CN103230917B - Forest floor combustible biodegradation agent and use thereof - Google Patents
Forest floor combustible biodegradation agent and use thereof Download PDFInfo
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- CN103230917B CN103230917B CN201310080358.5A CN201310080358A CN103230917B CN 103230917 B CN103230917 B CN 103230917B CN 201310080358 A CN201310080358 A CN 201310080358A CN 103230917 B CN103230917 B CN 103230917B
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Abstract
The invention discloses a forest floor combustible biodegradation agent and a use thereof, and belongs to the field of forest fire prevention. The forest floor combustible biodegradation agent is characterized in that each 10L of a spore suspension is added with synergists comprising 4.5 to 5.0 grams of Tween-80, 0.18 to 0.22 grams of manganese peroxidase, 0.77 to 0.83 grams of lignin peroxidase, 2.8 to 3.1 micrograms of KCl, 9.8 to 10.2 micrograms of NH4NO3, 4.8 to 5.2 micrograms of MgSO4, 9.8 to 10.2 micrograms of Na2SO4, 5.9 to 6.3 milligrams of phosphate, 4.9 to 5.3 milligrams of cysteine, 7.6 to 8.3 milligrams of glutathione, 2.4 to 3.0 milligrams of vitamin B1, 1.7 to 2.2 milligrams of vitamin B2 and 2.9 to 3.3 milligrams of vitamin B6. The forest floor combustible biodegradation agent can decompose floor combustible by a microbe regulation and control method, and can accelerate forest floor combustible decomposition thereby reducing forest fire insurance. The method solves the defect of single bacterium degradation, adopts a high-efficiency degradation bacterium composition-active enzyme combined formula, and accelerates forest floor combustible degradation efficiency.
Description
Technical field
The invention belongs to field of forest fire prevention, relating to a kind of degradation agent for clearing up forest Forest fuel and application thereof.
Background technology
The method of existing cleaning forest Forest fuel mainly contains controlled prescribed burning, eliminates (utilizing chemical reagent to remove combustible or logging residue in woods), labor cleaning etc.Although controlled prescribed burning is fast, easily causes runout, thus lose more than gain; Although and eliminate instant effect, the chemical agent sprayed easily pollutes forest; Artificial clear woods then waste of manpower, material resources, financial resources.
Summary of the invention
For the deficiency that the method for existing cleaning forest Forest fuel exists, the invention provides a kind of forest Forest fuel biodegradation agent and application thereof, the method utilizing microorganism to regulate and control decomposes Forest fuel, the decomposition rate of forest Forest fuel can be accelerated, thus reduce risk of forest fire method.
Forest Forest fuel degradation agent of the present invention comprises degraded bacterial classification spore suspension and synergist, wherein adds following synergist in every l0L spore suspension: Tween 80: 4.5-5.09, manganese peroxidase: 0.18-0.22g, lignin peroxidase: 0.77-0.839, KCl:2.8-3.lug, NH
4nO
,: 9.8-10.2Hg, MgSO4:4.8-5.2Hg, Na
2sO
4: 9.8-10.2 μ g, phosphoric acid are cruel: 5.9-6.3lng, cysteine: 4.9-5.3mg, the sweet skin of paddy Guang: 7.6-8.3mg, Cobastab
1: 2.4-3.0lng, Cobastab
2: 1.7-2.2mg, Cobastab
6: 2.9-3.3mg.
In above-mentioned formula, described degraded bacterial classification spore suspension is the mixture than coming mould Penicillin bilaiae suspension, Trichoderma atroviride Trichodgrma atroviride suspension and Trichoderma harzianum Trichoderma AarTianum suspension and mixing in proportion by same concentration, than come mould penicillin bilaiae suspension, Trichoderma atroviride Trichoderma atroviride suspension, Trichoderma harzianum Trichoderma harzianum suspension spore concentration be 10
8/ mL.
The present invention utilizes efficient degradation bacterial classification to combine and appropriate multienzyme active product, adds appropriate water and fully mixes the degradation agent be prepared into thoroughly.To be degraded forest Forest fuel, certain effect can be reached, overcome the shortcoming of single culture degraded.What adopt the combination of efficient degradation bacterial classification and organized enzyme to combine joins ten thousand, accelerates the degradation efficiency of forest Forest fuel.For China's forest fire protection provides new method in Control Technology field, enrich the theory and technology of forest fire protection research.
