CN103436450B - Alkaline-tolerant and halophilic aspergillus strain and application thereof in environmental management - Google Patents
Alkaline-tolerant and halophilic aspergillus strain and application thereof in environmental management Download PDFInfo
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Abstract
The invention belongs to the field of microbial engineering and technology, and specifically relates to an alkaline-tolerant and halophilic aspergillus strain and an application thereof in environmental management. The strain is preserved in China General Microbiological Culture Collection Center on April 20th, 2012, with a preservation name of aspergillus JL and a preservation number of CGMCC 6025. The strain has the characteristic of degrading biological substances such as straws in a high salt and alkali environment and can be used for improvement of saline lands and treatment of environmental pollutions.
Description
Technical field:
The invention belongs to microbial project and technical field.Be specifically related to a kind of to there is the alkaline-resisting Aspergillus amstelodami bacterial strain addicted to salt characteristic, and the application in alkaline land improving and environmental improvement.
Background technology:
Microorganism is the biology that nature living environment is extremely wide, no matter protokaryon or eukaryote, and no matter how severe envrionment conditions is, the earth almost can not find nowhere their existence.Addicted to salt or (with) alkaline-resisting fungi is the Microbial resources be separated from high salt, high-alkali or high salinity coexisted environment, due to its there is important research, applicating and exploitation is worth and always receives much concern.At present, along with the Microbial resources addicted to the alkaline-resisting bacterium of salt are excavated, people are just starting these various harm be applied in high salinity environment addicted to the alkaline-resisting bacterium of salt to eliminate and Pollution abatement, as the reparation transformation in saltings, the purifying treatment of mud sewage, and agricultural wastes recycle etc.
For a long time; scientific circles generally believe to only have prokaryotic organism just can survive at extreme environment; but from 2000; after Gunde-Cimerman etc. (2000) isolate fungi from natural salt alkaline earth, people just start target tightening to excavating new halophilic fungi resource (Casamayor et al.2002 in hypersaline environment; Petrovi et al.2002; Zalar etal.2005).Debaryomyces hansenii, Hortaea werneckii and Wallemia ichthyophaga are the halophilic fungis be separated from natural hypersaline environment, and research finds that the salt concn upper limit of their growths is up to 5.2mol/L (Norkrans.1966; Prista et al.2005;
etal.2008).Along with the excavation of a large amount of halophilic fungi, it is found that some Aspergillus (Aspergillus) fungi has salt tolerance at its Perfect stage (Eurotium), as (2003) such as Nevo obtain 3 kinds of salt tolerant aspergillus Aspergillus versicolor, Aspergillusniger and Eurotium herbariorum from the Dead Sea and littoral separation.Afterwards, Butinar etc. (2005) have also screened serial salt tolerant aspergillus: Eurotium amstelodami, Eurotium repens, Eurotium herbariorum, Eurotium rubrum, an Eurotiumchevalieri and novel species Eurotium halotolerans, and wherein E.amstelodami and E.halotolerans can grow under containing the culture medium condition of 27.5%NaCl.But regrettably, the alkaline-resisting fungi found at present, particularly addicted to the alkaline-resisting fungi commutation of salt to less.In many saline-alkali environments, coexist due to saline and alkaline, these are restricted undoubtedly with its range of application of fungi being advantage addicted to salt or salt tolerant merely.
The enzyme of soil microorganisms and secretion thereof is as the activity unit of soil, can participate in directly and promote the biochemical reactions of soil inertia organic composition, they are the prerequisite of material cycle and Conversion of energy in soil ecosystem, are also that can soil ecosystem Developmental maturity and system resource the important symbol of high-efficiency continuous utilization.The microflora of saltings particularly in heavy salinized soil is extremely simple, and be in its non-activated state, although in straw-returning process, add the degraded that certain bacteria preparation can accelerate stalk, add soil with organic matter content, obviously improve soil physical and chemical property, to maintaining the good ecotope system in farmland and crop alimentary system, reduce environmental pollution, the aspect successful such as raise productivity and improve the quality, but this research team finds that these microbial inoculums can play good effect in normal soil, and in salt affected soil, due to the salt of high density and the environment of high ph-values, these bacterial strains are difficult to play due effect.Therefore, anti-(resistance to) the saline and alkaline organism of fermentation of screening is the key point determining that saltings High-efficiency straw utilizes.
