KR20160089074A - Producing method of polyhydroxyalkanoates by mixed culture - Google Patents

Abstract

The present invention relates to a composition for producing polyhydroxyalkanoate from biomass or polysaccharide substrates, the composition containing, as an active ingredient, a mixed culture medium of a Saccharophagus degradans strain and a Bacillus cereus strain, and to a method for producing polyhydroxyalkanoate using the same. According to the present invention, the polyhydroxyalkanoate can be produced at high yield with a simple method through the mixed culture of strains.

Description

TECHNICAL FIELD The present invention relates to a method for producing polyhydroxyalkanoates by using a mixed culture,

The present invention relates to a method capable of producing a polyhydroxyalkanoate using a mixed culture of a strain.

As the global exhaustion of fossil fuels and concerns over environmental pollution increase, research is underway to produce stable and sustainable energy and plastics. As part of the development of alternative energy and plastics, the technology of producing alcohol and biodegradable plastic polyhydroxyalkanoates (PHA) from biomass resources is receiving the greatest attention.

Conventional alcohol and biodegradable plastic First generation alcohol and PHA using sugar starch such as sugar cane and corn starch such as sugar cane are produced for the production of PHA. However, this is due to competition with food and livestock feed, I am in trouble. Thus, second generation alcohols using lignocelluloses, which are the most abundant and reproducible resources on the planet, are under development, but lignocellulose is a lignin and lignin, which is a degradable aromatic polymer, As a complex of cellulose and hemicellulose, a complicated process is required to remove lignin, which is costly.

On the other hand, PHA is a polyester that accumulates in the form of an energy storage in the body of microorganisms. Poly (3-hydroxybutyrate) [(P (3HB)], which is one of the polyesters, is produced by Lemoigne in 1925 by Bacillus megata PHA is a biodegradable polymer with excellent properties comparable to synthetic polymers and can be used to solve serious pollution problems caused by the degradation of synthetic polymers when used in food packaging and disposable product packaging. .

However, since PHA requires a high manufacturing cost, it is difficult to replace conventional non-degradable plastic. Therefore, it is necessary to develop a new production process that can reduce manufacturing cost.

1. INTERNATIONAL JOURNAL OF ENVIRONMENTAL SCIENCES Volume 1, No 5, 2011.

Therefore, the present inventors have completed the present invention by developing a method for producing PHA at a high yield in a cost-effective manner by a simple method through mixed culture of strains.

Therefore, an object of the present invention is to provide a method for producing polyhydroxyalkanoate from a biomass or polysaccharide substrate, which comprises a mixed culture of a strain of Saccharophagus degradans and a strain of Bacillus cereus as an active ingredient, And to provide a composition for production.

Another object of the present invention is to provide a method for producing a polyhydroxyalkanoate comprising saccharifying and fermenting a biomass or polysaccharide substrate using the composition of the present invention.

In order to achieve the object of the present invention, the present invention provides a method for producing a polyhydroxyalkanoate from a biomass or polysaccharide substrate, which comprises a mixed culture of a Saccharophagus de Grassense strain and a Bacillus cereus strain as an active ingredient, Lt; / RTI >

In one embodiment of the present invention, the Saccharophagus de Grudace is a deposit number ATCC 43961, and the bacillus cereus may be a strain of NCBI gene bank number: KF 801505.

In one embodiment of the present invention, the biomass may be a lignocellulosic biomass or a seaweed biomass.

In one embodiment of the present invention, the polysaccharide is selected from the group consisting of cellobiose, alpha-cellulose, sigma cellulose, carboxymethyl cellulose, avicel, agarose, xylan, pectin, agar, alginate, fucoidan and may be selected from the group consisting of fucoidan, laminarin, chitin, pullulan, and filter paper.

The present invention also provides a method for producing a polyhydroxyalkanoate comprising saccharifying and fermenting a biomass or polysaccharide substrate using the composition of the present invention.

In one embodiment of the present invention, the Saccharophagus de Grudace is a deposit number ATCC 43961, and the bacillus cereus may be a strain of NCBI gene bank number: KF 801505.

In one embodiment of the present invention, the biomass may be a lignocellulosic biomass or a seaweed biomass.

In one embodiment of the present invention, the polysaccharide is selected from the group consisting of cellobiose, alpha-cellulose, sigma cellulose, carboxymethyl cellulose, avicel, agarose, xylan, pectin, agar, alginate, fucoidan and may be selected from the group consisting of fucoidan, laminarin, chitin, pullulan, and filter paper.

