WO2013177471A2 - Dépolymérisation enzymatique et solubilisation de charbon prétraité chimiquement et de constituants dérivés du charbon - Google Patents

Dépolymérisation enzymatique et solubilisation de charbon prétraité chimiquement et de constituants dérivés du charbon Download PDF

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WO2013177471A2
WO2013177471A2 PCT/US2013/042536 US2013042536W WO2013177471A2 WO 2013177471 A2 WO2013177471 A2 WO 2013177471A2 US 2013042536 W US2013042536 W US 2013042536W WO 2013177471 A2 WO2013177471 A2 WO 2013177471A2
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coal
enzyme
derived constituents
mnp
derived
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WO2013177471A3 (fr
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Michael A. Urynowicz
Zaixing HUANG
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University Of Wyoming
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C12P1/02—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using fungi
    • C—CHEMISTRY; METALLURGY
    • C10—PETROLEUM, GAS OR COKE INDUSTRIES; TECHNICAL GASES CONTAINING CARBON MONOXIDE; FUELS; LUBRICANTS; PEAT
    • C10L—FUELS NOT OTHERWISE PROVIDED FOR; NATURAL GAS; SYNTHETIC NATURAL GAS OBTAINED BY PROCESSES NOT COVERED BY SUBCLASSES C10G, C10K; LIQUEFIED PETROLEUM GAS; ADDING MATERIALS TO FUELS OR FIRES TO REDUCE SMOKE OR UNDESIRABLE DEPOSITS OR TO FACILITATE SOOT REMOVAL; FIRELIGHTERS
    • C10L9/00—Treating solid fuels to improve their combustion
    • C10L9/02—Treating solid fuels to improve their combustion by chemical means
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/16—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing two or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/188—Heterocyclic compound containing in the condensed system at least one hetero ring having nitrogen atoms and oxygen atoms as the only ring heteroatoms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y111/00—Oxidoreductases acting on a peroxide as acceptor (1.11)
    • C12Y111/01—Peroxidases (1.11.1)
    • C12Y111/01013—Manganese peroxidase (1.11.1.13)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/0102—Tannase (3.1.1.20)

Definitions

  • Embodiments of the present invention relate generally to the enzymatic depolymerization and solubilization of highly complex structural biopolymers found in coal and, more particularly, to the use of chemical pretreatment agents followed by subsequent enzymatic conversion to significantly improve the depolymerization and solubilization of highly complex structural biopolymers found in coal.
  • Coal can be described as a coal complex polymer or macromolecule consisting of a condensed aromatic carbon-atom lattice surrounded by a typical "fringe” formed by functional side groups. It can also be described as a heterogeneous mixture composed of a macromolecular network with varying degrees of cross-linking. Coal consists of modified lignin, as well as cellulose and melanoidin-type materials which are considered to be the "backbone" of the macromolecular network. Cross-linkage is generally dominated by alkyl and aryl ether groups, especially in low-rank coal, with oxygen functional groups, while the degree of aromaticity tends to increase with coal rank.
  • Manganese peroxidase (MnP, Enzyme Commission Number (EC) 1.1 1.1.7) is one of the most common and efficient extracellular lignin-modifying heme-peroxidases secreted by "classic" white-rot fungi (See, e.g., Hofrichter, M., 2002, Enzyme and Microbial Technology 30, 454-466; Martinez, 2002, Enzyme and Microbial Technology 30, 425-444; Hatakka, A. et al., 2003,. Manganese peroxidase and its role in the degradation of wood lignin. In Mansfield SD, Saddler JN (eds) Applications of Enzymes to Lignocellulosics, ACS Symposium Series 855.
