CN102559508A

CN102559508A - A Trichoderma viride producing cellulose-degrading enzymes and its application in the degradation of urban greening waste

Info

Publication number: CN102559508A
Application number: CN2011102354312A
Authority: CN
Inventors: 赵心清; 孙鸿金; 白凤武
Original assignee: Dalian University of Technology
Current assignee: Dalian University of Technology
Priority date: 2011-08-17
Filing date: 2011-08-17
Publication date: 2012-07-11
Anticipated expiration: 2031-08-17
Also published as: CN102559508B

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to trichoderma viride; in addition, the invention also relates to application of the trichoderma viride. Trichoderma viride strain for high yield of cellulase, wherein the strain is trichoderma viride in trichodermaTrichodermavirideThe accession number M20, 4.2.2011 is preserved in China general microbiological culture collection center, and the preservation number is CGMCC No. 4732. The strain can grow by taking various cellulose compounds as the only carbon source, including sodium carboxymethyl cellulose and microcrystalline celluloseCellulose, corn stalks, corn cobs, fallen leaves, turfgrass, and the like. The trichoderma viride related by the technology can secrete a plurality of cellulose hydrolases, can be used for treating urban greening waste, can obtain high reducing sugar in a short time, can be used for producing single-cell protein and fuel ethanol, and has important significance for solving a series of troublesome problems of energy shortage, environmental pollution and the like faced by human at present.

Description

The viride of one strain production of cellulose degrading enzyme and the application in the degraded of urban afforestation waste thereof

Technical field

The invention belongs to microbial technology field, particularly a kind of trichoderma viride; The invention still further relates to the application of this trichoderma viride in addition.

Background technology

Mierocrystalline cellulose is one type of maximum renewable resources of occurring in nature storage, is example with the stalk, and the whole world is annual produces about 1000～2,000 hundred million tons of agricultural crop straw, yet several hundred million tons of Mierocrystalline celluloses is only arranged by the human use.China is a large agricultural country, according to rough Statistics, and annual about 700,000,000 tons of the agricultural crop straw that produces of China.Owing to have many high energy hydrogen bonds in the Mierocrystalline cellulose, so its hydrolysis, utilize difficulty, most cellulose substances is all thrown aside.A large amount of Mierocrystalline celluloses has only sub-fraction to be used for aspects such as weaving, feed and papermaking; The overwhelming majority gets back in the natural carbon cycle as fuel or through the natural decomposition of mikrobe.These agricultural waste materials not only are not fully used, and its burning has caused pollution to environment again.Therefore reasonable development and these abundant natural resource of scientific utilization have become major fields of research and development.

Though the utilization of agricultural waste such as stalk receives generally and payes attention to that the bio-transformation of urban afforestation waste does not also obtain enough concerns.Scale along with urbanization progressively enlarged in recent years; And the continuous development of urban afforestation, the urban afforestation waste has become the important component part of municipal wastes, it is reported; The fallen leaves about 400 tons can be produced every day in Nanjing, and the Qingdao City will produce more than 600 ton of fallen leaves every day.How to handle these greening wastes, and problem such as free from environmental pollution needs to be resolved hurrily.Greening wastes such as dead leaf are transported to the refuse tip more and burn or bury, and can produce a large amount of floating dust and carcinogenic substance after the waste incineration, have a strong impact on environmental health and residents ' health.

On the other hand, fallen leaves and turfgrass contain abundant cellulose components, can be used as the production that renewable resources is used for bioenergy, solve China's bioenergy resources of production problem of shortage.Have the scholar to study of the degraded of wooden mould degraded and aspergillus to fallen leaves respectively, but the two research is the degraded of material, exists degradation time long withered grass; Problems such as reducing sugar is low; In addition, do not consider the processing of degradation bacteria behind degraded fallen leaves and turfgrass yet, and the preconditioning technique of raw material.Utilize high warm and humid technology of separating turfgrass that report is also arranged, but this arts demand special devices, Heating temperature is up to 480 ℃, and it is more to consume energy, and biological treatment mild condition, operation more easily.

Trichoderma viride is the wooden enzyme bacterium that can be used for administering waste agronomic crop pollution and cellulase high-yield that receives common concern in recent years gradually; This belongs to the multiple agricultural waste of bacterium degradable; Comprise corn cob, corn straw, straw etc.; But domestic research to viride is deep not enough, and is also less to its applied research.