A composition for producing polyhydroxyalkanoate from a biomass or polysaccharide substrate and a polyhydroxyalkanoate using the same as an effective ingredient, which comprises a mixed culture of a Saccharophagus de Grudace strain and a Bacillus cereus strain. ≪ / RTI > According to the method of the present invention, polyhydroxyalkanoate can be produced at a high yield by a simple process at a low cost, compared with the conventional method, and is useful for various industries that can produce alternative energy and plastic from biomass resources Can be used.

Fig. 1 is a GC analysis result obtained by pure culture of Saccharophagus de Grondans using red algae as a carbon source.
FIG. 2 is an FT-IR spectrum of cells obtained by pure culture of Saccharophagus de Grudens using various kinds of cellulose as a carbon source.
FIG. 3 is a graph showing the results of a pure culture (A) of Saccharophagus de Grondans and a mixed culture (B) of Saccharophagus de Grassense and Bacillus cereus using agarose and xylan as carbon sources. FT-IR spectrum of the cells.
4 is an FT-IR spectrum of the cells obtained by pure culture of Saccharophagus de Grondans using red algae as a carbon source.
FIG. 5 is an FT-IR spectrum of a cell obtained by pure culture of Saccharophagus de Grondans using corn leaves as a carbon source.

The present invention is characterized in that it has been established that a PHA can be obtained at a high yield while reducing production costs compared to the prior art.

The method of producing PHA identified in the present invention is characterized in that a mixed culture of Saccharophagus de Grudace and Bacillus cereus is used. That is, when a mixed culture of these two strains is used, Experimental results show that PHA can be obtained in high yield from mass or polysaccharide substrates.

Furthermore, the inventors of the present invention have confirmed that these two strains can not only glycosylate biomass such as corn leaf, rice straw, seaweed, or various polysaccharides such as cellulose, xylan and agarose but also ferment and directly produce PHA .

Further, the present inventors have identified optimal process conditions for producing PHA at a high yield by using the culture of the strain. Thus, the present invention provides a method for producing PHA by culturing a mixture of Saccharophagus de Grudaceus strain and Bacillus cereus strain, Massaging the polysaccharide matrix, and saccharifying and fermenting the mass or polysaccharide substrate.

The Saccharophagus de Grondans used in the present invention is a strain harvested at a river mouth and decomposes more than ten kinds of polysaccharides constituting woody and seaweeds. The wild-type Saccharophagus de Grondans is deposited with the international depository institution under accession number ATCC 43961 And obtained from the above-mentioned organs and used. In addition, recombinant Saccharophagus de Grondans may also be used. In some cases, the recombinant Saccharophagus de Grondos can be used by genetic engineering and mutagenesis. In the present invention, bacillus cereus was used together with Saccharophagus de Grondense. Through such mixed cultivation, degradation of polysaccharides and conversion of the degraded sugars to PHA can be performed more rapidly, ultimately increasing production of PHA .

In addition, the method of the present invention is characterized in that it is not necessary to use a saccharifying enzyme in the method of producing PHA unlike the prior art, and the biomass is not required to be subjected to a separate pretreatment, For a period of 20 hours or more, or for 20 to 200 hours.

The saccharophagus degustatus may preferably be a strain No. ATCC 43961, and the bacillus cereus may preferably be a strain of accession KF 801505. In addition, in the case of the present invention, the saccharophagus de Grassense and bacillus cereus are mixed, and the growth is stabilized due to the symbiosis relationship between the two strains, which effectively increases the cell concentration.

In the present invention, the biomass may include cellulose, lignocellulose, seaweed biomass, and the like. The algae biomass may include algae, green algae, and brown algae, though not limited thereto.

The term " cellulosic " or " lignocellulosic biomass " refers to organic matter (wood or non-wood) derived from plants and includes crop wastes such as corn tiller, straw, rice straw, But are not limited to, these. But are not limited to, wood, wood energy crops, wood waste and residues such as coniferous trees, bark waste, sawdust, paper / pulp industry waste steam, wood fibers and the like. In addition, it may include grasses such as loess grasses, garden garbage (for example, lawn mowers, fallen leaves, shrubs and bushes), vegetable-treated wastes and the like. The cellulose feedstock may be selected from the group consisting of certain types of plant biomass such as non-timber plant biomass, crops, grasses, sugar processing residues, agricultural residues such as soybean tallow, corn tiller, rice straw, Corn fiber, recycled wood pulp fiber, sawdust, hardwood, conifer, or a combination of these. It may also include waste materials, newsprint, cardboard, sawdust, and the like.