Summary of the invention:
The technical problem to be solved in the present invention is to provide a kind of alkaline-resisting Aspergillus amstelodami addicted to salt type (Aspergillus glaucus) bacterial strain JL, and preservation number is CGMCC6025.
Provided by the inventionly alkaline-resistingly be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, depositary institution address addicted to the strain of salt type aspergillus glaucus on April 20th, 2012: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; Preservation name is called Aspergillus amstelodami JL, and Classification And Nomenclature is Aspergillus amstelodami (Aspergillus glaucus); Deposit number is CGMCC NO.6025.
The alkaline-resisting Aspergillus amstelodami bacterial strain addicted to salt type of the present invention has addicted to salt characteristic: bacterial strain suitable growth is at Na salt environment, and the most suitable growth sodium salt concentration is 10-20%(grams per liter), under hypersaline environment condition, bacterial strain can long-term surviving.
The alkaline-resisting Aspergillus amstelodami bacterial strain addicted to salt type of the present invention also has alkaline-resisting characteristic simultaneously: under alkalescence (pH=7-12) culture medium condition, and bacterial strain can by organic acid such as secretion citric acid, oxysuccinic acid etc., and adjust ph is to suitable growth neutrallty condition.
The alkaline-resisting Aspergillus amstelodami bacterial strain addicted to salt type of the present invention passes through secretion lytic enzyme under saline and alkaline condition, efficient degradation organic substance.
Invention also provides a kind of alkaline-resisting Aspergillus amstelodami bacterial strain addicted to salt type and can be used for environmental pollution treatment.
The present invention also provides a kind of alkaline-resisting Aspergillus amstelodami bacterial strain addicted to salt type simultaneously and can be used for alkaline land improvement.
Accompanying drawing illustrates:
Fig. 1, aspergillus colony morphology characteristic
Fig. 2, conidiophore and conidium
Fig. 3, born of the same parents' outer fiber element enzyme and dextranase activity detect
The organic acid GC-MS of Fig. 4, alkaline environment thalline secretion detects
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described in further detail.
Embodiment
Embodiment 1, Isolation and ldentification addicted to salt alkaline profeinace strain
The separation of bacterial strain: the pedotheque of isolated strains is from Zhenlai County, Jilin Province.Choose this ground to lie waste for many years and the plot of height salinization, get the following soil of upper soll layer 2cm, it is for subsequent use that laboratory taken back by the vial loading sterilising treatment.Adopt method of dilution butteron on plate, on the PDA substratum soil supernatant clarified after dilution being coated interpolation NaCl to 250.0g/L, (pH=10) cultivates 4 days, takes turns screening and separating to a strain addicted to the alkaline-resisting fungal bacterial strain (the alkaline-resisting Aspergillus amstelodami bacterial strain addicted to salt type of the present invention is numbered CGMCC6025) of salt through 3.
Strain morphology is identified: what screening obtained is inoculated on Cha Shi nutrient agar addicted to salt alkaline profeinace strain, cultivates one week for 25 DEG C, observes colonial morphology and its conidial fructification of microscopy and hypha form.25 DEG C on Cha Shi agar plate, cultivate 1 week after, thalline develops into blackish green or greyish-green bacterium colony, and circular colonies radius is about 3.5-4cm, and aerial hyphae is flourishing, center is open and flat, border extended white mycelium, the speed of growth is very fast, and aging thalline darkens, period, part is special turns to conidiophore, is combined into macroscopic yellow particle, is non-uniformly distributed in bacterium colony top layer (Fig. 1); Getting bacterium colony top layer mycelia with choosing pin, making slide, carrying out microscopic examination: mycelia is upright, smooth surface, the spherical unit cell of conidium, life at product falx top, very easily come off, after coming off, producing falx top is semisphere baldness (Fig. 2).Be Aspergillus amstelodami through Morphological Identification.
