CN103013887A - Bacillus pumilus KMXU56 and inoculant thereof - Google Patents
Bacillus pumilus KMXU56 and inoculant thereof Download PDFInfo
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- CN103013887A CN103013887A CN2012105759048A CN201210575904A CN103013887A CN 103013887 A CN103013887 A CN 103013887A CN 2012105759048 A CN2012105759048 A CN 2012105759048A CN 201210575904 A CN201210575904 A CN 201210575904A CN 103013887 A CN103013887 A CN 103013887A
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse
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Abstract
The invention relates to a bacillus pumilus KMXU56 and an inoculant thereof, belonging to the technical field of environmental microorganism. The preservation number of the baacillus pumilus KMXU56 is CGMCC No.6595. The inoculant containing the bacillus pumilus KMXU56 is prepared by comprising the steps of activating strains, preparing fermentation seed solution and preparing the inoculants. Strains separated and selected from water hyacinth residues under natural fermentation conditions have good capability of decomposing cellulose, and can be survived under the conditions of high and intermediate temperature range (40-70DEG C); the inoculant prepared by fermental cultivation and micropropogation is added into water hyacinth residue mixture, and the water hyacinth residue mixture is converted into bio-organic fertilizer by conducting composting fermentation under natural conditions. The water hyacinth residue bio-organic fertilizer has good fertilizer efficiency and water retention capacity, is green and environment-friendly, and pollution-free, and has good economic benefits and social benefits.
Description
Technical field:
The present invention relates to a kind of bacillus pumilus KMXU56 and Inoculant thereof, belong to the environmental microorganism technical field.
Background technology:
Herba Eichhorniae has another name called Herba Eichhorniae, and belong to the Pontederiaceae Herba Eichhorniae and belong to, be a kind of perennial floating plant that originates in South America.Herba Eichhorniae natural disposition happiness light is adapted to the torrid zone, subtropical climate (27-30 ℃ is optimum growth temp), fast growth, and under the condition of being fit to, biomass namely doubled in per five days; The unit biomass is large, and the per hectare water surface reaches 2,000,000 strains, closes the 300-400 ton; Endurance is extremely strong, no matter be in poor nutrition, or at Hyper-eutrophic, reservoir, even in peracidity and high alkalinity water body, and Herba Eichhorniae can both survive (Sheng Jing etc., 2010).In recent years, along with the aggravation of the many water body eutrophication degrees of China, the distribution area of Herba Eichhorniae has the trend of continuous increase, and it covers common report in rivers, lake, reservoir big area, and therefore, Herba Eichhorniae is abundant in china natural resources.Because the Herba Eichhorniae big area covers, easily cause the detrimentally affects such as Dissolved Oxygen in Water reduction, species diversity minimizing, waterborne traffic obstruction, a lot of areas, annual south all need actively to take measures it is salvaged prevents and kill off, often directly abandon after but Herba Eichhorniae is salvaged, cause serious secondary pollution and the wasting of resources (Liu Hongjiang etc. 2011).
