CN1510142A - Production of flavicidic acid by microorgan degradation - Google Patents

Production of flavicidic acid by microorgan degradation Download PDF

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Publication number
CN1510142A
CN1510142A CNA021581894A CN02158189A CN1510142A CN 1510142 A CN1510142 A CN 1510142A CN A021581894 A CNA021581894 A CN A021581894A CN 02158189 A CN02158189 A CN 02158189A CN 1510142 A CN1510142 A CN 1510142A
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coal
cgmcc
acid
degradation bacteria
xanthohumic
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CN1236065C (en
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袁红莉
陈文新
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China Agricultural University
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China Agricultural University
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Abstract

A process for preparing the xanthonic acid by microbial degradation includes such steps as mixing raw coal with auxiliaries chosen from natural plant carbon source, natural plant protein source, urea and KH2PO4, adding the degradating bacteria chosen from Panicillium sp. LNP6 (CGMCC No.0866) and Bacillus sp.BJB2 (CGMCC No.0867), and fermenting. Its advantages are high activity, small molecule and high oxygen and nitrogen content of product, no environmental pollution, and use of residual dregs are organic fertilizer.

Description

The xanthohumic acid method is produced in microbiological deterioration
Technical field
The present invention relates to a kind of method of utilizing the low-yield coal of microbiological deterioration to produce xanthohumic acid.
Background technology:
Xanthohumic acid is molecular weight less (it is generally acknowledged less than 500D) in the humic acids, active high component, aspect such as can be widely used in worker, farming, cure, herd, as improve the plant drought ability, as chemical fertilizer, pesticide synergistic agent, plant growth stimulator, medicine industry raw material etc.
Chemical process is all adopted in xanthohumic acid production at present, mainly extracts from weathered coal:
(1) uses the sulfuric acid acetone method
Weathered coal behind the wet concentration and aqueous acetone are mixed, make it to suspend, add the sulfuric acid stirring reaction again through stirring.To react supernatant liquor after the natural subsidence and remove solvent, obtain the xanthohumic acid product.Because of weathered coal xanthohumic acid content is low, must xanthohumic acid content be brought up to more than 38% through wet concentration; For the pH that keeps extracting solution about 1.5, consume a large amount of sulfuric acid, and production equipment requires acidproof; Solvent is an acetone, and is volatile during recovery, and reaction vessel requires airtight.
(2) ion-exchange-resin process
The metal ion that replaces in the Xanthohumate with strong acid ion exchange resin discharges xanthohumic acid.
This method requires xanthohumic acid content>30% in weathered coal, and granularity<80 orders fully reacting with resin, and is separated with ion exchange resin by screening; The granularity that also requires resin is between the 30-60 order, and consumption is big; Needing with relatively large concentration during regeneration is about 8% hydrochloric acid that enter the mouth; Also to use soft water during reaction; Will feed pressurized air during reaction and stir, energy consumption is big; Ash content from coal dust is arranged in the xanthohumic acid that obtains, will keep higher temperature (70-80 ℃) during de-ash.
There is abundant lignite resource in China.Brown coal incendiary thermo-efficiency is low, and causes environmental pollution easily, therefore extracts xanthohumic acid highly significant from brown coal.Prior art is to extract from brown coal with chemical means.Method is: extract with alkali lye earlier, then with the supernatant liquor acidifying, be settled out xanthohumic acid wherein.But this method extraction yield is low, and no industrial production is worth.And the xanthohumic acid flocculation limit that chemical method is produced is low, and is active low, and produces the secondary refuse of contaminate environment,
Summary of the invention
The problem to be solved in the present invention is with the microbial method low-yield coal of degrading, and obtains xanthohumic acid.
The invention provides a kind of accessory formula mixes with feed coal, add the efficient degrading bacterial strain of seed selection of the present invention, coal is fermented, the xanthohumic acid that is combined in the coal is discharged, become the component of water soluble, and can make part macromole humic acids become the small molecules xanthohumic acid.
Concrete grammar is as follows:
1, batching:
Feed coal is mixed with auxiliary material, add entry and stir;
2, fermentation:
Plant a kind of degradation bacteria in above-mentioned batching, inoculum size is 10% (every gram wet feed of solid koji or liquid culture: V/W or W/W), and fermentation was to 7~9 days;
Or in above-mentioned batching, plant first kind of degradation bacteria, and inoculum size is 5-10% (every gram wet feed of solid koji or liquid culture: V/W or W/W), cultivates and plants second kind of degradation bacteria (inoculum size is the same) after 2 days again, fermentation was to 6~7 days;
3, extract:
Add entry, separate and contain the xanthohumic acid supernatant.
