CS231793B1 - Process of microbial utilization of lignocellulosic complex - Google Patents

Abstract

Method of microbiological utilization of lignocellulosic complex. The invention relates to the field of chemistry and biochemistry of lignin, carbohydrates as well as environmental protection. The purpose of the invention is to break down the lignocellulosic complex both under the conditions of the experiment and as a technological step of breaking down the components of lignocellulosic waste arising during the production of pulp, also under the condition of progressive technology of thermomechanical processing. The mentioned effect is achieved with the help of microscopic fungi from the group of aquatic hyphomycetes, which have the ability to degrade lignin and produce cellulolytic, xylanolytic and β-mainanolytic enzymes, while obtaining their biomass. The biomass of aquatic hyphomycetes can be obtained in an amount of about 30% of the dry weight in relation to the starting lignocellulosic complex. The invention is used in technical microbiology, taxonomy, chemistry and biochemistry of lignin and carbohydrates and in environmental protection.

Description

The present invention relates to the microbial utilization of the lignocellulose complex, cellulose, lignin and hemicelluloses by means of microscopic fungi from the group of aqueous hyphomycetes.

Biological utilization of the lignocellulosic complex represents an important technological problem not only in terms of utilization of the material in question for nutritional purposes, but also from an ecological point of view, since the release of water-soluble fragments of lignin, cellulose and hemicellulose occurs in the state of the art. Biological utilization of said complex material requires the presence of organisms. capable of producing enzyme systems cleaving and utilizing the lignocellulose complex such as cellulose (β-1,4-glucan glucanohydrolase EC 3.2.1.4), cellobiohydrolase (EC 3.2.1.—), xylanase (, 6-1,4-xylan xylanohydrolase EC 3.2 .1.8), β-mannanase (./3-1,4-mannan mannanohydrolase EC 3.2.1.78), enzymes capable of cleaving the aromatic components of lignin (tyrosinase EC 1.10.3.1, lactase EC 1.10.3.2, peroxidase EC 1.11.1.7) but (a) glucosidases (β-glucosidase EC 3.2.1.21, α-glucosidase EC 3.2.1.20) responsible for the cleavage of the aromatic constituents of lignin bound by α - and β-glucosidic linkages (such as β-glucopyranosides of valine, eugenol, -OH-3-O-methyl benzylmethylketone, pir, resinol and the like) [Joniak D., Košíková B .: Chemical Rumors 28, 110 (1974), Košíková

B., Joniak D .: Cellulose Chem. Technol 1.0, 695 (1976)). The production of these enzymes is described in several organisms, but is not observed in the overall complex [fr. U.S. Patent 2,385,730, Ciaramitaro D., Steelink

C.: Phytochemistry 14, 543 (1975); Rosch R., Liese W., Berndt H., Arch. Microbiol 67, 28 (1969)].

However, the ability of microorganisms to produce a complex enzyme system degrading lignin, cellulose, and wood-cellulose hemoglobin components is substantially more widespread than would be inferred from a small number of publications in this field. Using a gel method for testing microorganisms for polysaccharide hydrolase (and no. 186 059) and lignocellulosic material models based on β-glucosides-vanillin, eugenol, isoeugenol, 4-OH-3-O-methyl-benzylmethyl ketone and di-5S. Pinoresinol-D-glucopyranoside, methyl-4,6-O-vanilidene α-D-glucopyranoside and similar derivatives of anisylidene, syringylldene, veratylidene and - [1- (4-OH, 3-O-methylbenzylidene)] and ether derivatives of 3- O-anisyl-D-glucose, 3-O-veratryl-D-glucose, and water-soluble lignin prepared from tissue cultures of Norway spruce (Picea excela Link) [Zemek J., Strmen J., Hricova D .: Z. Pflanzenphysiol 77, 95 (1976)] and [Brauns FE, Brauns DA: The Chemistry of Lignin (1960), Academic Press New York, p. 87], and aa] water-soluble lignin-rich waste obtained from thermo-mechanical pulping of wood pulp [Košíková B ., Joniak D., Polcin J .: Cellul. Chem. Technol have found significant utilization of the lignocellulosic complex especially in the microscopic fungi from the group of aquatic hyphomycetes.