Studies have shown that, Herba Eichhorniae has higher utility value.Gajalakshmi and Abbasi (2002) result of study shows, the green manure that utilizes the Herba Eichhorniae hybrid composting to produce produces obvious facilitation effect to the growth of plant, and is safe and feasible.Murugesan etc. show take Herba Eichhorniae, straw as raw material culturing edible fungus, result, and the income that Herba Eichhorniae is processed is significantly higher than straw and processes.Trans-utilization by Herba Eichhorniae reduces Herba Eichhorniae quantity, and making it turns waste into wealth, and is the good approach of the water body harm of control Herba Eichhorniae and secondary pollution.Existing studies show that, Herba Eichhorniae are one of the highest waterplant of potassium content (Reddy et al., 1991; Zhou Wenbing etc., 2004), complete stool butt potassium content can reach more than 5%, and Herba Eichhorniae also has stronger nitrogen receptivity, and nitrogen content reaches 3%.This shows, the Herba Eichhorniae plant is fabulous nutrients resource.And in the numerous Disposal Measures of Herba Eichhorniae, use in the Herba Eichhorniae farmland is the most economical and effective, environmental friendliness, a kind of processing mode simple to operate.Because to the asynchronism that Herba Eichhorniae is salvaged and plant growth may exist in time, Herba Eichhorniae is often become organic fertilizer application by simple and easy composting.The as a supplement good measure of Potassium in Soils, the Herba Eichhorniae fertilizer is applied in the south China development potentiality in the farmland huge.Along with the expansion of Dianchi Lake cucurbit cultivated area, the generation of Herba Eichhorniae slag also sharply increases.The processing of Herba Eichhorniae slag has become restriction with disposal and has utilized Herba Eichhorniae to remove one of restricted condition of the eutrophication in the Dian Chi.The Herba Eichhorniae slag muck fertile with other treatment processs, such as landfill, burn and compare, have a construction investment few, working cost is low, the advantages such as basic non-secondary pollution.And the Herba Eichhorniae slag is energy soil utilization after compost treatment, can take full advantage of its organic and nutritive elements such as nitrogen, phosphorus, is the disposal options that is fit to China's national situation.But the development of compost batch production is subject to the puzzlement of the Cost Problems that brings because of compost time and place always, and the rapid of compost has important practical significance.
The fertile inoculation of Herba Eichhorniae slag muck has become one of important means that improves compost quality and fermentation efficiency.At present, existing multiple-microorganism is applied to the compost inoculation.But the most water of decomposition cucurbit of these bacterial strains organic matter is indifferent, neither be isolated from the Herba Eichhorniae slag, and organic decomposition efficiency is descended, and affects compost maturity.Therefore, in the urgent need to seek a class can middle temperature, again can the condition at high temperature under, the organic temperature broad spectrum of metabolism microorganism, and, this quasi-microorganism is inoculated in the compost fast decoupled Herba Eichhorniae slag fiber in compost, realize the quick composting of Herba Eichhorniae slag, reduce further environmental pollution, reduce cost, shorten and produce fermentation time.
The present invention relates to a kind of bacillus pumilus KMXU56 and Inoculant thereof of growth temperature wide spectrum, in the fertile processing of Herba Eichhorniae slag muck, accelerate digest process and cellulose decomposition.By literature search, have no the open report identical with the present invention.
Reference:
[1]?Reddy?K.R.,?Agami?M.,?D'Angelo?E.M.,?Tucker?J.C，Influence?of?K?supply?on?growth?and?nutrient?storage?of?water?hyacinth.?Bioresource?Technol.?,?1991,?37?(1)：79–84
[2] Liu Hongjiang keeps root, and Zhu Puping contains Jing, Zhang Yuefang, and Zheng Jianchu, farmland application of water cucurbit uses and the environmental organism journal 2011,17 (4): 521-526 to the impact of rice dry matter production and distribution
[3] contain Jing, keep root, Zhu Puping, Zhou Wei, Xue Xinhong, the farmland utilization research of high eutrophication vegetation water cucurbit, Chinese Ecological Agriculture journal, 2010,18 (1): 46-49
[4] thank to the Jianchang, Zhou Jianmin, Harder R. potassium and Chinese agriculture. Nanjing: press of Hohai University, 2000:105-106
[5] Zhou Wenbing, Liu Dahui, Zhu Duanwei, different amendments be on the impact of pig manure process and nutrient situation thereof, Hua Zhong Agriculture University's journal, 2004,23 (4): 421 – 425
Summary of the invention:
The object of the present invention is to provide a kind of bacillus pumilus (Bacillus pumilus) KMXU56 and Inoculant thereof.