  • the enzyme has been shown to efficiently oxidize a number of recalcitrant polymers (e.g., polycyclic aromatic hydrocarbons, organohalogens, nitroaromatic compounds, and natural substances like lignins, milled wood and humic substances) derived from low rank coal or low-rank coal and other persistent aromatics in cell-free reaction systems (in vitro)
  • recalcitrant polymers e.g., polycyclic aromatic hydrocarbons, organohalogens, nitroaromatic compounds, and natural substances like lignins, milled wood and humic substances
  • recalcitrant polymers e.g., polycyclic aromatic hydrocarbons, organohalogens, nitroaromatic compounds, and natural substances like lignins, milled wood and humic substances
  • recalcitrant polymers e.g., polycyclic aromatic hydrocarbons, organohalogens, nitroaromatic compounds, and natural substances like lignins, milled
  • the fungus, PaecHomyces variotii is known to produce a variety of enzymes including tannase.
  • Manganese peroxidase belongs to the class II peroxidase group of the plant peroxidase superfamily that is characterized by a protoporphyrin IX (heme) as a prosthetic group in the active center (Welinder, 1992, Current Opinion in Structural Biology 2, 388-393; Poulos et al., 1978, J. Biol. Chem. 253, 3730-3735; Piontek et al., 1993, FEBS Letters 315, 1 19-124; and Hofrichter et al., 2010, supra.).
  • heme protoporphyrin IX
  • the catalytic cycle of the enzyme behaves like other well-known heme peroxidases such as lignin peroxidases (LiP, EC 1.11.1.14) and the peroxidase of Coprinopsis cinerea (CiP, EC 1.1 .1.7), except that MnP uses Mn 2+ ions as the preferred electron donor.
  • the catalytic cycle is activated by H 2 0 2 .
  • the native MnP is oxidized to intermediate forms which then oxidize Mn 2+ to Mn 3+ and return it to its native form.
  • Manganese(lll) is highly reactive and both chelated and stabilized by organic acids such as oxalate or malonate (See, e.g., Wariishi et al., 1992, J. Biol. Chem. 267, 23688-23695; and Hofrichter et al., 2001 , Appl. and Environ. Microbiol. 67, 4588-4593).
  • Chelated Mn 3+ ions act as strong, diffusible redox mediators that are able to attack organic bonds in large biopolymers non-specifically.
  • Embodiments of the present invention overcome the disadvantages and limitations of prior art by providing a method for improving the enzymatic depolymerization and solubilization of highly complex structural biopolymers found in coal.
  • the method for depolymerizing and solubilizing coal and coal-derived constituents includes: treating the coal with an aqueous solution including at least one oxidizing agent, forming thereby treated coal and coal-derived constituents; and exposing the treated coal and coal-derived constituents to an aqueous solution including at least one enzyme effective for reacting with coal and coal-derived constituents.
  • the method for depolymerizing and solubilizing coal and coal-derived constituents includes: treating the coal with an aqueous solution including at least one acid, forming thereby coal and coal-derived constituents; and exposing the treated coal and coal-derived constituents to an aqueous solution including at least one enzyme effective for reacting with coal and coal-derived constituents.
  • the method for depolymerizing and solubilizing coal and coal- derived constituents includes: treating the coal with an aqueous solution including at least one base, forming thereby treated coal and coal-derived constituents; and exposing the treated coal and coal-derived constituents to an aqueous solution including at least one enzyme effective for reacting with coal and coal-derived constituents.
  • Benefits and advantages of the present invention include, but are not limited to, providing a method for improving enzymatic depolymerization and solubilization of highly complex structural biopolymers found in coal.
  • FIGURE 1 is a graph of the total organic carbon (TOC) from chemical pretreatments after 24 hours for sodium hydroxide (SH), nitric acid (NA), hydrogen peroxide (catalyzed) (HP), potassium permanganate (PP); low concentration (C1 ) medium concentration (C2) and high concentration (C3), where the data points represent the means of three replicates.
  • SH sodium hydroxide
  • NA nitric acid
  • HP hydrogen peroxide
  • PP potassium permanganate
  • C1 low concentration
  • C2 medium concentration
  • C3 high concentration
  • FIGURE 2 is a graph of the total organic carbon solubilized from coal following pretreatment with hydrogen peroxide at various concentrations, illustrating optimized concentrations of hydrogen peroxide (uncatalyzed) maximizing the amount of total organic carbon, while higher concentrations over-oxidize the resulting organic carbon, with the controls being coal and de-ionized water (no oxidant).