Embodiment 2, bacterial strain Aspergillus amstelodami JL identify addicted to salt, salt tolerance and the suitableeest salt concn
The PDA substratum (pH7.0) of preparation gradient NaCl content, its salt concn (massfraction) is increased to 31%(saturation concentration by 1%), every grade increases by 2%, not add the PDA of NaCl for contrast.By the inoculation of equivalent in substratum central position, 25 DEG C, cultivate 1 week.Found that, bacterial strain, not containing not growth (growing very slow) on the substratum of NaCl, grows relatively slow under 1-5% concentration; Under 5%-19% concentration, strain growth is accelerated, but speedup slows down within the scope of 9-19%, and bacterium colony size is close; Under 21-27% concentration, strain growth is sharply slack-off, and 29-31% concentration hypothallus does not almost grow, and therefore, this bacterial strain belongs to addicted to salt and the fungi of high salt tolerance characteristic.
In order to determine the suitableeest salt concn of strain growth, with above-mentioned series salt gradient PD(potato liquid nutrient medium, do not add agar) (pH=7.0) cultivate bacterial strain, 25 DEG C are shaken training after one week, collected by centrifugation mycelia, repeatedly rinse 4 times with deionized water, dry at 105 DEG C, measure dry mycelial weight, compare and find that the dry weight of bacterial strain under 9-15% concentration is maximum, the salt concn of its most suitable growth is 9-15%.
Embodiment 3, bacterial strain Aspergillus amstelodami JL grow pH and measure
The soda acid gradient PDA substratum of preparation pH1.0-13.0, adds NaCl to final concentration (massfraction) 9%, connects bacterium, cultivates the growing state recording bacterial strain after 1 week for 25 DEG C.Research finds, bacterial strain all can grow at pH3-11, but very fast with pH5-9 range growth, and pH10-12 can grow, but the speed of growth is obviously slack-off, and therefore, this bacterial strain belongs to halophilic fungi.
In order to determine the optimal pH scope of this strain growth, utilize above-mentioned pH gradient to cultivate with PD liquid nutrient medium further, 25 DEG C are shaken training after 1 week, collected by centrifugation mycelia, repeatedly rinse 4 times with deionized water, dry, compare dry mycelial weight at 105 DEG C.Bacterial strain is within the scope of pH6-8, and dry cell weight is maximum, and therefore, the optimal pH scope of this bacterial strain is 6-8.
The analysis domain qualification of embodiment 4, the outer lytic enzyme of bacterial strain born of the same parents
Prepare and be primary carbon source with 5% stalk (paddy rice, corn or Chinese sorghum) pulverized, be matrix with 80% Saline-Sodic soil (taking from Zhenlai County, Jilin Province), take 10% flyash (thickness ratio=1:5) as the solid medium of dispersion gas-permeable medium, with the mixture of 5%PD substratum numerous nutrition for thalline expands, adjust ph to 7.0 after sterilising treatment, inoculation Aspergillus strain, 25 DEG C of fermentation 3-4 weeks in simple and easy fermentation system.Observe stalk fermentation effect, compared with the stalk not adding this bacterial strain, bacterial strain Aspergillus amstelodami JL obviously accelerates the degraded of stalk.Then infiltrate fermented product 2 hours with sterilized water, centrifugal reservation supernatant, is bacterial strain extracellular enzyme solution, and use 90%(saturation concentration) albumen in ammonium sulfate precipitation supernatant liquor, load dialysis tubing dialysed overnight, concentrate with polyoxyethylene glycol, finally measure the enzymic activity such as cellulase, dextranase by DNS method.Result shows, the remarkable degraded cellulose of Aspergillus amstelodami JL fermented liquid and dextran (Fig. 3), therefore, this bacterial strain may be used for the degraded of stalk in environment, thus environmental pollution processes.