The object of the present invention is achieved like this:
Bacterial strain KMXU56 of the present invention is that the strain that separation screening obtains from pick up from the Herba Eichhorniae compost sample that becomes thoroughly decomposed in the refreshing auspicious agriculture company limited in Kunming compost fermentation workshop has preferably decomposition of cellulose ability, the strain of i (bacillus) pumilus that can under middle high temperature range (40 ℃ ~ 70 ℃) condition, survive, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on September 21st, 2012 and (is called for short CGMCC, address: No. 3, No. 1 institute in BeiChen West Road, Chaoyang District, BeiJing City, Institute of Microorganism, Academia Sinica, postcode 100101), Classification And Nomenclature is bacillus pumilus (Bacillus pumilus), preserving number CGMCC No.6595.
Bacterial strain KMXU56 of the present invention has following characteristic: KMXU56 bacterial strain thalline is shaft-like, and circle is terminal, and is single or be short chain and arrange 1.2～1.5 * 2.0～4.0 microns.Can move, Gram-positive, 1.0～1.2 * 1.5～2.0 microns of gemma, ellipse, middle life or inferior end are given birth to.This bacterium energy hydrolyzed starch, degraded mannosans, xylan, Mierocrystalline cellulose.
Contain the Inoculant of bacillus pumilus KMXU56, obtain through following steps:
(1), actication of culture: the bacterial classification inoculation of getting bacillus pumilus KMXU56 is in the LB liquid nutrient medium: Tryptones 10g/L, yeast extract 5g/L, NaCl 10g/L among the pH7.0, cultivates 3-5d under 30-35 ℃ of condition;
(2), fermentation seed liquid preparation: the bacterial classification inoculation of activation is carried out the one-level enlarged culturing in the LB liquid nutrient medium, as nutrient solution OD
600Stopping to cultivate greater than 1.5 o'clock, get first order seed; First order seed 8% is seeded to and carries out the secondary enlarged culturing in the LB liquid nutrient medium by volume, as nutrient solution OD
600Stopping to cultivate greater than 2.6 o'clock, get fermentation seed liquid; One-level, secondary culture condition are 30-35 ℃, and rotating speed is 120-180r/min, and incubation time is 5-7d;
(3), the preparation of Inoculant: in water content is the rice husk heap of 40%-50%, add urea and sucrose, behind the 24h, wheat bran, Semen Maydis powder and the fermentation seed liquid fermentation of banking up of in rice husk heap, evenly splashing again, during this time turning, to temperature-stable when the envrionment temperature, stop fermentation, tunning is dry, make Inoculant; Wherein:
A. the addition means of urea and sucrose is: take by weighing urea and sucrose by rice husk dry weight mass ratio 0.5% and 2% respectively, with urea and sucrose and water by weight the 1:20 wiring solution-forming, in the rice husk of evenly the splashing heap;
B. the addition means of wheat bran, Semen Maydis powder and fermentation seed liquid is: the fermentation seed liquid that will obtain by the step (2) of the wheat bran of rice husk heap dry weight 1%, 2% Semen Maydis powder and 2% mixes, splash after puddling evenly in the rice husk heap, rice husk waste height 0.6-1.8m, floor space＜50m
2, cover the fermentation of banking up of ventilation property coverture;
C. when leavening temperature reach 60-65 ℃ and continue 36-48h after, carry out the 1st turning, later on by again turning 4-8 time of the condition of the 1st turning, until temperature-stable after envrionment temperature, at the 45-55 ℃ of lower 4h of drying, gets solid-state Inoculant with tunning.
The invention has the advantages that:
1, the bacterial classification bacillus pumilus KMXU56 of seed selection of the present invention can be suitable for Herba Eichhorniae slag solid state fermentation, and the Inoculant adding that this bacterial strain is made is stirred in the Herba Eichhorniae slag, can carry out biological fermentation to it, changes biological organic fertilizer into.
2, strain of i (bacillus) pumilus KMXU56 successful, harmless, pollute without environment and to have important economic worth and application prospect, be fit to large-scale application in the Herba Eichhorniae slag muck fertilizer of water field of big area plantation.