The red algae may be selected from the group consisting of, for example, Gelidium amansii, Gracilaria verrucosa, Bangia atropurpurea, Porphyra suborbiculata, Porphyra yezoensis, Galaxaura falcata, (Eg, Scinaia japonica), Gelidium divaricatum, Gelidium pacificum, Lithophylum okamurae, Lithothammion cystocarpideum, Amphiroa anceps, Amphiroa beauvoisii, Corallina officinalis, Corallina pilulifera, Marginisporum aberrans, Carpopeltis prolifera, Grateloupia filicina, Grateloupia elliptica, Dog gambling Grateloupia lanceolanta, Grateloupia turtuturu, Phacelocarpus japonicus, Gloiopeltis furcata, Hypnea charoides, Hypnea japonitca, Chondrus cryptspus, Chondracanthus tenellus, Gracilaria textorii, Lomentaria catenata, Heterosiphonia japonica, Chondria crassicaulis, and Chamaecyparis spp. (Porphyra yezoensis Ueda), Cottonii, Grateloupia lanceolata, Pterocladia tenuis, Acanthopeltis japonica, Gloiopeltis tenax, Irish but are not limited to, moss, gambling (Pachymeniopsis elliptica), Ceramium kondoi, Ceramium boydenii, Gigartina tenella, Campylaephora hypnaeoides and the like.

Examples of the green algae include Enteromorpha, Spirogyra spp., Codium fragile, Codium minus, Caulerpa okamurai, Nostoc commune, and the like.

The brown algae include, for example, seaweeds such as Laminaria japonica, Undaria pinnatifida, Hizikia fusiforme, Analipus japonicus, Chordaria flagelliformis, Ishige okamurai, Scytosiphon lomentaria, Ecklonia stolonifera, Eisenia bicyclis, Costaria costata, Sargassum fulvellum, Sargassum horneri, Leptoptera, Lepidoptera, Lepidoptera, Lepidoptera, Sargassum thunbergii).

The polysaccharide may be selected from the group consisting of cellobiose, alpha-cellulose, sigma cellulose, carboxymethyl cellulose, avicel, agarose, xylan, pectin, agar, alginate, fucoidan fucoidan, laminarin, chitin, pullulan, and filter paper may be used.

As described above, the present invention can provide a method for efficiently producing and producing a polyhydroxyalkanoate using the two culture strains of the present invention. According to one embodiment of the present invention, The results were compared with those of Saccharophagus degustaceus strain and Bacillus cereus strain alone and the case of using these two strains together. Further, when analyzed by using various biomass or polysaccharide as a carbon source, It was confirmed that a PHA with high yield could be obtained. In particular, when xylan was used as a carbon source by using a mixed culture of Saccharophagus degustrad and Bacillus cereus, the accumulation rate of PHA in the cell was 30% Respectively.

Therefore, it was found that PHA can be obtained in the greatest amount when using a mixed culture of xylan as a carbon source and Saccharophagus de Grassense and Bacillus cereus.

Further, the present invention can provide a composition for producing polyhydroxyalkanoate from a biomass or polysaccharide substrate, which comprises a mixed culture of a Saccharophagus de Grudace strain and a Bacillus cereus strain as an active ingredient.

The composition according to the present invention may contain various nutritional media, buffer solutions and the like, preferably at least 1% of seawater salt for growth of the strains, 0.5% NH ₄ Cl, 50 mM Tris-HCl pH 7.6, 0.1% yeast extract, 0.2% carbon source) can be used.

The culture of the strain may contain a crushed cell wall fraction, a live cell, a dead cell, a dried bacterium or a culture solution of two strains of the present invention as an active ingredient. The culture may be a culture medium itself containing a strain cultured in a liquid medium, A filtrate (centrifuged supernatant) obtained by removing the strain by filtration or centrifugation of the culture solution, and the like.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

< Example  1>

Preparation of culture medium of strain

Bacillus cereus strain B3-D (NCBI gene bank number: KF801505) was isolated from the laboratory and used. The Saccharophagus degradans 2-40 (ATCC 43961) was purchased from ATCC. (ATCC 43961) and Bacillus cereus (KF801505) were inoculated into 50 ml of seawater salt medium (2.3% sea water salt, 0.5% NH ₄ Cl, 50 mM Tris-HCl pH 7.6, 0.1 % Yeast extract, 1% carbon source) and cultured in a 28 ° C shaking incubator. Solid culture was also performed with liquid culture through shaking culture as described above. In the case of solid medium, 1.5% agar was further added to the culture solution and cultured in a 28 ° C incubator.

For the experiment of Example 2 below, the carbon source was added at a concentration of 10 g / L of glucose, cellobiose, alpha-cellulose, sigma cellulose, carboxymethyl cellulose, avicel, agarose, xylan, Two strains were cultured or mixed, respectively.