This supernatant can directly dilute utilization, will can obtain the xanthohumic acid powder after this supernatant drying; Residue can further extract or directly be used as humic acid fertilizer.
Described feed coal comprises brown coal, weathered coal and coal gangue;
Described auxiliary material comprise natural phant carbon source from agricultural byproduct (as in sawdust, rice bran, the straw (wheat, corn or rice straw etc.) one or more) and natural plant protein source (as soya-bean cake, peanut cake), and urea, KH 2PO 4
Feed coal and ratio of adjuvant are: coal: auxiliary material (natural phant carbon source): auxiliary material (natural plant protein source): urea: KH 2PO 4=40-60: 20-40: 10-20: 2: 0.1 (weight part)
Described degradation bacteria is Penicillium notatum Penicillium sp.LNP6 CGMCC 0866 and/or bacillus sp.BJB2 CGMCC 0867
Preferred fermentation process is to plant 5-10% Penicillium notatum Penicillium sp.LNP6CGMCC 0866 (every gram wet feed of solid koji or liquid culture: V/W or W/W) in batching earlier, cultivate and plant bacillus sp.BJB2 CGMCC 0867 (inoculum size is the same) after 2 days again, fermentation was to 6~7 days;
The present invention's beneficial effect against existing technologies is:
1, can obtain xanthohumic acid from multiple sources such as brown coal, weathered coal and coal gangues, and the rate ratio chemical method improves obviously;
2, the gained xanthohumic acid is active strong, because biological Degradation, gained xanthohumic acid molecule obviously diminishes, and oxygen and nitrogen content increase, and flocculation limit is big;
3, fermentation, leaching process do not use chemical solvents, can not produce the secondary refuse of contaminate environment;
4, auxiliary material is an agricultural byproduct, and residue can be made fertilizer and use, and can realize the sustainable use of the energy.
The explanation of related microorganism material
1, microbial name
(1) Penicillium notatum
Be Penicillium (Penicillium s p.), bacterial strain called after: Penicillium s p.LNP6
(2) genus bacillus
Be bacillus (Bacillus sp.), bacterial strain called after: Bacillus sp.BJB2
2, microbial preservation number
(1) Penicillium notatum Penicillium sp.LNP6 CGMCC 0866
(2) bacillus sp.BJB2 CGMCC 0867
3, microorganism mycology feature
(1) morphological specificity
Penicillium notatum:
Bacterium colony is round, bacterium colony is surperficial is fine and close fine and soft shape, is seeded on the czapek's solution, can form obvious bacterium colony in one day, cultivates a few days ago that bacterium colony is white in color, and the 3rd day begins to become yellow, and the substratum back side also produces tangible yellow soluble pigment.Along with the prolongation of incubation time, yellow is deepened gradually, after seven days, produces yellow to the safran conidium.
The inserted sheet culture method is observed and is found that the conidiophore of this bacterium is tangible single-wheel broom shape branch, and the conidium ellipse is so be defined as Penicillium.
Genus bacillus:
The bacterium colony circle, white, refractive index is strong.Gram-positive, the thalline size is 1-1.5 μ m * 4.0-5.0 μ m, cultivates on bacteria culture medium, and beginning in 16 hours can produce gemma, and gemma is middle giving birth to.
So be defined as genus bacillus.
(2) spawn culture separation method
Penicillium notatum separates from coal washery, colliery, Fuxin, Liaoning, and genus bacillus separates in the coal gangue of Mentougou, is normal temperature type bacterium (the mould growth temperature is 25-28 ℃, and genus bacillus is 28-30 ℃).Mould can be with general liquid fermenting or solid koji enlarged culturing, and genus bacillus is cultivated with general bacterial liquid fermentation method and gets final product.
The strain separating method
Sampling is made bacteria suspension with 8.5% salt solution, is that the minimum medium of sole carbon source is coated with separation in order to brown coal, waits to grow picking dominant bacteria behind single bacterium colony, and further checks the ability of its brown coal of degrading.Method be with inoculation on solid plate, 28-30 ℃ of cultivation, (mould three days behind the formation bacterium colony, genus bacillus 1 day) on bacterium colony, adds through (121 ℃ of high-temperature sterilizations, brown coal 30min), continue to cultivate observe brown coal and be degraded and form the situation of black drop, speed that occurs with drop and formation amount are as screening index.
Find that through screening a strain mould can form obvious drop in three days, a bacillus can obviously form drop in one day.Penicillium notatum is carried out ultraviolet mutagenesis twice, obtain a plant mutant strain, be numbered P6 (Penicillium sp.).The ability of this mutant strain degraded brown coal obviously improves, and beginning degradable brown coal generated the black drop in 36 hours, and its cell-free filtrate of cultivating through 3 days shaking tables brown coal of also degrading produce the black soluble material.
4, effect
This two strains bacterium has the effect of stronger degraded brown coal.Dull and stereotyped this mould of cultivating can will place the Inner Mongol brown coal degraded on its bacterium colony to produce the black drop since 36 hours.Genus bacillus can begin to place the brown coal degraded on its bacterium colony to produce the black soluble material at 20 hours, and atrament infiltration substratum, and tangible black is dyed at the substratum back side.