Summary of the invention

The viride that the purpose of this invention is to provide a strain production of cellulose degrading enzyme; This bacterial strain can be grown in Mierocrystalline celluloses such as Microcrystalline Cellulose, Xylo-Mucine, corn straw are the substratum of sole carbon source, and the fermented liquid that is produced has the ability of degraded cellulose.Another object of the present invention is the application of viride in the degraded of urban afforestation waste of a strain production of cellulose degrading enzyme; The efficient technology of handling the urban afforestation waste of exploitation; Reduce environmental pollution and refuse treatment burden on the one hand; Improve processing efficiency on the other hand, and obtain high-load reducing sugar.

The viride bacterial strain of the present invention's one plant height cellulase-producing, this bacterial strain is the viride in the Trichoderma Trichoderma viride, numbering M20 was preserved in Chinese common micro-organisms DSMZ on April 2nd, 2011, and preserving number is CGMCC No.4732.

Described trichoderma viride M20 belongs to the imperfect fungi door; Hyphomycetales separated in the soil below Shanxi stalk soil is piled up in 2009, extracted trichoderma viride M20 genomic dna; With ITS universal primer (forward primer ITS1:5 '-TCCGTAGGTGAACCTGCGG-3 '; Reverse primer ITS4:5 '-CCTCCGCTTATTGATATGC-3 ') carry out pcr amplification, sequence carried out Blastn homology comparison, its with Trichoderma viride(the GenBank accession number: GU325689.1) have higher similarity (99%), so the called after trichoderma viride.

The M20 ITS sequences: 5'- 3 '.

Said trichoderma viride cultural method: dull and stereotyped activation, M20 grows on the PDA flat board, the dull and stereotyped compound method of PDA: 200g peeling potatoes; Be cut into piece, boiled 30 minutes, filtered juice is got with filtered through gauze in the back; Add 1% glucose then, 2% agar is settled to 1 liter then; Culture temperature and time: 30 ℃, 3～5 days;

When spore has just grown, with the rifle of 1000 μ L end to end nose circle get 4 ferfas and fall to being inoculated in the seed culture medium, get into seed culture;

Seed culture medium wherein: Semen Maydis powder (40 order) 3g soybean cake powder 1.5g MgSO ₄7H ₂O 0.05g K ₂HPO ₄0.2g, water 100ml, pH nature, 50 milliliters of the bottle dress substratum of every bottle of 250ml;

2 days M20 seed culture time, shaking speed 150rpm, 30 ℃ of shaking table temperature.

Liquid produces enzyme and cultivates: utilize aforementioned inorganic salt to produce the enzyme substratum, seed culture was got seed liquor 1～2ml in fermention medium after 2 days, and shaking speed is 150rpm, 30 ℃ of shaking table temperature, and initial fermention medium pH 6.0 is best.

The application of said trichoderma viride M20 fermented liquid in the degraded of urban afforestation wastes such as fallen leaves, turfgrass.

Said trichoderma viride M20 is as the application of Mierocrystalline cellulose production bacterium, and the method for utilizing M20 hydrolysis fallen leaves and turfgrass to obtain reducing sugar is specially: A, the preparation of bacterial classification seed; B, the submerged fermentation of cellulase liquid; C, fallen leaves and turfgrass pre-treatment and hydrolysis; D, the acquisition of reducing sugar.

The bacterium colony of trichoderma viride M20 of the present invention is circular on solid medium, and bacterium colony begins to be white, grows green spores through 3～5 days bacterium colonies from the center, becomes green fully through all left and right sides bacterium colony, similar with bibliographical information.This bacterial strain can be grown in Mierocrystalline celluloses such as Microcrystalline Cellulose, Xylo-Mucine, corn straw, corn cob are the substratum of sole carbon source, also well-grown on the flat board that with the xylan is sole carbon source.

The relevant strain trichoderma viride that separates stalk accumulation place pedotheque of the present invention from Shanxi; It has hydrolysis ability preferably to fallen leaves and turfgrass; Shown that it utilizes manufacture order cellular proteins and alcohol fuels such as discarded fallen leaves and greening rubbish; Solve the energy and shortage of resources that current mankind faces, and the important application of a series of thorny problems such as environmental pollution of bringing of greening castoff burning.

Description of drawings

Fig. 1 is that M20 cellulase activity in the different concns Microcrystalline Cellulose is tried hard to;

Fig. 2 did not handle leaf enzymolysis figure as a result with diluted acid;

Fig. 3 is the leaf enzymolysis handled of diluted acid figure as a result;

Fig. 4 is the turfgrass enzymolysis do not handled with diluted acid figure as a result;

Fig. 5 is the turfgrass enzymolysis handled with diluted acid figure as a result.