< Example  2>

PHA Analysis of production

The production rate of PHA was analyzed in the experiment group in which the carbon sources were different from each other and each strain was cultured alone or mixed in Example 1. For this purpose, the dry cell weight of the cultured cells was measured. That is, 10 ml of the culture solution was centrifuged and lyophilized to measure the mass.

PHA formation was confirmed by FT-IR and gas chromatography (GC, 6890N, Agilent, Inc., HP-5 (30 m * 320 μm * 0.25 μm)). For GC analysis, 2 ml of the solution prepared in the ratio of methanol: H ₂ SO ₄ : internal standard (benzoic acid) = 850: 150: 2 g and 2 ml of chloroform were added to 10 mg of the sample for pretreatment, After cooling for 5 hours, 1 ml of distilled water was added, and the mixture was stirred to inject the lower layer into the GC, and the amount of produced PHA was measured. Table 1 shows the PHA content (%) in the dried cells according to the GC results, and the representative GC analysis results obtained when the red algae was used as the carbon source are shown in FIG. As a result of GC analysis, it was confirmed that the produced PHA was P (3HB). The confirmation results are shown in FIGS. 2 to 5 by FT-IR analysis results. In the case of ester carbonate of characteristic PHA peak at 1727-1745 cm ^-1 wavelength (C = O) band was detected. In addition, the same experiment as in Example 1 was carried out with a concentration of 10 g / L.

Carbon source Culture method PHA content in dried cells (%) Glucose (glucose) Pure cultivation of Saccharophagus de Grondans 22.4 Cellobiose Pure cultivation of Saccharophagus de Grondans 20.8 Alpha-cellulose
(α-cellulose) Pure cultivation of Saccharophagus de Grondans 11.8 Sigma cellulose
(Sigma cellulose) Pure cultivation of Saccharophagus de Grondans 13.7 Carboxymethylcellulose
(CMC) Pure cultivation of Saccharophagus de Grondans 12.7 Avicel Pure cultivation of Saccharophagus de Grondans 14.6 Agarose &lt; / RTI &gt; Pure cultivation of Saccharophagus de Grondans 18.1 Agarose &lt; / RTI &gt; Sakarog Pargus de Grudans and Bacillus
Mixed cultures of Cereus 19.7 Xylan (xylan) Pure cultivation of Saccharophagus de Grondans 22.7 Xylan (xylan) Sakarog Pargus de Grudans and Bacillus
Mixed cultures of Cereus 34.5 Mugwort Pure cultivation of Saccharophagus de Grondans 19.9 Mugwort Sakarog Pargus de Grudans and Bacillus
Mixed cultures of Cereus 28.8

As a result of the analysis, as shown in Table 1, PHA was produced in pure culture of Saccharophagus de Grondans or in a mixed culture with Bacillus cereus. In particular, when xylan was used as a carbon source, other carbon sources The PHA accumulation rate in the cells was relatively higher than that in the used cells. In particular, it was confirmed that the mixture of Saccharophagus de Grassense and Bacillus cereus was mixed and the amount of PHA produced was 34.5% when xylan was used as a carbon source.

Therefore, it was found that PHA could be produced with high yield by culturing these strains under a specific carbon source under these conditions.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (8)

A composition for producing a polyhydroxyalkanoate from a biomass or polysaccharide substrate, which comprises a mixed culture of a Saccharophagus de Grondos strain and a Bacillus cereus strain as an active ingredient. The method according to claim 1,
Wherein the Saccharophagus de Grudace is a strain of Accession No. ATCC 43961 and the Bacillus cereus is a strain of NCBI gene bank number: KF 801505. The method according to claim 1,
Wherein the biomass is lignocellulosic biomass or seaweed biomass. The method according to claim 1,
The polysaccharide may be selected from the group consisting of cellobiose, alpha-cellulose, sigma cellulose, carboxymethylcellulose, avicel, agarose, xylan, pectin, agar, alginate, fucoidan, laminarin, Chitin, pullulan, and filter paper. &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt; A process for preparing a polyhydroxyalkanoate, comprising saccharifying and fermenting a biomass or polysaccharide substrate using the composition of claim 1. 6. The method of claim 5,
Wherein said Saccharophagus de Grudace is a deposit number ATCC 43961 and said Bacillus cereus is a strain of NCBI gene bank number: KF 801505. 6. The method of claim 5,
Wherein the biomass is lignocellulosic biomass or seaweed biomass. 6. The method of claim 5,
The polysaccharide may be selected from the group consisting of cellobiose, alpha-cellulose, sigma cellulose, carboxymethylcellulose, avicel, agarose, xylan, pectin, agar, alginate, fucoidan, laminarin, Chitin, pullulan, and filter paper. &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt;

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