Compared with prior art, the invention has the beneficial effects as follows:
1, compared with controlled prescribed burning, although it is comparatively slow to take effect, expense is relatively high, and it is safer, more environmental protection.
2, compared with eliminating, although the method takes effect relatively slow, it is environmental protection more, more economical.
3, with artificial clear forest form ratio, the method is environmental protection more, more saves human and material resources, financial resources, can also provide nutrient for forest.
Accompanying drawing explanation
Fig. 1 is the changes of four fungal strains in degradation process MnP enzymatic activity;
Fig. 2 is the changes of four fungal strains in degradation process FPA enzymatic activity;
Fig. 3 is the changes of four fungal strains in degradation process CMCase activity.
Detailed description of the invention
Elaborated technical scheme of the present invention for Daxinganling District woodland below, but do not limited protection scope of the present invention.
(1) bacterium source
In the fallen leaves that sample collection rots from Daxing'an Mountainrange sylvan life, rotten timber.
(2) filter paper hole method enrichment primary dcreening operation stalk cellulose degradation bacteria
1. culture medium
(1) He Qixunshi minimal medium: KH
2pO
41.0g, NaCl0.1g, MgSO
47H
2o0.3g, NaNO
32.5g, FeCl
30.01g, CaCl0.1g, water 1000mL, about pH7.2.
(2) filter paper culture medium: add agar powder 18g/L in He Qixunshi minimal medium, good dull and stereotyped rear paving a slice triangle filter paper is on culture medium.
(3) seed culture medium: adopt potato-agar medium (PDA).
2. method
Soak the fallen leaves, the rotten wood sample that rot that gather with sterilized water, at shaking table vibration 30min, sample thief suspension, is diluted to 10 with sterilized water
-1, 10
-2, 10
-3concentration gradient, draw diluted concentration be 10
-3dilution, be coated on filter paper culture medium, be placed in 28 DEG C of incubators and cultivate 24-48h, pick out well-grown on filter paper culture medium, and have the fungus colony that degraded hole occurs, and on PDA culture medium (potato dextrose agar), carry out separation and purification further, after the bacterial strain inclined plane inoculating after separation and purification is cultivated 2-4d on seed culture medium, put 4 ° of C refrigerator cold-storages, stand-by.
This experiment is by a series of means such as enrichment, separation, purifying, from Daxinganling forest area Litter-fall, separation and purification goes out 32 strain bacterium, by the judgement to its growing way and color, rejects 13 strain growing ways bad, color is not bright-coloured, and remaining 19 strains are as the aimed strain of primary dcreening operation.
Carried out shaking training by the conical flask fluid nutrient medium of 19 strain bacterium access PDA of remainder, it is as shown in table 1 that 5d observes its feature.
Table 1 bacterial strain shakes training 5d feature
Note: in table, the label of 19 strain bacterial strains is self-defined bacterial strain label
(3) filter paper degradation experiment
1. culture medium
(1) liquid fermentation medium: KH
2p0
41.0g, NaCl 0.1g, MgSO
47H
2o0.3g, NaNO
32.5g, FeCl
30.01g, CaCl 0.1g, filter paper bar 1.0g, pH are 7.2-7.4.
2. method
The bacterial strain obtained will be separated, be placed in liquid seed culture medium 28 DEG C of shaking tables cultivation 5d and prepare bacterium liquid, the filter paper bar of 1x6cm is added in the He Qixunshi minimal medium of 40mL, and access lmL bacterium liquid and carry out constant-temperature shaking culture 10d, judge effect according to filter paper bar breaking degree: (1) is destroyed serious: degraded wild Oryza species is half clear shape; (2) destruction is more serious: the agglomerating pasty state of degraded wild Oryza species; (3) destroy medium: filter paper indefinite form; (4) destroy slight or unchanged: degraded wild Oryza species filter paper neatly expands and to bend or without significant change.Choose the bacterial strain large to filter paper destructive power, carry out next step research.The results are shown in Table 2.
The filter paper degradation effect of table 2 strain culturing 10d
(4) Forest fuel sample degradation experiment
1. sample source and process
(1) originate: pick up from Daxing'an Mountainrange Tahe Forestry Bureau show peak forest field
(2) process: the Forest fuel of collection is cut into 2-3cm segment, is crushed to 60 orders after oven dry, sodium hydroxide solution to the solid-liquid weight ratio adding 2% is 1: 4, in 85 DEG C of water-baths, process 1h, be washed to pH for neutral, dry for standby.