Embodiment:

Below in conjunction with specific embodiment the present invention is done further explain, but the present invention is not limited to specific embodiment.

Embodiment 1:

Trichoderma viride M20 belongs to the imperfect fungi door, and hyphomycetales separated in the soil below Shanxi stalk soil is piled up in 2009, and this bacterial strain is well-grown on the PDA solid medium, and 30 ℃ of culture temperature are cultivated and can be obtained ripe green spores in 3-5 days.The dull and stereotyped compound method of PDA: the 200g peeling potatoes, be cut into piece, to boil 30 minutes, filtered juice is got with filtered through gauze in the back, adds 1% glucose then, and 2% agar is settled to 1 liter then, the pH nature.

Extract trichoderma viride M20 genomic dna; With ITS universal primer (forward primer ITS1:5 '-TCCGTAGGTGAACCTGCGG-3 '; Reverse primer ITS4:5 '-CCTCCGCTTATTGATATGC-3 ') carry out pcr amplification, sequence carried out Blastn homology comparison, its with Trichoderma virideHave higher similarity (99%), so the called after trichoderma viride. Trichoderma viride, numbering M20 was preserved in Chinese common micro-organisms DSMZ on April 2nd, 2011, and preserving number is CGMCC N0.4732.

Trichoderma viride M20 can 0.1～2% Microcrystalline Cellulose, Xylo-Mucine, fallen leaves, turfgrass, corn straw etc. be the sole carbon source growth, the urban afforestation waste therefore can be used for degrading.

Embodiment 2:

Trichoderma viride M20 is not adding under any inorganic salt situation, in the substratum of 2% fallen leaves and turfgrass, grows, and promotes fallen leaves and turfgrass to decompose, and therefore can be effective to the processing of urban afforestation waste.

Embodiment 3:

Trichoderma viride M20 produces in the enzyme substratum at inorganic salt and carries out liquid submerged fermentation, and medium component is: 0.05% (NH ₄) ₂SO ₄, 0.05% L-aspartic acid, 0.1% KH ₂PO ₄, 0.05% KCl, 0.041% MgSO ₄7H ₂0,0.01% CaCl ₂, 0.05% yeast powder, 0.1% ~ 4% Mierocrystalline cellulose, wherein Mierocrystalline cellulose comprises Microcrystalline Cellulose, straw powder, corn cob meal, straw powder, bran etc.M20 induces the cellulase activity that is produced the highest with Microcrystalline Cellulose.With 0.1,0.2,0.3,0.4,0.5; 1,1.5,2,2.5,3; 3.5 4 % Microcrystalline Celluloses are the sole carbon source growth, can produce cellulase, wherein record the cellulase filter paper enzyme activity than big except that 0.5% and 1% Microcrystalline Cellulose group, other experimental group are all smaller.Lower Microcrystalline Cellulose requirement can reduce the cost of cellulase-producing.The cellulase measuring method: (specification is 1 * 6cm) accurately to take by weighing Whatman filter paper (Cat No. 1,001 110) 50mg; It is curled into a tube comparison tubes of putting into 25ml, adds in fermented liquid supernatant liquid 0.5 ml to the 25ml tube comparison tubes of the centrifugal 5min of 8000rpm, add pH again and be 4.8 citric acid-sodium citrate damping fluid 1ml filter paper is rolled into a ball submergence; 50 ℃ of water-bath 1h; Add 3ml DNS immediately and boil 5min, the 540nm place surveys absorbancy, calculates reducing sugar content.Enzyme live unit definition be PM to generate the required enzyme amount of 1.0 μ mol glucose (in reducing sugar) be 1 unit, use IUml ^-1Expression.

Table 1 is that cellulase activity compares under the different carbon sources, and Fig. 1 is the comparison of cellulase activity under the different concns Microcrystalline Cellulose, and visible Microcrystalline Cellulose is an optimum carbon source, and straw powder is reasonable natural substrate.

Table 1 M20 is the cellulase activity that produces in the substratum of sole carbon source at different Mierocrystalline celluloses

Mierocrystalline cellulose	Microcrystalline Cellulose	The straw powder	Corn cob meal	Straw powder	Wheat bran
						Relative enzyme (%) alive	100	6	12	36	11

Embodiment 4:

M20 carries out liquid submerged fermentation and carries out cellulase production, and it is following that the bacterial classification seed prepares concrete route:

1, dull and stereotyped activation, M20 grows on the PDA flat board, the dull and stereotyped compound method of PDA: the 200g peeling potatoes, be cut into piece, to boil 30 minutes, filtered juice is got with filtered through gauze in the back, adds 1% glucose then, and 2% agar is settled to 1 liter then.Culture temperature and time: 30 ℃, 3～5 days.