  • FIGURE 3 is a graph of the HPSEC elution profiles of water-soluble aromatic fragments released from coal (PRB) after combined chemical and enzymatic (1 U ml "1 MnP) treatment at ambient temperature, wherein curve A represents HN0 3 + MnP; curve B represents H 2 0 2 + MnP; curve C represents KMn0 4 + MnP; and curve D represents NaOH + MnP, where the dotted lines represent the enzymatic controls without any chemical pretreatment; the dashed lines represents low chemical concentrations; the thin lines represent medium chemical concentrations; and the thick lines represent high chemical concentrations, and where the data points represent the means of three replicates with standard deviation values of ⁇ 5%.
  • FIGURE 4 is a graph of the cumulative CO 2 production per gram of coal as a function of time in days with Paecilomyces variotii, a fungus known to generate a wide variety of enzymes including tannase, as the sole aerobic microorganism contacting a coal sample, for several hydrogen peroxide concentrations.
  • embodiments of the present invention include the use of chemical pretreatment agents for the subsequent enzymatic conversion of coal and coal derived constituents, the enzymes by themselves having little effect on the untreated coal controls.
  • the nature of pretreatment agents and their applied concentrations were found to have significant impact on subsequent enzymatic conversion of coal.
  • Four agents were investigated: HN0 3 , catalyzed H 2 O 2 , KMn0 4 , and NaOH.
  • hydrogen peroxide generated the greatest quantity of total organic carbon from the coal samples employed.
  • Chemical pretreatment in accordance with embodiments of the present invention creates two fractions: treated coal and coal derived constituents.
  • the coal is the solid fraction and the coal- derived constituents are the aqueous fraction (the coal that has been solubilized).
  • the enzymatic treatments are shown to enhance the solubilization of the solid fraction (the chemical treated coal) and alter the coal-derived constituents (the coal that was solubilized from the chemical treatment).
  • the coal-derived constituents are transformed from higher molecular weight compounds that aren't readily biodegradable to lower molecular weight compounds that tend to be more readily biodegradable.
  • MnP Manganese Peroxidase
  • MnP manganese peroxidases from white rot fungi like Phlebia radiata, Clitocybula dusenii and Bjerkandera adusta may be produced on a large scales (e.g., total volumes of 300 L with maximum activities of -2000 U L "1 ).
  • the enzymes are stable and able to effectively depolymerize and solubilize humic acids derived from low-rank coals (See, e.g., Hofrichter et al., 1997, supra; and Nueske, J. et al., 2002, Enzyme and Microbial Technol. 30, 556-561.).
  • Other fungi including Paecilomyces variotii, discussed hereinbelow, generate a wide variety of enzymes effective for acting on coal and coal-derived constituents.
  • Powder River Basin (PRB) coal pretreated by various chemical agents including two oxidants (catalyzed and uncatalyzed hydrogen peroxide and permanganate), one acid (nitric acid) and one base (sodium hydroxide), was followed by treatment using cell-free enzymatic reaction systems (in vitro) for depolymerization, for example MnP.
  • MnP cell-free enzymatic reaction systems
  • the released fragments were characterized by size exclusion chromatography and fluorescence excitation-emission matrix (EEM) spectroscopy.
  • TOC Total organic carbon
  • Fluorescence spectrometry can be used to distinguish humic-like and fulvic acid-like organic matter from protein-like and aromatic/polycyclic aromatic hydrocarbons (PAHs) substances (See, e.g., Tang et al., 2011 , Chemosphere 82, 1202-1208; and Jaffrennou et al., 2007, J. Fluorescence 17, 564-572.).
  • PAHs protein-like and aromatic/polycyclic aromatic hydrocarbons
  • Humic and fulvic acid-like intensities were quantified at emission wavelengths of 420 and 440 nm and at excitation wavelengths of 330 and 240 nm, respectively.
  • the aromatic compounds with one and two rings are located at emission wavelengths from 300 to 350 nm and at excitation wavelengths from 280 to 330 nm, while PAHs with three to five rings emit between 370 and 480 nm and at excitation wavelengths from 360 to 460 nm (Jaffrennou et al., supra).