With these two strains bacterium mixed fermentation degraded Inner Mongol brown coal, water-soluble xanthohumic acid content reaches more than 30% in the tunning.
Embodiment
Embodiment 1
1) batching
45% brown coal+14% sawdust+14% soya-bean cake+25% straw (corn or rice straw)+2% urea+0.1%KH 2PO 4
Brown coal (from Huolin Gol colliery, Inner Mongol) are crushed to about 50 orders, and other ingredient powders are broken to about 2mm;
2) fermentation
Various material are mixed, stir, add water (urea and the KH of 2 times of weight 2PO 4Dissolve in the water and add), leave standstill after 30 minutes and stir, insert Penicillium notatum (every gram wet feed of solid koji or liquid culture: V/W or W/W) with 10% inoculum size, cultivated 2 days for 28 ℃~30 ℃, once since stirring in the 12nd hour per 5~6 hours.Insert sporeformer (inoculum size is the same) after 2 days again, standing for fermentation to 6~7 day.
3) extraction step: fermentation finishes, by expecting (doing): water=1: 20 adding tap water, room temperature (or keeping 60 ℃) two hours, intermittently stir, sedimentation two hours separates supernatant, and residue adds the equal-volume flooding once again, merge twice supernatant and be xanthohumic acid, can drying obtain xanthohumic acid powder or directly dilution utilization.If can extract (xanthohumic acid) 30-35%, if can extract (xanthohumic acid) 40-44% at 60 ℃ with room temperature (about 20 ℃).Humic acid fertilizer is further extracted or directly be used as to the residue available bases.
4) to the evaluation and the specificity analysis of product xanthohumic acid
1919, Oden at first proposed humic acids is divided into water miscible xanthohumic acid, soluble ulmic acid of ethanol and the soluble Ulmic acids of alkali.1984, Xue Hongji etc. pointed out: xanthohumic acid is that alkali is solvable, the solvable and water soluble of acid humic acid substance.1996, Wang Tianli etc. discussed with regard to the condition that defines of xanthohumic acid, and defining of xanthohumic acid of proposition should be with water-soluble humic acids as unique conditional.When being equipped with xanthohumic acid, limit the pH value (2) after the acidifying with the alkali-soluble acid analysis legal system.Leavened prod is directly to use water extraction in this research, so be the xanthohumic acid product.Because auxiliary material is an organism, measures in the product and contain 5-6% amino acid, 3-5% nucleic acid.And gained xanthohumic acid product carried out following specificity analysis: Infrared spectroscopy shows: this product is at 1720cm -1, 1620cm -1, 1400cm -1, 1250cm -1There is absorption peak at the place, conforms to the absorption spectrum of Gongxian (now being the Gong Yi city) xanthohumic acid.All solvable under diluted acid (0.01mol/L HCl), neutrality and diluted alkaline (0.01mol/L NaOH) condition.So show it is xanthohumic acid.
5) strain separating method
From the sampling of limit, ditch, coal washery, colliery, Fuxin, Liaoning, make bacteria suspension with 8.5% salt solution, in order to brown coal the minimum medium (K of sole carbon source 2HPO 40.5, KH 2PO 40.5, MgSO 4.2H 2O 0.5, (NH 4) 2SO 41, CaCl 2.2H 2O 0.1, brown coal 2g, H 2O 1L) is coated with separation, waits to grow picking dominant bacteria behind single bacterium colony, and further check the ability of its degraded brown coal.Method be with inoculation on solid plate, 28-30 ℃ of cultivation, (mould three days behind the formation bacterium colony, genus bacillus 1 day) on bacterium colony, adds through (121 ℃ of high-temperature sterilizations, brown coal 30min), continue to cultivate observe brown coal and be degraded and form the situation of black drop, speed that occurs with drop and formation amount are as screening index.
Find that through screening a strain mould can form obvious drop in three days, a bacillus can obviously form drop in one day.Penicillium notatum is carried out ultraviolet mutagenesis twice, obtain a plant mutant strain, be numbered P6 (Penicillium sp.).The ability of this mutant strain degraded brown coal obviously improves, and beginning degradable brown coal generated the black drop in 36 hours, and its cell-free filtrate of cultivating through 3 days shaking tables brown coal of also degrading produce the black soluble material.
6) strain identification
According to colony characteristics, mycelia have every and [determine that P6 is mould (Penicilliumsp.); According to bacterium colony, thalli morphology and can produce features such as gemma and determine that bacterium is genus bacillus (Bacillus sp.).
Embodiment 2
1) batching
56% brown coal (from the Sanmenxia Gorge, Henan)+20% soya-bean cake+12% rice bran+10% sawdust+2% urea+0.1%KH 2PO 4
2) operation steps
Various material are mixed, stir, and (moistening to expect, loose is principle, urea and KH to add suitable quantity of water 2PO 4Dissolve in the water and add), leave standstill after 30 minutes and stir, insert Penicillium notatum (every gram wet feed of solid koji or liquid culture: V/W or W/W) with 10% inoculum size, cultivated 2 days for 28 ℃~30 ℃, once since stirring in the 12nd hour per 5~6 hours.Insert sporeformer (inoculum size is the same) after 2 days again, standing for fermentation to 6~7 day.Fermenting finishes extracts xanthohumic acid 18-25% with method described in the embodiment 1 at 20 ℃, and the residue water further extracts or directly be used as humic acid fertilizer.
The evaluation of strain separating cultural method and product xanthohumic acid and characteristic analysis method are with embodiment 1.