2, when spore has just grown, with the rifle of 1000 μ L end to end nose circle get 4 ferfas and fall to being inoculated in the seed culture medium, get into seed culture.

Seed culture medium: Semen Maydis powder (40 order) 3g soybean cake powder 1.5g MgSO ₄7H ₂O 0.05g K ₂HPO ₄0.2g, water 100ml, pH nature.50 milliliters of the bottle dress substratum of every bottle of 250ml.

3, liquid produces enzyme and cultivates:

After the seed culture 2 days, get seed liquor 1～2ml in fermention medium, enzymatic production culture medium prescription: (NH ₄) 2SO4 0.5g, peptone 0.5g, KH ₂PO ₄1g, KCl 0.5g, MgSO ₄.7H ₂0 0.5g, CaCl ₂, 0.1g, yeast powder 0.5g, Microcrystalline Cellulose 5g, water 1000 ml, pH 6.0,, shaking speed is 150rpm, 30 ℃ of shaking table temperature were cultivated 7 days.

Embodiment 5:

The fermented liquid of M20 is specially the degraded and the product reducing sugar effect of fallen leaves with turfgrass:

A, bacterial classification seed preparation: the seed that adopts embodiment 4 preparations;

B, the submerged fermentation of cellulase liquid; Cellulase source: M20 fermentative prodn.Condition: shake 30 ℃ of bottled liquid measure 400ml/1000ml shaking speed 150rpm temperature, the initial pH 6 of substratum, centrifuging and taking supernatant behind the fermentation 7d, preservation in 4 ℃ of refrigerators.

C, fallen leaves and turfgrass pre-treatment and hydrolysis; Substrate: fallen leaves, turfgrass were pulverized 40 mesh sieves, utilized 0.7% dilute sulphuric acid, handled 1h for 121 ℃, and solid-to-liquid ratio is 1:12.Get untreated fallen leaves and turfgrass and be contrast.

D, the acquisition of reducing sugar: the ratio of substrate and enzyme liquid is 1:60, whenever at a distance from sampling in 2 hours, measures concentration of reduced sugar after the hydrolysis.

The reducing sugar test method is the DNS method.The prescription of DNS is following:

First liquid: dissolving 6.9g crystalline phenol with distilled water diluting to 69 mL, adds 6.9g NaHSO _{3 again in the NaOH of 15.2 mL 10%.}

Second liquid: take by weighing the 255g Seignette salt, be added among 300 mL, 10% NaOH, add 880 mL 1% again

3,5-dinitrosalicylic acid solution.

First and second liquid phases are mixed promptly obtaining yellow DNS reagent, be stored in the brown reagent bottle.At normal temperatures, place

Use after 7～10 days, effective in half a year.

Get 1 mL hydrolyzed solution and join in the 25 mL scale test tubes, add 1.5 mL DNS reagent again, shake up, boiling water bath 5 min, then immediately with the cold water cooling, the adding distil water constant volume is to 25 mL.Get 200 μ L after shaking up and on ELIASA, measure 540 nm place absorbancys, calculate total reducing sugars concentration in the sample according to typical curve and extension rate.

4. hydrolysis temperature is 50 ℃.

Fallen leaves and turfgrass utilize result such as Fig. 2-Fig. 5 of cellulase fermentations liquid hydrolysis different time.

The ratio of substrate and enzyme liquid is the experimental group that 1:20 handles, the dry weight before and after contrast leaf and the turfgrass hydrolysis, and calculating degradation rate, degradation rate are the amount that alleviates of dry weight and the ratio of former dry weight, the result is following:

Handled degradation rate after the leaf enzyme effect in 12 hours with diluted acid: 2%;

Do not handle degradation rate after the leaf enzyme effect in 12 hours: 1.7% with diluted acid;

Handled degradation rate after the turfgrass enzyme effect in 12 hours with diluted acid: 2.6%;

Do not handle degradation rate after the leaf enzyme effect in 12 hours: 1.9% with diluted acid.

It is thus clear that fallen leaves and turfgrass are handled without diluted acid also can reach higher degradation rate, so the cellulase in the M20 fermented liquid can be used for the degraded of urban afforestation waste.