  • Chen et al. (2003) divided the matrix into five regions: aromatic protein I, aromatic protein II, fulvic acid-like, soluble microbial by-product-like and humic acid-like regions.
  • the EEM spectroscopy is extensively used to determine protein-like, fulvic acid-like, humic acid-like and aromatic/PAH (1-5 rings) substances (Tang et al., supra; Jaffrennou et al., supra).
  • Organism, culture conditions and enzyme preparation are identical to Organism, culture conditions and enzyme preparation:
  • the inoculum for this study was prepared from white-rot fungus Bjerkandera adusta on agar plates (basal medium plus 1.5% agar) incubated at 24°C for 12 days.
  • the basal medium contained 10 g glucose, 2 g KH 2 P0 4 , 0.5 g MgS0 4 -7 H 2 0, 0.1 g CaCI 2 , 0.5 g NH 4 tartrate, 0.3 g yeast extract, 2 g sodium acetate, 0.015 g FeS0 4 -7 H 2 0 and 25 mg MnCI 2 , per liter. Prior to sterilization, the pH was adjusted to 4.5.
  • the fungus was precultured in 500-ml culture flasks containing 200 ml basal medium at 24°C for 10 to 12 days on a rotary shaker (100 rpm). After suitable levels of biomass growth were attained, the fungal mycelia in the precultures were homogenized and used as inoculum for a 10-liter stirred-tank bioreactor. After growth, sterile samples were taken every second or third day, and the MnP activity as well as the pH of the culture liquid were determined.
  • the enzyme-containing culture liquid was harvested, separated from the fungal biomass by filtration (filter GF6; Schleicher & Schuell, Dassel, Germany) and concentrated 10-fold at 10°C in a Pall-Filtron tangential flow system (Dreieich, Germany) using a 10-kDa cutoff filter cassette.
  • the crude enzyme liquid was used in the present conversion studies.
  • Coal samples were obtained from the Powder River Basin (PRB) located 31 miles west of the Powder River on the Montana side of the Montana-Wyoming state line.
  • the sample well (SL-5) was located in the Canyon Aquifer at coordinates 45.01 189° North and 106.27149° West, lying within the Upper Wyodak Formation.
  • Coal samples were collected on June 10, 2005, from a depth ranging between 408 feet and 431 feet. The coal was dried and ground, and the portion of the coal particles passing through a 60 mesh (0.25 mm) sieve was retained for the chemical enzyme treatment studies.
  • Hydrogen peroxide is decomposed by the soluble Fe (II) or other transition elements to hydroxyl radicals (Fenton reaction) that are strong, nonspecific oxidants capable of reacting with most organic compounds (See, e.g., Watts et al., 1994, J. Hazard. Mat. 39, 33-47).
  • each reaction vial was 1 ml.
  • the vials were centrifuged at 16,000 rpm for 10 min., the supernatant liquid was separated from the coal, and the coal was then resuspended in distilled water and centrifuged at the same speed and duration. This washing process was repeated 10 times to remove any residual chemical agents. The washed coal was then used in enzymatic reactions. The supernatant was filtered through 0.45 pm syringe filters prior to TOC analysis.
  • Pretreated samples were centrifuged to separate the liquid from the solid, each aliquot of liquid then being filtered through a 0.45 microsyringe filter, and analyzed for Total organic carbon (TOC) with a Shimadzu TOC analyzer (TOC-VCSN, Japan).
  • TOC Total organic carbon
  • the enzymatic depolymerization of pretreated coal was carried out in the same 1.5-ml HPLC vials at ambient temperature for 24 h.
  • the reaction system was comprised of a sodium malonate buffer (50 mM, pH 4.5), MnCI 2 (25 mM), 1 total Unit MnP (1 U ml “1 ), dimethylformamide (0.05%) and H 2 0 2 (stock solution 150 mM, 2 ⁇ h " ). Magnetic stir bars (8 x 2 mm) were used to mix the solution during the incubation.