Thermomechanical leach represents a significant progress in reducing waste water pollution compared to pulp production by conventional methods. With this technology, the wood mass components dissolve in an amount of 2 to 5% due to the loosening of its structure by steaming and refining. In view of the planned implementation of this technology, the present process according to the invention promises to address the biological disposal of this type of waste as well, since the wastewater treatment overviews of thermo-mechanical leachate abroad show that precipitation and flocculation methods have little effect on soluble organic substances. L .: Perspectives of Technol. pulp production, 7th all-cotton day, Banská Bystrica (1978)].

The principle of the invention is that by cultivating the following strains of microscopic fungi from the group of aquatic hyphomycetes, namely:

Alatospora acuminata Alatospora flagellata Articulospora tetracladia Calcarispora hiemalis Culicidospora gravida Dendrospora erecta Gyoerffyella specula Gyoerffyella rotula Lemomniera aquatica Lemonniera centrosphaera Tricladium splendes

CCMF - 732 CCMF - 501 CCMF - 113 CCMF - 110 CCMF - 120 CCMF - 404 CCMF - 401 CCMF - 400 CCMF - 124 CCMF - 149 CCMF - 313 individually or mixtures thereof in aqueous medium at pH 5.0 to 6.5 and 15 to 25 ° C in the presence of lignocellulosic complex components such as lignin, cellulose, xylan, mannan, are enzymatically cleaved for 24 to 144 hours, and the following organisms are able to utilize the resulting low molecular weight enzymatic degradation products to produce biomass in an amount of 20 to 30% w / w to the dry weight of the lignocellulose complex.

The advantage of this process is the complex treatment of the lignocellulosic complex under low energy conditions (temperatures up to 25 ° C) as well as soluble components, which enables biological treatment of waste water containing lignocellulosic components resulting from pulp production, even with the use of perspective thermo-mechanical wood processing technologies. directly in conditions of ecosystems of watercourses. In view of the complexity of bio-waste utilization, the presence of amylolytic enzymes in these organisms and the ability to cleave lichenate and other (1-> 4.3) -S-D-glucanes, particularly in the case of Tricladium splendes, are also important advantages. All these strains are furthermore producers of extracellular and proteolytic enzymes.

Example 1

Alatospora acuminai CCMF-732, Alatospora flagellata CCMF-501, Articulospora tetracladium CCMF-113, Culicidcspora gravida CCMF-120, Gyoerffyella rotula CCMF-400, Gyoerffyella speciosa CCMF-401, Lemonniera aquatica CCMF-149, Lemonniera aquatica CCMF-313 are seeded onto a gel prepared by cross-linking hydroxyethylcellulose (1 g) with 2-chloromethyl oxirane (and no.

183 169], immersed in half its height in 5 ml of distilled water enriched with mineral constituents according to Vogel [Vogel HJ: Microbiol. Ger. Bull. 13, 42 (1958}) at pH 5.5 The cultivation is carried out at 20 ^DEG C. for 3 days, when the cellulytic activity of the gel is run off, and the thus prepared biomass of aqueous hyphomycetes is used as inoculum on the soil prepared from (1). -> 4] - β-D-xylan (1.2 g) (ex Fagus silvatica) cross-linked with 2-chloromethyl oxirane (α # 186 059) The gel is immersed in water with minerals as described for hydroxyethylcellulose, the same are also After 3 days, the biomass obtained is used to inoculate a gel prepared by cross-linking (1 → 4) -, SD-mannan (1 g) (ex Phytelaphas macrocarpe) (and # 186 059) and the cultivation is carried out as in the case The biomass thus prepared is used as liquid inoculum containing mineral substances (Vogel) as well as water-soluble lignin (0.5 g / 100 ml) prepared by extraction from the spruce tissue biomass of Picea excelsa Link for 20 days. . The cultivation is carried out for 5 days at 20 DEG C. The biomass thus prepared (0.6 gj is used as a mixed inoculum of soil prepared from 2.5 g thermomechanical extract and 0.2 g ammonium sulfate in 1 liter of water (pH) 5.8} and cultivation is performed for 5 days at 20 ° C submerged without shaking. After this time, the proportion of aromatic components of the lignin cellulose complex falls below 3.5% of the starting value, the proportion of the sugar components decreases below 2% of the starting value.