2. culture medium
(1) liquid fermentation medium: KH
2pO
41.0g, NaCl 0.1g, MgSO
47H
2o0.3g, NaNO
32.5g, FeCl
30.01g, CaCl 0.1g, Forest fuel sample 20g, pH are 7.2-7.4.
3. method
(1) mensuration of Forest fuel resolution ratio
The method of Wang Yuwan etc. is adopted to carry out the mensuration of holocellulose content (cellulose, hemicellulose total content), lignin content.The results are shown in Table 3-table 4.
Table 3 strains for degrading 10d lignin result
Table 4 holocellulose Degrading experiment result
(5) enzyme assay
The preparation of crude enzyme liquid: the fresh mycelia that oese picking is cultivated on PDA is in liquid seed culture medium, 28 DEG C, 170r/min cultivates 60h, spore growth situation is examined under a microscope with blood counting chamber, inoculum concentration by 1% is in 300mL culture medium, 28 DEG C, 170r/min liquid fermentation and culture, every 24h sampling and measuring enzyme activity, cultured liquid fermentation culture medium is first used two-layer filtered through gauze, filtrate is again in 4 DEG C, and the centrifugal 10min of 10000xg, gets the crude enzyme liquid that supernatant is preparation.
The mensuration of carboxymethylcelluloenzyme enzyme CMCase (EC 3.2.1.4) and Filter paperlyase FPA (EC 3.2.1.91) enzymatic activity: get the enzyme liquid 0.5mL after suitably dilution, join the tool plug scale test tube without starch Xinhua filter paper and 1.5mL 0.05mol/L pH5.0 citrate buffer solution containing 50mg process, 50.Be incubated 1h in C water-bath, press DNS method immediately and measure content of reducing sugar.
Note: (DNS preparation of reagents: by 3 of 7.5g, the NaOH of 5 one dinitrosalicylic acids and 14g is dissolved in and is dissolved with in 216g sodium potassium tartrate tetrahydrate hot solution, add the phenol that 5g dissolves in advance in 50 ° of C water-baths again, and 5g sodium sulfite, 1000mL is settled to after abundant dissolving, be placed in brown bottle, place and use after 3-5 days).
The mensuration that manganese peroxidase MnP (EC 1.1 1.1.13) enzyme is lived adopts phenol red method.
Measurement result:
(1) lignoenzyme change
As shown in Figure 1, in whole sweat, the MnP enzymatic activity of four fungal strains secretions all reached maximum at the 12nd day, and bacterial strain JZMll2, bacterial strain THl2 331, bacterial strain HZllFX441, bacterial strain HZl3FX532 enzymatic activity are respectively 0.065U/mL, 0.062 U/mL, 0.027 U/mL and O.168 U/mL.In whole sweat, bacterial strain JZMll2, bacterial strain THl2 331 and bacterial strain HZllFX441 enzymatic activity much smaller compared with bacterial strain HZl3FX532.All there are two minor peaks in the MnP enzyme of four strain bacterium secretions, wherein peak value is all more obvious for the first time.First time, peak value appeared at the 12nd day, and second time peak value appears at the 24th day.
(2) cellulase change
As shown in Figures 2 and 3, in whole sweat, FPA with the CMCase Enzyme activities trend of four fungal strain secretions is not identical.Wherein, the PFA enzymatic activity of bacterial strain JZMll2, bacterial strain THl2331, bacterial strain HZl3FX532 all reached maximum at the 12nd day, enzymatic activity is respectively 0.657U/mL, 0.540 U/mL and 0.374 U/mL, the PFA enzymatic activity of bacterial strain HZllFX441 reached maximum at the 24th day, and enzymatic activity is 0.027 U/mL; The CMCase enzymatic activity of bacterial strain JZMl12 and bacterial strain HZllFX441 reached maximum at the 8th day, and the CMCase enzymatic activity of bacterial strain HZl3FX532 and bacterial strain THl2 331 reached maximum at the 12nd day.