  • the H 2 0 2 was delivered precisely and slowly with an infusion pump (KDS220, KD Scientific, Bath, UK) to prevent enzyme inactivation by heme-bleaching (Hofrichter et al., 2010, supra). After the prescribed incubation time of 24 h, the vials were centrifuged to retain the liquid supernatants, which were filtered through cellulose syringe filters (0.45 pm, Restek, Bellefonte, Pennsylvania) for further analysis.
  • KDS220 KD Scientific, Bath, UK
  • Samples contained water only, dimethylformamide (DMF) without MnP, and MnP without DMF, as well as MnP and DMF without chemical pretreatment agents as controls.
  • DMF was found to support the depolymerization of humic acids (See, e.g., Hofrichter et al., 1997, supra.).
  • the enzymatic control contained MnP, DMF and coal without chemical pretreatment agents.
  • HPSEC high-performance size-exclusion chromatography
  • the elution solvent consisted of 80% salt in an aqueous buffer of sodium chloride (3.44 g ⁇ 1 ) and dipotassium phosphate (2 g ⁇ 1 ), and a 20% organic buffer of acetonitrile.
  • the aqueous buffer was adjusted to pH 10.0.
  • Polystyrene sulfonate sodium salts (0.891- 976 kDa, Polymer Standard Service, Ashton, Maryland, USA) were used as molecular weight standards.
  • the elution was performed at a flow rate of 1 ml min "1 and analyzed at a wavelength of 280 nm, where aromatic substances typically exhibit maximum absorbance.
  • the injection volume was 25 ⁇ for both standards and samples. 6.
  • the 3D-EEM was performed on a Varian Cary Eclipse Fluorescence Spectrophotometer (Agilent, Santa Clara, California). The samples were scanned under emission 3D mode. The scanning emission (Em) spectra from 290 to 590 nm were obtained at 2 nm increments by varying the excitation (Ex) wavelengths from 225 to 450 nm at 2.5 nm increments. The scan rate was 9600 nm min "1 . Slit bandwidths of 5 nm for both emission and excitation were used at all times.
  • Higher-rank coals are classified according to fixed carbon on a dry weight basis, while the lower-rank coals are classified according to gross calorific value on a moist, mineral-matter-free basis.
  • the coal was determined to be subbituminous B coal based on its heating value of 9576.3 Btu/lb on a moist, mineral-matter-free basis.
  • FIG. 1 TOC analyses indicate that up to 1000 mg/L (PP-C3) of total organic carbon was released by chemical treatment reagents within 24 h. Except for sodium hydroxide, the TOC values are positively correlated to the chemical agent concentrations. It may be observed that potassium permanganate had the highest on a per mol/L increment basis commensurate with TOC, followed by nitric acid and catalyzed hydrogen peroxide.
  • FIGURE 2 is a graph of the total organic carbon solubilized from coal following pretreatment with hydrogen peroxide at various concentrations, illustrating optimized concentrations of hydrogen peroxide (uncatalyzed) maximizing the amount of total organic carbon while higher concentrations over-oxidize the resulting organic carbon. It may be observed from this FIGURE that the TOC far exceeds that from FIG. 1.
  • FIGURE 3 shows the HPSEC elution profiles of the various chemical pretreatments followed by exposure to the fungal MnP as compared with the enzymatic controls treated with MnP, but without any chemical pretreatment.
  • all of the chemical pretreatments enhanced the subsequent enzymatic conversions, and with the exception of KMN0 4 , the concentration of the pretreatment agents had a significant effect on the subsequent enzymatic treatments.
  • HNO 3 was the most effective pretreatment agent of the four tested, followed by H 2 0 2 .
  • the medium and high concentrations of each reagent exerted the most distinct effects and roughly doubled the fragment release at twice the concentration (from 1 .67 to 3.33 M for HN0 3 and 1.62 to 3.24 M for H 2 0 2 ).
  • the lowest chemical concentration had no significant effect on the release of water-soluble aromatics with results almost indistinguishable from the controls (FIGS. 2A and 2B).