Embodiment
The used equipment of embodiment is the general-purpose equipment of market sale.
Embodiment 1: the isolation and purification of bacillus pumilus KMXU56
Take by weighing sample fertilizer 0.5 g that the Herba Eichhorniae slag muck fertilizer in the refreshing auspicious agriculture company limited in Kunming compost fermentation workshop becomes thoroughly decomposed, add 20 mL sterilized waters, mixing, sample is suspended, get suspension 1 mL and be inoculated in the 250 mL shaking flasks that contain 100 mL liquid seed culture mediums, 45 ° of C, 180 r/min cultivate 5 d.Get 1 mL nutrient solution gradient dilution to 10
2, 10
4, 10
6, 10
8Coat 47 ℃ of cultivation 2 d on the solid medium.Full, the obvious single colony inoculation of transparent circle of bacterium colony is in liquid nutrient medium on the picking flat board, and 47 ° of C, 180 r/min cultivate 2 d again.Repeating liquid culture separates with solid plate 3 times.), the eugonic single bacterium colony of picking, on bacteria culture medium, the separation and purification 3 times of repeatedly ruling, the again bacterium colony film-making that form is consistent, simply dye, at oily Microscopic observation thalli morphology, observe the somatic cells form under many visuals field, after confirming that thalli morphology is consistent, be seeded to the bacterium slant culture to abundant, 4 ℃ of lower preservations, for subsequent use.The single strain that primary dcreening operation is obtained carries out cellulose decomposition evaluation and differing temps activity experiment, and finishing screen is selected a strain 40 ℃ ~ 70 ℃ existence growths, and effective cellulolytic bacterial strain KMXU56.
Embodiment 2: the evaluation of bacterial strain KMXU56
Physiological and biochemical property: physiology and chemistry feature experimental determining method and the evaluation of bacterial strain, gramstaining is the result show, bacterial strain KMXU56 is Gram-positive.It is all positive that bacterial strain KMXU56 knows that catalase, glucose produce acid, Starch Hydrolysis test, malonate utilization, nitrate reduction, Citrate trianion utilization; Glucose aerogenesis, methyl red experiment, V-P mensuration, indole reaction, H
2The S experiment is all negative; According to " the outstanding Bacteria Identification handbook of uncle, its feature and bacillus pumilus (Bacillus pumilus) characteristic matches.The 16S rRNA gene sequencing of bacterial strain KMXU56 adopts the PCR technology, successfully amplifies the 16SrRNA gene of bacterial strain KMXU56, then Cloning and sequencing.The sequence length of measuring is 1458 bp, the 16S rRNA gene order that obtains is compared by GENBANK database (http://www.ncbi.nlm.nih.gov/genbank/), the result shows that similarity is the bacterial strain of bacillus pumilus (Bacillus pumilus) at the bacterial strain more than 99%, this strain number called after KMXU56, and be deposited in (address: No. 3, No. 1 institute in BeiChen West Road, Chaoyang District, BeiJing City, China Committee for Culture Collection of Microorganisms common micro-organisms center on September 21st, 2012, Institute of Microorganism, Academia Sinica, postcode 100101) preservation, Classification And Nomenclature is bacillus pumilus Bacillus pumilus, preserving number CGMCC No.6595.