In whole sweat, FPA, CMCase enzymatic activity of bacterial strain HZl3FX532 and bacterial strain HZllFX441 is much smaller compared with bacterial strain JZMll2 and bacterial strain THl2 331.At Forest fuel sample degradation in earlier stage, fungi mainly consumes materials such as utilizing the cellulose of the non-wood existed in sample and the flammable carbohydrate that can directly utilize, so cellulosic height determines the decomposition rate in early stage, this may be bacterial strain JZMll2 and bacterial strain THl2 331 major reason that early stage, on average decomposition rate was higher.FPA and the CMCase enzymatic activity of bacterial strain JZMl12 reaches peak value the 8th day and the 12nd day respectively, and FPA and the CMCase enzymatic activity of bacterial strain THl2 331 is all reached peak value at the 12nd day.
The enzymatic activity of the FPA that four fungal strains produce is all low compared with the enzymatic activity of CMCase.FPA and the CMCase enzymatic activity maximum enzyme activity value of bacterial strain JZMl12 is respectively 0.657 U/mL and 0.757 U/mL, FPA and the CMCase enzymatic activity maximum enzyme activity value of bacterial strain THl2 331 is respectively 0.540 U/mL and 0.830 U/mL, and the CMCase enzymatic activity of two strain bacterium is all obviously greater than the enzymatic activity of FPA.
(6) strain idenfication
Observe bacterium colony and bacterial classification morphological feature, and carry out strain idenfication with reference to " Fungal identification handbook " and Chinese fungi will (the 35 volume).
(1) JZM112: Trichoderma atroviride Trichoderma atroviride
Feature: colony growth is very fast, diameter 7-9cm, conidium is subsphaeroidal, and to short ellipse, full ripe conidium becomes dark green, and full ripe conidium wall is level and smooth, usually 2.6-3.8x2.2-3.4 μm.
(2) THl2 331: Trichoderma harzianum Trichoderma harzianum
Feature: colony growth is very fast, diameter 5-8cm, conidium is subsphaeroidal to short ellipse, and full ripe conidium is light green, 2.5-3.5x2.1-3.O μm.
(3) HZl3FX532: than carrying out mould Penicillin bilaiae
Feature: conidiophore results from aerial hyphae on PDA, falx stem have every, top is not expanded, 31.7-49.2x1.6-2.4 μm, and wall is level and smooth; The remarkable single-wheel of penicillus is raw; Bottle stalk often takes turns 3-8,6.3-8.7x2.4-2.6 μm, ampuliform, and stalk neck is short; Conidium is spherical, subsphaeroidal to oval, 2.4-2.8x2.0-2.4 μm, and wall is smooth to slightly coarse; Conidia chain presents irregular cylindric.
(7) degradation agent is produced
1, the preparation of conidia powder and spore quantitative determination thereof
(1) preparation of potato agar culture medium (PDA): potato (peeling) 200g, glucose 18-20g, agar 18-20g, water 1000mL, peeling potatoes is cut into small pieces, about the 1000mL that adds water in pot boils half an hour, and by filtered through gauze, get its filtrate to add after glucose and agar dissolve completely and add water to 1000mL, pH value nature, be sub-packed in after conical flask in 121 DEG C of sterilizings 30 minutes, pour in flat board at ultra-clean operating desk, wait to be cooled.
(2) wheat bran medium preparing: get commercially available, without go mouldy wheat bran sieve remove fine powder (50 mesh sieve), take 8g wheat bran, wood chip 4g, add in 250mL triangular flask, add about 8mL and mix thoroughly, in 121 DEG C of sterilizings 30 minutes.
(3) on ultra-clean operating desk, four strain bacterium are inoculated on wheat bran culture medium, are placed in incubator, 32 DEG C of cultivations, cultivate 8-10d, in incubation, require that every day is stirred once, until stop when having spore to produce stirring.Namely wheat bran spore is obtained after cultivation completes.
The collection of (4) four strain bacterium conidia powder and drying
Under wheat bran culture medium the collection of the conidia powder of four strain bacterium by wheat bran solid matrix after vacuum freezedrying process, make its moisture content be down to less than 8%, utilize vibratory sieve, conidia powder is separated with wheat bran and wood chip matrix, obtain conidia powder, be stored in the refrigerator of 4 ° of C for subsequent use.
(5) spore count measures:
Prepared by A, spore suspension: get conidia powder 1g, add 5mL sterilized water, this spore suspension is placed in sterilized 50mL triangular flask, several sterile glass balls are placed in advance in bottle, filter with the absorbent cotton of sterilizing after shake well, and with aseptic water washing filter residue 2-3 time, finally make filtrate volume reach 10mL.