Technology reducing sugar output disclosed by the invention can reach 4.35 g/L, after the hydrolyzed solution appropriateness concentrates, can be used for culturing yeast manufacture order cellular proteins as feed, and resulting reducing sugar also can be used as the raw material of alcohol fuel fermentation.In addition, the urban afforestation waste residue after the processing can be used as fertilizer, improves added value.

Claims

1. A Trichoderma viride strain with high cellulase production, the strain is Trichoderma viride in the genus Trichoderma, numbered M20, preserved in the China General Microorganism Culture Collection Center on April 2, 2011, and the preservation number is CGMCC No .4732.

2. the Trichoderma viride strain of a strain high-yield cellulase according to claim 1, is characterized in that: described Trichoderma viride M20 belongs to Deuteromycota, Hyphospora, below the accumulation of straw soil in Shanxi in 2009 Trichoderma viride M20 genomic DNA was isolated from soil, and PCR amplification was performed with ITS universal primers (forward primer ITS1: 5'-TCCGTAGGTGAACCTGCGG-3', reverse primer ITS4: 5'-CCTCCGCTTATTGATATGC-3'), and the sequence Perform Blastn homologous comparison, which has a high similarity with Trichoderma viride.

3.根据权利要求1所述的一株高产纤维素酶的绿色木霉菌株，其特征是：所述M20的ITS序列：5’-AGGAATACGAGCATTTCAGCTCCCAAACCCAATGTGAACCATACCAAACTGTTGCCTCGGCGGGGTCACGCCCCGGGTGCGTCGCAGCCCCGGAACCAGGCGCCCGCCGGAGGGACCAACCAAACTCTTTTCTGTAGTCCCCTCGCGGACGTTATTTCTTACAGCTCTGAGCAAAAATTCAAAATGAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGCCAGTATTCTGGCGGGCATGCCTGTCCGAGCGTCATTTCAACCCTCGAACCCCTCCGGGGGGTCGGCGTTGGGGACCTCGGGAGCCCCTAAGACGGGATCCCGGCCCCGAAATACAGTGGCGGTCTCGCCGCAGCCTCTCCTGCGCAGTAGTTTGCACAACTCGCACCGGGAGCGCGGCGCGTCCACGTCCGTAAAACACCCAACTTCTGAAATGTTGACCTCGGATCAGGTAGGAATACCCGCTGAACTTAAGCATATCAAAAGCGGGAGGAA-3’。

4. The cultivation method of the Trichoderma viride strain of a high-yield cellulase described in any one of claims 1-3 is characterized in that: plate activation, M20 grows on the PDA plate, PDA plate preparation method: 200g potato peeled , cut into pieces, boiled for 30 minutes, then filtered with gauze to get the filtered juice, then added 1% glucose, 2% agar, and then set the volume to 1 liter, and the culture temperature and time were 30°C, 3-5 days;

When the green spores have just grown, use a 1000 μL gun with a round end to take 4 colonies and inoculate them into the seed medium and enter the seed culture;

Among them, seed medium: 3g corn flour (40 mesh), 1.5g bean cake powder, 0.05g MgSO ₄ 7H ₂ O, 0.2g K ₂ HPO ₄ , 100ml water, natural pH, 50ml culture medium in each 250ml bottle ;

The M20 seed culture time is 2 days, the shaker speed is 150rpm, and the shaker temperature is 30°C;

Liquid enzyme production culture: After the seed culture is over, take 1-2ml of seed liquid into the enzyme production medium, the formula of the enzyme production medium is: (NH ₄ )2SO4 0.5g, peptone 0.5g, KH ₂ PO ₄ 1g, KCl 0.5 g, MgSO ₄ .7H ₂ 0 0.5g, CaCl ₂ , 0.1g, yeast powder 0.5g, microcrystalline cellulose 5g, water 1000 ml, pH 6.0, shaker speed 150 rpm, shaker temperature 30°C, fermentation culture 7 days.

5. According to any one of claims 1-3, the application of a Trichoderma viride strain fermented liquid with high cellulase production in the degradation of urban greening wastes such as fallen leaves and lawn grasses.

6. According to the application of a Trichoderma viride strain with high cellulase yield described in any one of claims 1-3 as the application of cellulose producing bacteria, the method of utilizing M20 to hydrolyze fallen leaves and lawn grass to obtain reducing sugars is specifically: A, bacterial classification Seed preparation; B, liquid submerged fermentation of cellulase; C, pretreatment and hydrolysis of fallen leaves and turfgrass; D, obtaining reducing sugar.