Embodiment 3: the determination of activity of bacillus pumilus KMXU56 decomposition of cellulose
Real bacterial strain bacillus pumilus KMXU56 bacterial strain is seeded to LB substratum (Tryptones 10g/L in gnotobasis, yeast extract 5g/L, NaCl 10g/L, pH7.0) on, supported 3 days 37 ℃ of lower training activation, then in gnotobasis, with inoculating needle picking one ring KMXU56, be seeded to and be equipped with in the sterilized LB substratum of 600ml, at 180rpm, 37 ℃ of lower shaking culture 3 days get fermented liquid.Fermented liquid is through centrifugal 10 min of 4000 r/min, and supernatant liquor is as crude enzyme liquid.With 0.5 mL suitably the enzyme liquid of dilution behind 70 ℃ of water bath with thermostatic control preheating 2 min, add 1% carboxymethylcellulose sodium solution that 1.5 mL prepare with acetate buffer solution pH 4.8,70 ℃ of water bath with thermostatic control enzymolysis 30 min, the sodium hydroxide that adds 1 mL concentration and be 1 mol/L shakes up with termination reaction.Then DNS 5 min in boiling water bath that add 2 mL with the cold water termination reaction that flows, measure its light absorption value, and calculate enzyme activity behind the reference standard curve under 540 nm after the cooling.The result shows that enzyme activity is higher, reaches 95% ~ 89% under 40 ℃ ~ 70 ℃ of the cellulases that the bacterial strain KMXU56 that produces enzyme produces.Outside this temperature range, enzyme activity is lower.
Embodiment 4:
Contain the Inoculant of bacillus pumilus KMXU56, obtain through following steps:
(1), actication of culture: the bacterial classification inoculation of getting bacillus pumilus KMXU56 is in the LB liquid nutrient medium: Tryptones 10g/L, yeast extract 5g/L, NaCl 10g/L among the pH7.0, cultivates 5d under 35 ℃ of conditions;
(2), fermentation seed liquid preparation: the bacterial classification inoculation of activation is carried out the one-level enlarged culturing in the LB liquid nutrient medium, as nutrient solution OD
600Stopping to cultivate greater than 1.4 o'clock, get first order seed; First order seed 8% is seeded to and carries out the secondary enlarged culturing in the LB liquid nutrient medium by volume, as nutrient solution OD
600Stopping to cultivate greater than 2.6 o'clock, get fermentation seed liquid; One-level, secondary culture condition are 35 ℃, and rotating speed is 150 r/min or 180r/min, and incubation time is 7d;
(3), be to add urea and sucrose in 50% the rice husk heap in water content, behind the 24h, the fermentation of banking up of in the rice husk heap, evenly splash again wheat bran, Semen Maydis powder and fermentation seed liquid, during this time turning, to temperature-stable when the envrionment temperature, stop fermentation, tunning is dry, make Inoculant; Wherein:
A. the addition means of urea and sucrose is: take by weighing urea and sucrose by rice husk dry weight mass ratio 0.5% and 2% respectively, with urea and sucrose and water by weight the 1:20 wiring solution-forming, in the rice husk of evenly the splashing heap;
B. the addition means of wheat bran, Semen Maydis powder and fermentation seed liquid is: the fermentation seed liquid that will obtain by the step (2) of the wheat bran of rice husk heap dry weight 1%, 2% Semen Maydis powder and 2% mixes, splash after puddling evenly in the rice husk heap, rice husk waste height 1.2m, floor space＜50m
2, cover the fermentation of banking up of ventilation property coverture;
C. when leavening temperature reach 60 ℃ and continue 42h or 48h after, carry out the 1st turning, later on by again turning 4-8 time of the condition of the 1st turning, until temperature-stable after envrionment temperature, at 45 ℃ or 55 ℃ of lower 4h of drying, gets solid-state Inoculant with tunning.
Claims (2)
1. a bacillus pumilus (Bacillus pumilus) KMXU56, its preserving number is: CGMCC No.6595.
