Method for decomposition of the metallorganic matter of graptolite-argillite by microbial consortium

Abstract

The present invention describes a method, which consists in decomposition of graptolite-argillite organometallic matter in anaerobic environment by a stable adapted microbial consortium, accompanied by bioleaching of metals and methane generation. Supporting experimental data are presented and the effect of betaine in biodegradation of argillite organometallic compounds is demonstrated. Microbial communities provoking these processes are characterized.

Claims

1. A ethod for decomposition of organometallic matter of graptolite-argillite by a microbial consortium which leads to release of biogenic methane, the method comprising a step of using a liquid growth medium R2A plus betaine for producing methane, and bioleaching of metals occurs in an anaerobic medium.

2. The method according to claim 1, wherein the metals leached are nickel and cobalt.

3. The method according to claim 1, wherein for the release of biogenic methane from the organometallic material, accompanied by metal leaching a microbial community inherent to argillite is used.

4. The method according to claim 3, wherein when inoculating fresh argillite samples with microbial community inherent to argillite, a new adapted consortium with better biodegrading capability will be achieved that gives a higher methane yield.

Description

THE LIST OF DRAWINGS AND OTHER ILLUSTRATIVE MATERIAL

[0021] FIG. 1Dynamics and yield of methane evolution from argillite using the growth medium R2A plus betaine: a) with indigenous microbial consortium of argillite, non-adapted to growth medium; b) with microbial consortium adapted to growth medium.

[0022] FIG. 2Determination of the origin of the methane by isotopic analysis ((.sup.13C method).

[0023] FIG. 3Bioleaching of metals from argillite in various growth media; Y-axis represents the yield of metal from its maximum concentration in argillite (enrichment value).

[0024] FIG. 4Argillite sample prepared for cultivation experiment with particles dimensions of 1-2 cm.

[0025] FIG. 5Change of external characteristics of argillite on cultivating in growth medium: a) in experiments with methane evolution a blackish suspension was formed; b) in experiments where methane evolution was modest or nonexistent, the growth medium remained transparent. 1Section of argillite drill core, 2reactor with growth medium and microbial consortium, 3reactor with growth medium, argillite and microbial consortium.

[0026] FIG. 6Species detected by pyrosequencing from the communities in various growth media with primer pair BSR357-BSF8 suitable for the bacterial 16S rRNA V2 region [McKenna, et al., 2008]: a) percentage of different taxa (operational taxonomic unit, OTU); b) the part of most important taxa in the community.

[0027] FIG. 7Species detected by pyrosequencing from the communities in various growth media with primer pair Arch349F V2-A934B suitable for the archaeal 16S rRNA V2 region [Takai et al., 2000; Grosskopf et al., 1998]: a) percentage of different taxa (operational taxonomic unit, OTU); b) the part of most important taxa in the community.

[0028] Example 1. With the method described in the invention, methane generation into the gas phase was initiated with an indigenous to argillite non-adapted consortium and medium R2A plus betaine in anaerobic cultivation experiment in argon atmosphere in a 500 mL test flask (FIG. 5) at a temperature of 37 C. and at pH 7.5. The gas phase pressure was measured by manometric system OxiTop (WTW, Germany), and the gas phase composition was analyzed with a gas chromatograph GC-2014 (Shimadzu, Japan; methane measurement range 10 ppb-30%). Using 25 g of crushed argillite as a substrate (with particle dimensions of 1-2 cm) within 90 days 417 ml of gas with methane content from 15 to 28%, with a yield of 3.1 liters of methane per 1 kg of argillite was obtained (FIG. 1a). 671 ml of biogenic gas with methane content of up to 37.5% was evolved as a maximum, which means a yield of 6.4 liters methane per 1 kg of mineral (argillite). On day 77 the culture media were sampled for liquid phase to determine the metal content by the flame-AAS method (ISO 8288). 26.2% cobalt and 9.14% of nickel of the maximum concentrations of these metals in the original sample had been leached into cultivation medium (FIG. 3). On the same day samples were taken from cultivation media for identification of microorganisms. Samples were centrifuged (5000 rev/min, 10 min) to separate the biomass of microorganisms, from which in turn the DNA was isolated with DNA Powersoil kit (MoBio, USA) and sequenced by 16S rRNA gene, using the pyrosequencing technology of 454 Life Sciences and primers according to the reference [Uuring Eesti argilliidist . . . , 2014]. In the growth medium R2A plus betaine with a primer pair BSR357-BSF8 suitable for amplification of bacterial 16S rRNA gene, bacterial genus Ureibacillus accounted for 87.43%, class Clostridia, order D8A-2 for 2.72%, and genus Thermacetogenium, Firmicutes bacterium for 3.07% of all taxa (FIG. 6). With a primer pair Arch349F-A934B suitable for amplification of archaeal 16S rRNA gene, archaeal genus Methanosarcina accounted for 3.69% and bacterial order Bacillacae for 36.25%, bacterial genus Desulfotomaculum for 16.7% and bacterial class Clostridia for 10.5% of all taxa identified (FIG. 7).

[0029] Example 2. Using freshly ground argillite and growth medium R2A plus betaine a new experiment was launched with a sample taken from the cultivation medium of Example 1 on day 129 (5% inoculum) in anaerobic conditions in argon atmosphere in a 1000 mL test flask 3 (FIG. 5a)) at a temperature of 37 C. and at pH 7.5. The gas phase pressure was measured by manometric system OxiTop (WTW, Germany); the gas phase composition was analyzed with gas chromatographs GC-2014 (Shimadzu, Japan; methane measurement range 10 ppb-30%) and Varian Inc., Model CP-4900 (methane measurement range 1-100%). Using 50 g of crushed argillite as a substrate (with particle dimensions of 1-2 cm) 7.920.39 liters of methane (35417 mol) per kg of argillite was evolved (FIG. 1b). Methane originated from the organic fraction of argillite, because the average values for .sup.13C ( V-PDB) for methane from the samples containing argillite and medium and from the samples without argillite, containing only medium (blank samples) were 51.994.60 and 72.865.35, correspondingly (FIG. 2). The biodegradable part of argillite organic matter amounted to 19.860.98% of the total organic matter. Thus with adapted microbial consortium and growth medium R2A plus betaine, using freshly ground argillite, 1.4 times more methane was obtained than has been previously extracted from similar black shales. Methane release from cultivation medium started immediately without a lag-phase (FIG. 1b), and argillite was disintegrated into fine suspension material 3 (FIG. 5a)

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