B, spore count: spore suspension sterilized water is diluted to 10
-2, 10
-3, draw appropriate dilution in the counting chamber of blood counting chamber, then add cover glass, leave standstill about 5 minutes, first under low power lens, find counting chamber, then convert high power lens to and observe and count.With lattice tally in 16 during counting, by diagonal orientation, get the spore count of 4 middle lattice (i.e. 100 little lattice) of upper left, lower-left, upper right, bottom right.
C, 1g conidia powder miospore amount
After measured, the conidia powder of the three strain object bacteria (bacterial strain JZMll2, bacterial strain THl2331 and bacterial strain HZl3FX532) that this experiment obtains, spore amount is respectively: 1.2 × 1011/g, 4.2x1010/g, 6x1010/g.
(6) preparation of degradation agent
The conidia powder of refrigeration is mixed with quantitative spore suspension, is diluted to 10 by unified for suspension spore concentration
8/ mL, three kinds of bacterium liquid are mixed and made into mix bacterium agent in proportion by same concentration, add following synergist by a certain percentage: Tween 80 5g in every 10L spore suspension; Manganese peroxidase 0.2g; Lignin peroxidase 0.8g; KCl 3 μ g; NH
4n0
310 μ g; MgS0
45 μ g; Na2S0
410 μ g; Phosphate 6mg; Cysteine (oxidized form) 5mg; Glutathione 8mg; Cobastab
13mg; Cobastab
22mg; Cobastab
63mg.
Above-mentioned degraded bacterial classification is present in the forest Forest fuel of Daxinganling District.Utilize above-mentioned formula, to the effect in the main sylvan life Forest fuel degraded in three kinds, Daxinganling District 30t as table 5.
Table 5 microbial inoculum is evaluated three kinds of combustible crop type ground cover field degradation efficiencies
The principle of applied microbiology of the present invention and forest fire science, techniques and methods, by series of studies such as the separation to Daxinganling District combustible degradation bacteria, purifying, strain excellent screening, degradation bacterial agent preparation, indoor and outdoor Degrading experiments, establish and be suitable for Daxinganling forest area microorganism fireproof techniques and methods, solve current Daxinganling District forest Forest fuel to degrade a slow difficult problem, for China's forest fire protection provides new method in Control Technology field, enrich the theory and technology of forest fire protection research.
Claims (5)
1. a forest Forest fuel biodegradation agent, it is characterized in that described degradation agent comprises degraded bacterial classification spore suspension and synergist, wherein add following synergist in every 10L spore suspension: Tween 80: 4.5-5.0g, manganese peroxidase: 0.18-0.22g, lignin peroxidase: 0.77-0.83g, KCl:2.8-3.1 μ g, NH
4nO
3: 9.8-10.2 μ g, MgSO
4: 4.8-5.2 μ g, Na
2sO
4: 9.8-10.2 μ g, phosphate: 5.9-6.3mg, cysteine: 4.9-5.3mg, glutathione: 7.6-8.3mg, Cobastab
1: 2.4-3.0mg, Cobastab
2: 1.7-2.2mg, Cobastab
6: 2.9-3.3mg; Described degraded bacterial classification spore suspension is the mixture than coming mould Penicillin bilaiae suspension, Trichoderma atroviride Trichoderma atroviride suspension and Trichoderma harzianum Trichoderma harzianum suspension, is 10 than the spore concentration coming mould Penicillin bilaiae suspension, Trichoderma atroviride Trichoderma atroviride suspension and Trichoderma harzianum Trichoderma harzianum suspension
8/ mL.
2. forest Forest fuel biodegradation agent according to claim 1, is characterized in that adding following synergist in every 10L spore suspension: Tween 80: 5g, manganese peroxidase: 0.2g, lignin peroxidase: 0.8g, KCl:3 μ g, NH
4nO
3: 10 μ g, MgSO
4: 5 μ g, Na
2sO
4: 10 μ g, phosphate: 6mg, cysteine: 5mg, glutathione: 8mg, Cobastab
1: 3mg, Cobastab
2: 2mg, Cobastab
6: 3mg.
3. forest Forest fuel biodegradation agent according to claim 1, is characterized in that in described mixture, each bacterium is by mixing in proportion.
4. forest Forest fuel biodegradation agent according to claim 1 and 2, is characterized in that described cysteine is oxidized form.
5. forest Forest fuel biodegradation agent described in a claim 1 is for clearing up the application of forest Forest fuel.
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