Claims (7)

1, the method for the low-yield coal production of a kind of microbiological deterioration xanthohumic acid is characterized in that feed coal is mixed with auxiliary material, adds degradation bacteria strains, and coal is fermented, and obtains xanthohumic acid;
Concrete grammar is as follows:
1) batching: feed coal is mixed with auxiliary material, add entry and stir;
Described auxiliary material comprises natural phant carbon source, natural plant protein source, urea, KH 2PO 4
Feed coal and ratio of adjuvant are: coal: the natural phant carbon source: natural plant protein source: urea: KH 2PO 4=40-60: 20-40: 10-20: 2: 0.1 (weight part)
2) fermentation: plant a kind of degradation bacteria in above-mentioned batching, inoculum size is 10% (every gram wet feed of solid koji or liquid culture: V/W or W/W), and fermentation was to 7~9 days;
Or in above-mentioned batching, plant first kind of degradation bacteria, inoculum size is 5-10% (every gram wet feed of solid koji or liquid culture: V/W or W/W), cultivates after 2 days kind again and goes into second kind of degradation bacteria (inoculum size is the same), standing for fermentation to 6~7 day;
Described degradation bacteria is Penicillium notatum Penicillium sp.LNP6 CGMCC 0866 and/or bacillus sp.BJB2 CGMCC 0867;
3) extract: add entry, separate and contain the xanthohumic acid supernatant.
2, the method for the described production xanthohumic acid of claim 1, described first kind of degradation bacteria are that 0866, the second kind of degradation bacteria of Penicillium notatum Penicillium sp.LNP6 CGMCC is bacillus sp.BJB2 CGMCC 0867.
3, the method for claim 1 or 2 described production xanthohumic acids, wherein said natural phant carbon source be in sawdust, rice bran, the straw one or more.
4, the method for claim 1 or 2 described production xanthohumic acids, wherein said natural plant protein source comprises soya-bean cake, peanut cake.
5, the method for claim 1 or 2 described production xanthohumic acids, wherein said feed coal comprises brown coal, weathered coal and coal gangue.
6, Penicillium notatum Penicillium sp.LNP6 CGMCC 0866.
7, bacillus sp.BJB2 CGMCC 0867.
CN 02158189 2002-12-24 2002-12-24 Production of flavicidic acid by microorgan degradation Expired - Fee Related CN1236065C (en)