β

Example 2

As described in Example 1, except that the monoculture of Lemonniera aquatica CCMF-124 is used instead of the mixed inoculum, the soils are adjusted to pH 5 and the mixture (50 mg each) is used instead of water-soluble lignin from Picea excelsa Link. -glucoside of vanillin, (3-D-glucopyranoside-methyl-4,6-O-anisylidene and 3-O-veratryl-G-glucose in 20 ml of Vogla podium) The obtained Lemonniera aquatica 65 mg biomass is used as inoculum of liquid soil (250 ml) prepared from 0.5 g water-soluble lignin (Picea excelsa Link) enriched with 20 mg carboxymethylcellulose, 50 mg xylan (ex Fagus silvatica) and 50 mg ivory-β-D-mannan (pH 1 -> 4) (pH The cultivation is carried out at 25 ° C for 120 hours, with the content of lignin aromatic components falling below 3% of the starting value and the sugar components content below 2.1%.

Example 3

As described in Example 1, except that the Gyoerffyella rotula CCMF-400 strain is used instead of the mixed inoculum. Cultivation is carried out on soils at pH 5.6, at 15 ° C. The obtained biomass from the last passage through water-soluble lignin (55 mg) is used as an inoculum of soil prepared from 2.2 g of thermomechanical extract enriched with 0.4 g of xylan (ex Fagus silvatica), 0.1 g of KH2PO4, 0.3 g of ammonium sulfate in 200 ml of water (pH 6) and cultivation is carried out at 18 ° C for 144 hours. The biomass Gyoerffyella rotula 0.8 g is obtained, whereby the aromatic components of the lignin of the thermo-mechanical leachate decrease by · 96% and the sugar components of the starting soil by 97.5%.

The invention has application not only in the basic research of the chemistry and biochemistry of lignin and carbohydrates, but especially in the field of watercourse protection, since biological purification by said organisms effectively eliminates saccharide and lignin components without the addition of biogenic factors. without energy costs, all the microorganisms used are non-pathogenic and do not produce toxins. All microscopic fungi from the group of aquatic hyphomycetes represent a spontaneous microflora of river flows and therefore do not represent an ecosystem hazard.

Claims (1)

Method of microbial utilization of the lignocellulosic complex by means of microscopic fungi from the group of aquatic hyphomycetes, characterized in that the following strains are cultivated: Alatospora acuminata Alatospora flagellata Articulospora tetracladia Calcarispora hiemalis Culicidospora gravida Dendrospora erecta CCMF-732 CCMF-501 CCMF-113 CCMF-110 CCMF-120 CCMF-404 Gyoerffyella specioza Gyoerffyella rotula Lemonniera aquatica Lemonniera centrosphaera Tricladium splendes CCMF - 401 CCMF - 400 CCMF - 124 CCMF - 149 CCMF - 313 individually or in a mixture in an aqueous environment at pH 5.0 to 6.5 and 15 to 25 ° C in the presence of lignocellulose complex components such as lignin, cellulose, xylan, mannan at a concentration of 0.02 to 0.5% by weight for 24 to 144 hours. Severography, n. p., Plant 7, Mosl