Eggerthella is a bacterial filtrate of a bacterium of genus Eggerthella.

6. The isolation process according to claim 5, wherein the bacterial filtrate of a bacterium of genus Eggerthella is prepared following the method comprising a) cultivating a bacterium of genus Eggerthella in a medium, said medium comprising Biotrypcase, yeast extract, peptone, amino acids, meat extract, hemin, a vitamin selected from the group consisting of vitamin K1 and vitamin K3, and monosaccharides having 5 or 6 carbon atoms; b) incubating the culture medium comprising said bacterium at a temperature of 20 to 40° C. for at least 1 day; c) filtering the culture medium comprising said bacterium; and d) collecting the filtrate obtained after c).

7. The isolation process according to claim 1, wherein the archaeon of the order of commensal clade Methanomassiliicoccales is a Methanomethylophilus alvus archaeon.

8. The isolation process according to claim 7, wherein the archaeon Methanomethylophilus alvus is Methanomethylophilus alvus Mx-05, a strain deposited on Sep. 18, 2017 under the Budapest Treaty with the Leibniz-Institut (DSMZ, Braunschweig, Germany) under number DSM32684.

9. The isolation process according to claim 1, wherein the one or more compounds providing amino acids are selected from the group consisting of Biotrypcase, peptone, casamino acids, and yeast extract.

10. The isolation process according to claim 1, wherein the culture media of a) and b) also comprise coenzyme M.

11. The isolation process according to claim 1, wherein the bacterium of genus Eggerthella is selected from among Eggerthella lenta or the strain Eggerthella sp. Eg01-Mx05 deposited on Jul. 13, 2017 under the Budapest Treaty with the Leibniz-Institut (DSMZ, Braunschweig, Germany) under number DSM 32565.

12. A method of culturing an archaeon of the order of commensal clade Methanomassiliicoccales, pure or contained in a microbial consortium, said method comprising: a) inoculating into a culture medium either a biological sample comprising an archaeon of the order of commensal clade Methanomassiliicoccales or a pure archaeon of order Methanomassiliicoccales, wherein the culture medium comprises (i) a base medium comprising KH.sub.2PO.sub.4, NaCl, MgSO.sub.4.7H.sub.2O, NH.sub.4Cl, CaCl.sub.2.2H.sub.2O, sodium acetate, Cysteine-HCl, resazurin, a Widdel trace element solution, a selenite-tungstate solution, and a compound selected from the group consisting of 2% Na.sub.2S and 10% NaHCO.sub.3, to which are added (ii) one or more compounds providing amino acids, (iii) methylated compounds and (iv) a sterile extract of a bacterial culture of a bacterium of genus Eggerthella; b) placing the inoculum obtained at a) under anaerobic conditions in a gaseous atmosphere comprising dihydrogen H.sub.2; and c) incubating under mesothermal conditions to reach an exponential growth phase with release of methane in at least 2 days.

13. The method according to claim 12, wherein the culture medium further comprises coenzyme M.

14. The method according to claim 12, wherein the sterile extract of a bacterial culture of a bacterium of genus Eggerthella is a bacterial filtrate of a bacterium of genus Eggerthella.

15. The method according claim 12, wherein the archaeon of the order of commensal clade Methanomassiliicoccales is a Methanomethylophilus alvus archaeon.

16. The method according to claim 15, wherein the archaeon Methanomethylophilus alvus is Methanomethylophilus alvus Mx-05, a strain deposited on Sep. 18, 2017 under the Budapest Treaty with the Leibniz-Institut (DSMZ, Braunschweig, Germany) under number DSM32684.

17. The method of claim 12, wherein the one or more compounds providing amino acids are selected from the group consisting of Biotrypcase, peptone, casamino acids, and yeast extract.

18. The method according to claim 12, wherein the bacterium of genus Eggerthella is selected from among Eggerthella lenta or the strain Eggerthella sp. Eg01-Mx05 deposited on Jul. 13, 2017 under the Budapest Treaty with the Leibniz-Institut (DSMZ, Braunschweig, Germany) under number DSM 32565.

Description

EXAMPLES

(1) Composition of a Base Medium (BM) Per 1 Litre:

(2) TABLE-US-00003 TABLE 3 Name Quantity KH.sub.2PO.sub.4 0.5 g MgSO.sub.4•7H.sub.2O 0.4 g NaCl 5 g NH.sub.4Cl 1 g CaCl.sub.2•2H.sub.2O 0.05 g Na-acetate 1.6 g Cysteine-HCl 0.5 g Resazurin 1 mL from a stock solution (1 g/L) Widdel trace element solution 1 mL from a stock solution (1 g/L) Selenite-tungstate solution 1 mL

(3) After autoclaving, the addition is made of:

(4) TABLE-US-00004 2% Na.sub.2S (w/v) 0.1 mL per 5 mL of culture medium 10% NaHCO.sub.3 (w/v) 0.1 mL per 5 mL of culture medium Balch vitamin solution 0.1 mL per 5 mL of culture medium
Composition of the Balch Vitamin Solution (Balch et al, 1979)

(5) TABLE-US-00005 TABLE 4 (in mg per litre of distilled water) Biotin 2 Folic acid 2 Pyridoxine hydrochloride 10 Thiamine hydrochloride 5 Riboflavin 5 Nicotinic acid 5 Calcium pantothenate 5 Vitamin B12 0.1 p-aminobenzoic acid 5 Lipoic acid 5
Composition and Preparation of a Widdel Trace Element Solution

(6) Ferrous chloride was dissolved in hydrochloric acid. Double distilled water was added and the salts of the different trace elements. The pH was adjusted to between 7.1 and 7.3 with HCl or Na.sub.2CO.sub.3.