  • all three KMn0 4 concentrations had an enhancing effect on the ability of MnP to oxidize coal, the largest effect being observed in the medium concentration of KMn0 4 (FIG. 2C); after alkaline pretreatment, only minor differences were observed with MnP oxidation, as indicated by the HPSEC elution profile (FIG. 2D).
  • the oxidation potential for H 2 0 2 is 1.78 V, slightly higher than KMn0 4 (1.67 V); however, the decomposition of H 2 0 2 to hydroxy! radicals from Fenton reactions increases the oxidation potential to 2.8.
  • Research by others has revealed that catalyzed H 2 0 2 oxidizes toluene, nitrobenzene and chlorobenzene to phenol, cresols, biphenyls and benzaldehydes (See, e.g., Merz et al., 1949, J. Chem. Soc.
  • HN0 3 Based on the absorbance profiles from the HPSEC chromatograms (FIG. 2), HN0 3 , with twice as much of the absorbance intensity as catalyzed H2O2 pretreated coal, was considered the most promising pretreatment agent of the four tested. Deno and coworkers, 1981 , supra, documented that HN0 3 was able to cleave the aliphatic connectors between aromatic rings in coal. This would reduce the interconnectivity of the carbon clusters in the coal matrix which should favor MnP attack.
  • NaOH was the least effective of the pretreatment agents.
  • NaOH has been used to remove ash and sulfur in coal (See, e.g., Araya et al., 1981 , Fuel 60, 1127-1130; and Mukherjee et al., 2004, Fuel 82, 783-788.) and for the preparation of humic acid and fulvic acids (See, e.g., Hofrichter et al., 1996, supra; Juan et al., 1990, Fuel 60, 158-161 ; and Novak et al., 2001 , Reactive and Functional Polymers 47, 101-109.).
  • the mechanism of the MnP is that the activated enzyme oxidizes Mn 2+ to Mn 3+ ; that is, in turn, chelated by carboxylic acids such as malonic acid (See, e.g., Hofrichter, 2002, supra.).
  • This low- molecular weight diffusive redox-mediator then attacks the coal, particularly phenolic structures and amino-aromatic compounds, and returns to its reduced state (Mn 2+ ).
  • the mechanism is similar to the oxidation by permanganate from Mn0 4 " to Mn0 2 (Arndt, 1975). This similarity may explain the low fluorescence intensity at the aromatic/PAH regions with the higher concentration permanganate pretreated coals.
  • the humic and fulvic acid-like peaks occurred at Ex/Em wavelengths of 307.5/422 nm and 232.5/426 nm, respectively.
  • the observed increased peak intensity of the humic and fulvic acid-like peaks apparently offset the decrease in other regions and rendered medium permanganate treatment the highest absorbance in HPSEC analysis.
  • the NaOH pretreatments showed only aromatics/PAHs, regardless of the concentration of treatment agent applied, which correlated well with the HPSEC spectra.
  • the NaOH is used to prepare humic and fulvic acid for enzymatic reaction experiments (See, e.g., Hofrichter et al., 1996, supra; Juan et al., 1990, supra; and Novak et al., 2001 , supra.); it is not surprising then that no humic or fulvic acid-like peaks were visible in the EEM spectra.
  • the NaOH was the least effective pretreatment agent with respect to both HPSEC and EEM data.
  • FIGURE 4 is a graph of the cumulative C0 2 production per gram of coal as a function of time in days with Paecilomyces variotii as the sole aerobic microorganism contacting a coal sample, for several hydrogen peroxide concentrations.
  • the enzymes are produced by the Paecilomyces variotii, and C0 2 is being measured as opposed to the various types of organics generated.