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101913950A (en) * 2010-08-19 2010-12-15 范鸿基 Organic fertilizer preparation method and multi-strain breeding of biodegradable coal gangue
CN103588977A (en) * 2013-11-08 2014-02-19 云南联合药业有限责任公司 Method for extracting and preparing peat fulvic acid and drug application
CN103724060A (en) * 2013-12-31 2014-04-16 新疆科林思德新能源有限责任公司 Lignite biological fertilizer and production method thereof
CN107815433A (en) * 2017-12-08 2018-03-20 青岛益佰农肥业有限公司 A kind of complex micro organism fungicide and its application in fermentation humic acid lignite produces fulvic acid
CN108033860A (en) * 2018-01-09 2018-05-15 遵义粒满丰肥业有限责任公司 A kind of method of multiplicity reactivation weathered coal humic acid
CN109006364A (en) * 2018-10-10 2018-12-18 中国科学院沈阳应用生态研究所 A kind of planting lawn matrix and preparation method thereof
CN110747132A (en) * 2019-10-31 2020-02-04 内蒙古科技大学 White paecilomyces and application thereof in preparation of phthalic acid ester by dissolving lignite
CN110951626A (en) * 2019-12-27 2020-04-03 北京林业大学 Penicillium oxalicum capable of decomposing coal gangue
CN110999753A (en) * 2019-12-26 2020-04-14 北京林业大学 Coal gangue-based artificial ecological matrix and preparation method thereof
CN116589700A (en) * 2023-05-19 2023-08-15 内蒙古蒙土生态环保科技有限公司 Method for purifying fulvic acid

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101913950A (en) * 2010-08-19 2010-12-15 范鸿基 Organic fertilizer preparation method and multi-strain breeding of biodegradable coal gangue
CN103588977A (en) * 2013-11-08 2014-02-19 云南联合药业有限责任公司 Method for extracting and preparing peat fulvic acid and drug application
CN103588977B (en) * 2013-11-08 2015-10-28 云南联合药业有限责任公司 Method and the medicinal application of the yellow humic acid of peat are prepared in a kind of extraction
CN103724060A (en) * 2013-12-31 2014-04-16 新疆科林思德新能源有限责任公司 Lignite biological fertilizer and production method thereof
CN107815433B (en) * 2017-12-08 2021-03-12 青岛益佰农肥业有限公司 Compound microbial agent and application thereof in producing fulvic acid by fermenting humic acid lignite
CN107815433A (en) * 2017-12-08 2018-03-20 青岛益佰农肥业有限公司 A kind of complex micro organism fungicide and its application in fermentation humic acid lignite produces fulvic acid
CN108033860A (en) * 2018-01-09 2018-05-15 遵义粒满丰肥业有限责任公司 A kind of method of multiplicity reactivation weathered coal humic acid
CN109006364A (en) * 2018-10-10 2018-12-18 中国科学院沈阳应用生态研究所 A kind of planting lawn matrix and preparation method thereof
CN110747132A (en) * 2019-10-31 2020-02-04 内蒙古科技大学 White paecilomyces and application thereof in preparation of phthalic acid ester by dissolving lignite
CN110999753A (en) * 2019-12-26 2020-04-14 北京林业大学 Coal gangue-based artificial ecological matrix and preparation method thereof
CN110999753B (en) * 2019-12-26 2021-07-27 北京林业大学 Coal gangue-based artificial ecological matrix and preparation method thereof
CN110951626A (en) * 2019-12-27 2020-04-03 北京林业大学 Penicillium oxalicum capable of decomposing coal gangue
CN110951626B (en) * 2019-12-27 2021-05-25 北京林业大学 Penicillium oxalicum capable of decomposing coal gangue
CN116589700A (en) * 2023-05-19 2023-08-15 内蒙古蒙土生态环保科技有限公司 Method for purifying fulvic acid

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