(7) This trace element solution was used in a proportion of 1 mL per litre of culture medium.

(8) TABLE-US-00006 TABLE 5 Nitrilotriacetic acid 1.50 g MgCl.sub.2, 6H.sub.2O 2.50 g NaCl 1.00 g MnCl.sub.2, 4H.sub.2O 0.60 g FeCl.sub.2, 4H.sub.2O 100.00 mg CoCl.sub.2, 6H.sub.2O 100.00 mg CaCl.sub.2, 2H.sub.2O 100.00 mg ZnCl.sub.2 100.00 mg CuCl.sub.2, 2H.sub.2O 10.00 mg AlCl.sub.3 10.00 mg H.sub.3BO.sub.3 10.00 mg Na.sub.2MoO.sub.4, 2H.sub.2O 10.00 mg pH (adjusted with 10 M KOH solution) 6.50 H.sub.2O double distilled q.s. 1000 mL
Enriching with the Archeon Methanomethylophilus Alvus Mx-05

(9) The base medium was prepared under anaerobic conditions in a gaseous N.sub.2/CO.sub.2 atmosphere (80:20% v/v). Enrichments were performed on this base medium BM to which were added 1 g/L of yeast extract and 10 to 80 mM of methylated compounds (methanol, methylamines) as final electron acceptors. They were conducted in the presence of an anaerobic gaseous atmosphere formed of dihydrogen H.sub.2 as electron donor (2 bar), under mesothermal conditions (incubation at 20 to 40° C.).

(10) The culture medium subsequently used (containing the Balch vitamin solution) was the same medium with the exception that it was depleted of yeast extract (down to complete absence thereof (0 g/L yeast extract) as appropriate) so as to obtain a dominant population of the archaeon Methanomethylophilus alvus Mx-05.

(11) Verification of the abundance of the archaeon Methanomethylophilus alvus Mx-05 was performed by: Microscopy to determine cell forms and degree of purity of our enrichment; Measurement of methane production and H.sub.2 consumption via gas phase chromatography; qPCR to quantify the populations of bacteria and archaea, using described primers and protocols known to persons skilled in the art (Borrel et al, 2017); Sequencing to determine the dominant species of the enrichment.

(12) Dilution experiments in a liquid medium (Hungate tubes containing 5 mL of culture medium) or in solid medium (roll-tubes also containing 5 mL of culture medium) in the presence of H.sub.2 and methylated compounds were then carried out. They were conducted after prior filtration (0.45 μm filter) to remove most bacilli, the pure isolated archaeon Methanomethylophilus alvus Mx-05 being in the form of small-sized cocci with cells smaller than 500 μm in diameter. Growth of the archaeon Methanomethylophilus alvus Mx-05 was only obtained in the presence of a sterile extract of a culture of Eggerthella sp. prepared by filtration (0.22 μm filter) (see preparation of the filtrate below).

(13) Preparation of the Filtrate of Eggoerthella sp.

(14) Eggerthella sp. strain Eg01-Mx-05 (strain deposited on 13 Jul. 2017 under the Budapest Treaty with the Leibniz-Institut (DSMZ, Braunschweig, Germany) under number DSM 32565 was cultivated in the base medium containing 10 g/L Biotrypcase, 10 g/L yeast extract, 10 g/L peptone, 10 g/L casamino acids and 20 mM glucose. Incubation was conducted at 37° C. for one week. After filtering (0.22 μm filter) the culture medium of Eggerthella, 0.5 mL of filtrate was added per 5 mL of culture medium contained in the tubes used for dilution of the enrichment with the archaeon Methanomethylophilus alvus Mx-05.

(15) Isolation of the Archaeon Methanomethylophilus Alvus Mx-05 in Pure Form

(16) To obtain the pure, isolated archaeon Methanomethylophilus alvus Mx-05 in axenic culture, the serial dilutions in liquid or solid media were conducted using a base medium BM free of complex organic compounds (e.g. yeast extract, Biotrypcase, etc.) but in the presence of the Balch vitamin solution and Widdel Trace element solution, or on the contrary by adding these compounds (yeast extract, Biotrypcase, peptone, casamino acids in a proportion of 2 g/L). This last operation allowed easier detection of the presence of any heterotrophic contaminants in the last positive dilutions. After obtaining colonies in solid medium, these were sub-cultured in a liquid medium then re-diluted in a liquid medium, the last positive liquid dilution representing the collection strain to be studied.

(17) The pure, isolated strain of the archaeon Methanomethylophilus alvus Mx-05 was deposited with the Leibniz-Institut (DSMZ, Braunschweig, Germany) under number DSM32684 (Depositor: Université Clermont Auvergne, France) on 18 Sep. 2017.