The main organic acid of embodiment 5, bacterial strain alkaline-resisting process secretion
The soda acid gradient PDA substratum of preparation pH12.0, adds NaCl to final concentration (massfraction) 9%, connects bacterium, and 25 DEG C of cultivations are carried out suction filtration and thalline and fermented liquid separated after 30 days.Thalline is dried, pulverize rear methanol extraction, extracting solution Rotary Evaporators 40 DEG C of evaporates to dryness, merge with respective fermented liquid after evaporate to dryness, use sherwood oil again, ethyl acetate extracts respectively, and the material in fermented liquid is divided into the fermented liquid part after petroleum ether portion, ethyl acetate extraction part and extraction according to polarity difference.Carry out differentiating to carry out Structural Identification to its difference material with GC-MS, qualification result shows, Aspergillus amstelodami JL can secrete oxysuccinic acid (see figure 4), therefore, and the alkali in during this bacterial strain may be used for and saltings, thus saltings is transformed.
Claims (3)
1. an alkaline-resisting Aspergillus amstelodami bacterial strain addicted to salt type, it is characterized in that this bacterial strain preservation name is called Aspergillus amstelodami (Aspergillus glaucus) JL, preservation number is CGMCC NO.6025.
2. the alkaline-resisting application of Aspergillus amstelodami bacterial strain in alkaline land improvement addicted to salt type as claimed in claim 1.
3. the application of alkaline-resisting Aspergillus amstelodami bacterial strain degrading straw in environmental pollution treatment addicted to salt type as claimed in claim 1.
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| CN105420259B (en) * | 2016-01-06 | 2019-12-06 | 吉林大学 | Cellulase gene derived from extreme saline-alkali resistant aspergillus and application thereof |
| CN108949584B (en) * | 2018-07-30 | 2021-12-17 | 史洋 | Salt-resistant and alkali-resistant aspergillus strain, ITS sequence and application thereof |
| CN110499254B (en) * | 2019-07-22 | 2022-05-06 | 吉林大学 | Saline-alkali-resistant aspergillus ochraceus strain W1, and microbial inoculum and application thereof |
| CN111670915A (en) * | 2020-07-18 | 2020-09-18 | 山东胜伟盐碱地科技有限公司 | Microbial pesticide capable of being stored for long time in saline-alkali soil |
| CN112458009A (en) * | 2020-11-20 | 2021-03-09 | 东北农业大学 | Straw decomposition agent for improving saline-alkali soil and application thereof |
| CN112573953A (en) * | 2020-11-21 | 2021-03-30 | 黑龙江省农业科学院畜牧研究所 | Animal manure leavening agent for preparing fertilizer for improving saline-alkali soil and application thereof |
| CN112479759A (en) * | 2020-11-27 | 2021-03-12 | 沈阳农业大学 | Microbial decomposition agent and organic fertilizer for improving saline-alkali soil |
| CN112573951A (en) * | 2020-12-02 | 2021-03-30 | 黑龙江省农业科学院畜牧研究所 | Method for preparing bio-organic fertilizer by mixing sludge and straw |
| CN112573971A (en) * | 2020-12-07 | 2021-03-30 | 沈阳农业大学 | Soil microorganism improver containing furnace ash and application thereof |
| CN112574755A (en) * | 2020-12-25 | 2021-03-30 | 沈阳农业大学 | Microbial porous material preparation for improving saline-alkali soil, preparation method and application |
| CN112723922A (en) * | 2021-02-24 | 2021-04-30 | 黑龙江省农业科学院畜牧研究所 | Animal manure leavening agent and bio-organic fertilizer for improving saline-alkali soil |
| CN112939665A (en) * | 2021-03-05 | 2021-06-11 | 黑龙江省农业科学院畜牧研究所 | Method for preparing bio-organic fertilizer from biogas residue waste liquid of straw mixed agricultural product processing byproduct |
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| CN102517296A (en) * | 2011-11-25 | 2012-06-27 | 吉林大学 | Clone and application of extremely halophilic aspergillus ribosomal protein gene |
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| Halotolerant and halophilic fungi;Nina GUNDE-CIMERMAN等;《Mycological Research》;20090910;第113卷;1231-1241页 * |
| The genus Eurotium-members of indigenous fungal community in hypersaline waters of salterns;Lorena Butinar等;《FEMS Microbiology Ecology》;20040907;第51卷;155-166页 * |
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