  • the quantity of C0 2 produced correlates to the amount of bioavailable carbon in the biometer used for the measurement, which is an apparatus for measuring C0 2 evolution. In the present situation, the amount of bioavailable carbon is also directly related to TOO
  • the treated coal was separated from the liquid fraction which was added to biometer flasks (Bellco Glass, Inc., Vineland, New Jersey). Six ml of 0.5 M Sorensen phosphate buffer was added to each biometer flask to ensure the pH was maintained within an acceptable range for optimal microbial activity. Triplicate samples were set up unless otherwise stated. The following procedure was used to ensure that the biometers were inoculated consistently for all measurements, while minimizing the amount of additional carbon added to the system. First, the bacteria were grown and isolated on nutrient agar plates. A single colony of the organism was added to a 150-ml beaker of sterile nutrient broth and grown for 15 h at 30°C.
  • one ml of this bacterial culture was added to 150 ml of sterile nutrient broth and grown for approximately 30 h to an optical density reading of 1.5 at 600 nm.
  • An aliquot of 20 ml of cells was washed by centrifuging at 4000 rpm, removing the liquid, and re-suspending the centrifuged cells in a solution of phosphate-buffered saline to remove residual carbon from the unused nutrient broth.
  • One ml of the solution was inoculated into each biometer flask.
  • the ml of 0.05 M potassium hydroxide (KOH) solution was poured into the side arm of the biometer flask.
  • the C0 2 gas produced by the organism was trapped when it dissolved into the KOH solution.
  • a titration was performed on the KOH using 0.05 M hydrochloric acid to determine the amount of CO2 produced. After each titration, the side arm was refilled with 10 ml of fresh 0.05 M KOH.

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Abstract

L'invention concerne l'usage d'agents de prétraitement chimique sur la conversion enzymatique postérieure du charbon. À titre d'exemple, la manganèse-peroxidase (MnP) fongique produite par le champignon de pourriture blanche agarique Bjerkandera adusta, où l'enzyme MnP a peu d'effet sur les contrôles de charbon non traité, a été étudiée. On a trouvé que la nature des agents de prétraitement et leurs concentrations appliquées avaient un impact significatif sur la conversion enzymatique postérieure du charbon. On a étudié quatre agents : HNO3, H2O2 catalysé, KMnO4, et NaOH. On a trouvé que le peroxyde d'hydrogène générait la plus grande quantité de carbone organique total à partir des échantillons de charbon employés. Le traitement chimique et enzymatique combiné du charbon est approprié pour la dépolymérisation améliorée du charbon et des constituants dérivés du charbon et résulte en produits de liquéfaction chimiquement hétérogènes et complexes comme les acides humiques et fulviques, lesquels auront des ramifications importantes dans la génération de combustibles liquides et gazeux à partir des charbons comme alternatives de combustibles non dérivés du pétrole.
PCT/US2013/042536 2012-05-23 2013-05-23 Dépolymérisation enzymatique et solubilisation de charbon prétraité chimiquement et de constituants dérivés du charbon WO2013177471A2 (fr)

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CN107325851A (zh) * 2017-08-25 2017-11-07 太原理工大学 一种以液化煤为原料提高生物甲烷产量的方法
US9920253B2 (en) 2008-12-30 2018-03-20 Somerset Coal International Microorganism mediated liquid fuels
US10376837B2 (en) 2013-03-14 2019-08-13 The University Of Wyoming Research Corporation Conversion of carbon dioxide utilizing chemoautotrophic microorganisms systems and methods
US10557155B2 (en) 2013-03-14 2020-02-11 The University Of Wyoming Research Corporation Methods and systems for biological coal-to-biofuels and bioproducts

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US9920253B2 (en) 2008-12-30 2018-03-20 Somerset Coal International Microorganism mediated liquid fuels
US10376837B2 (en) 2013-03-14 2019-08-13 The University Of Wyoming Research Corporation Conversion of carbon dioxide utilizing chemoautotrophic microorganisms systems and methods
US10557155B2 (en) 2013-03-14 2020-02-11 The University Of Wyoming Research Corporation Methods and systems for biological coal-to-biofuels and bioproducts
WO2015094933A1 (fr) 2013-12-18 2015-06-25 Somerset Coal International Combustibles liquides formés par l'intermédiaire de microorganismes
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CN107325851A (zh) * 2017-08-25 2017-11-07 太原理工大学 一种以液化煤为原料提高生物甲烷产量的方法

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