2. the Inoculant that contains the described bacillus pumilus KMXU56 of claim 1 is characterized in that this Inoculant
Obtain through following steps:
(1), actication of culture: the bacterial classification inoculation of getting bacillus pumilus KMXU56 is in the LB liquid nutrient medium: Tryptones 10g/L, yeast extract 5g/L, NaCl 10g/L among the pH7.0, cultivates 3-5d under 30-35 ℃ of condition;
(2), fermentation seed liquid preparation: the bacterial classification inoculation of activation is carried out the one-level enlarged culturing in the LB liquid nutrient medium, as nutrient solution OD
600Stopping to cultivate greater than 1.3 o'clock, get first order seed; First order seed 8% is seeded to the LB liquid nutrient medium and carries out the secondary enlarged culturing by volume, as nutrient solution OD
600Stopping to cultivate greater than 2.5 o'clock, get fermentation seed liquid; One-level, secondary culture condition are 30-35 ℃, and rotating speed is 150-180r/min, and incubation time is 5-7d;
(3), the preparation of Inoculant: in water content is the rice husk heap of 40%-50%, add urea and sucrose, behind the 24h, wheat bran, Semen Maydis powder and the fermentation seed liquid fermentation of banking up of in rice husk heap, evenly splashing again, during this time turning, to temperature-stable when the envrionment temperature, stop fermentation, tunning is dry, make Inoculant; Wherein:
A. the addition means of urea and sucrose is: take by weighing urea and sucrose by rice husk dry weight mass ratio 0.5% and 2% respectively, with urea and sucrose and water by weight the 1:20 wiring solution-forming, in the rice husk of evenly the splashing heap;
B. the addition means of wheat bran, Semen Maydis powder and fermentation seed liquid is: the fermentation seed liquid that will obtain by the step (2) of the wheat bran of rice husk heap dry weight 1%, 2% Semen Maydis powder and 2% mixes, splash after puddling evenly in the rice husk heap, rice husk waste height 0.6-1.8m, floor space＜50m
2, cover the fermentation of banking up of ventilation property coverture;
C. when leavening temperature reach 60-65 ℃ and continue 42-48h after, carry out the 1st turning, according to again turning 4-8 time of preceding method, until temperature-stable after envrionment temperature, at the 45-55 ℃ of lower 4h of drying, gets solid-state Inoculant with tunning.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103468618A (en) * | 2013-09-25 | 2013-12-25 | 上海交通大学 | Cellulose-degrading bacteria with phosphate-dissolving capability and application thereof |
| CN104230582A (en) * | 2014-09-11 | 2014-12-24 | 红云红河烟草(集团)有限责任公司 | Bio-organic fertilizer containing bacillus pumilus |
| CN110885771A (en) * | 2019-11-22 | 2020-03-17 | 云南省烟草公司临沧市公司 | Biological agent for improving tobacco-planting soil micro-ecological environment level |
| TWI805161B (en) * | 2021-12-29 | 2023-06-11 | 財團法人工業技術研究院 | Cellulose-degrading bacteria agent, fermentation products from bacillus pumilus, methods of manufacturing the same, and methods of promoting biogas production |
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| WO2009022399A1 (en) * | 2007-08-10 | 2009-02-19 | Yoshichika Kameyama | Novel microorganism having gastric juice promoting action and composition secreted by the same |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103468618A (en) * | 2013-09-25 | 2013-12-25 | 上海交通大学 | Cellulose-degrading bacteria with phosphate-dissolving capability and application thereof |
| CN104230582A (en) * | 2014-09-11 | 2014-12-24 | 红云红河烟草(集团)有限责任公司 | Bio-organic fertilizer containing bacillus pumilus |
| CN110885771A (en) * | 2019-11-22 | 2020-03-17 | 云南省烟草公司临沧市公司 | Biological agent for improving tobacco-planting soil micro-ecological environment level |
| CN110885771B (en) * | 2019-11-22 | 2023-05-16 | 云南省烟草公司临沧市公司 | Biological microbial agent for improving micro-ecological environment level of tobacco planting soil |
| TWI805161B (en) * | 2021-12-29 | 2023-06-11 | 財團法人工業技術研究院 | Cellulose-degrading bacteria agent, fermentation products from bacillus pumilus, methods of manufacturing the same, and methods of promoting biogas production |
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Application publication date: 20130403 |