CN102191181A - Screening method of penicillium janthinellum and application thereof - Google Patents
Screening method of penicillium janthinellum and application thereof Download PDFInfo
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- CN102191181A CN102191181A CN 201010122178 CN201010122178A CN102191181A CN 102191181 A CN102191181 A CN 102191181A CN 201010122178 CN201010122178 CN 201010122178 CN 201010122178 A CN201010122178 A CN 201010122178A CN 102191181 A CN102191181 A CN 102191181A
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Abstract
The invention discloses a screening method of penicillium janthinellum and application thereof. The screening method comprises the following steps of: collecting an arsenic polluted soil sample; separating fungi from the soil sample in a laboratory; reacting the separated fungi with filter paper sheets soaked by arsenic with different concentrations on a solid potato-glucose-peptone (PGP) culture medium; and observing the colony growth conditions of the fungi under the stress of the arsenic with different concentrations so as to screen out the penicillium janthinellum. The penicillium janthinellum screened out by the screening method can be prepared into a corresponding microbial agent; the prepared microbial agent can reduce the arsenic content, particularly the effective arsenic content in soil to a certain extent through the enrichment and volatilization of the penicillium janthinellum after being applied to polluted soil or environments so as to guarantee the normal growth of crops and ensure that the arsenic content in the crops and farm products meets requirements on nuisanceless farm products.
Description
Technical field
The present invention relates to screening method and the application thereof of fungi, relate in particular to the screening method and the application thereof of penicillium janthinellum (Penicilliumjanthinellum).
Background technology
Arsenic is a kind of poisonous and carcinogenic metalloid element that extensively exists at nature, and arsenic contamination has become very one of serious environmental problem of whole world harm.It is reported that the whole world more than at least 5,000 ten thousand populations are being faced with the threat of endemic arsenic poisoning, wherein great majority are Asian countries.China is subjected to one of the most serious country of arseniasis harm, at 1956-1984 between two more than ten years, and an arseniasis incident surplus the whole nation once took place 30.China is again arsenic minerals big country, arsenic minerals extensively is distributed in provinces and regions such as the Hunan, Yunnan, Guangxi, Guangdong in the Central-South and west and south, arsenic minerals exploitation or smelting, contain the excessive input of agricultural material products such as arsenic agricultural chemicals, chemical fertilizer etc., can become the accumulation of arsenic in the soil building earth, and then affect the g and D of plant, animal, but also can enter human body by food chain, human existence and health are constituted a threat to.
As polluted soil restorative procedure commonly used at present comprises soil improvement agent method, molten local method, casting method, chemical irrigation and bioremediation technology.
Bioremediation technology is the form of utilizing biological (mainly being plant and microorganism) to be used for subduing, purifies the arsenic in the soil or changing arsenic, and quilt generally believes it is the technology of tool application prospect in the arsenic contamination improvement.The phytoremediation technology that wherein utilizes hyperaccumulative plant to remove arsenic in soil has been obtained breakthrough progress at nearest several years, Herba pteridis vittatae (Brakefern, Pteris vitta) and the plant of the tired arsenic of Da Ye Herba Pteridis multifidae ultraproducts such as (Pteris nervosa) be found in succession, and be used for the geographic soil remediation of arsenic contamination.In the bioremediation technology of heavy-metal contaminated soil, except using hyperaccumulative plant absorbing enriched metal, utilize plant, microorganism that heavy metal is converted into volatile organic compound, it is spilt in the atmosphere, thereby the heavy metal in the removal soil has also caused investigator's attention gradually.Utilize the effect of microorganism and plant as Terry etc., the selenium in the environment (Se) be converted into the lower gaseous form of bio-toxicity (methyl-selenide and dimethyl diselenide), directly or the tissue by plant it is evaporate in the atmosphere.After Meagher etc. changed the intravital mercury of bacterium (Hg) reductase gene over to mustard seed section plant Arabidopsis, the transgenic plant that obtain can tolerate, absorb the Hg in the edatope, and with Hg
2+Be reduced into Hg
0The back volatilization enters atmosphere.Arsenic compound in the soil also can change materials such as gasiform methyl arsenic into, migration and conversion in a plurality of ring layers.Therefore, utilize the enrichment to arsenic, utilization and the functions such as conversion, volatilization of certain micro-organisms, As is accumulated in the organism or with it forms volatile attitude arsenic compound, become one of potential arsenic in soil pollution remediation technology.
The fungi that arsenic is had a volatilization ability of present domestic report does not also have, and external report is also very few, and also just is confined to mostly in theory and breadboard research.
Summary of the invention
Technical problem to be solved by this invention provides a kind of screening method of penicillium janthinellum, penicillium janthinellum is applied to administer in the soil of arsenic contamination and reduces the accumulation of arsenic in crop and agricultural-food.
In order to solve the problems of the technologies described above, the invention provides a kind of screening method of penicillium janthinellum, comprising:
Gather the As polluted soil sample, in the laboratory, from pedotheque, isolate fungi, on solid-state PGP substratum,, observe the colony growth situation of fungi under different concns arsenic is coerced, filter out penicillium janthinellum thus isolated fungi and the filter paper effect that is soaked with different concns arsenic.
Preferably, this method specifically comprises:
Pedotheque is isolated fungi through coating behind the gradient dilution on Martin's substratum that arsenic content is 400～600mg/L;
With isolated fungi through purifying repeatedly, cultivate after, cut-off directly is that the circular fungi bacterium piece of 8～10mm or square fungi bacterium piece that the length of side is 8～10mm are put on the described PGP substratum, be that the circular filter paper sheet that the diameter that is soaked with the different concns arsenic solution is 2～4mm is placed at 1～3cm place respectively apart from this bacterium piece, the scope of arsenic concentration is set to 1000～10000mg/L; Described PGP cultivation after 5 days, is filtered out penicillium janthinellum by the growing state of observing bacterial strain based on 24～26 ℃ of following cultivations.
Preferably, the composition of Martin's substratum comprises: glucose, peptone, KH
2PO
4, MgSO
47H
2O, agar and water, the weight ratio of each composition are 24: 8: 2: 1: 60: 4000;
The composition of PGP substratum comprises: potato, glucose, peptone and water, the weight ratio of each composition are 60: 8: 1: 400.
In order to solve the problems of the technologies described above, the invention provides the authentication method of a kind of penicillium janthinellum to the resistance of arsenic, comprising:
Under the indoor cultivation condition, measure biological accumulation and the volatilization ability of penicillium janthinellum to arsenic, and the variation of penicillium janthinellum biomass under the culture condition of the solution that contains different concns arsenic, the resistance of penicillium janthinellum identified thus to arsenic.
Preferably, under the indoor cultivation condition, measure biological accumulation and the volatilization ability of penicillium janthinellum, specifically comprise arsenic:
The preparation total arsenic content is the PGP substratum of 50mg/L, and sterilization is after 14～17 minutes down at 120～125 ℃, and inserting 0.1ml penicillium janthinellum biomass is 10
4The bacteria suspension of cfu/ml, culture temperature is 24～27 ℃, rotating speed is 138～142rpm, cultivate after 5 days, carried out centrifugal treating 8～12 minutes, clean mycoplasma repeatedly 4 times with ultrapure water with 3800～4200rpm rotating speed, wash residual substratum off, this mycoplasma being dried to constant weight under 48～52 ℃, then described mycoplasma is weighed, is 4～6: 1 HNO with 11～13ml volume ratio
3, HClO
4Mixed solution disappears this mycoplasma under 158～162 ℃ of conditions and boils 10 hours, adopts the total arsenic content in this mycoplasma of atom fluorimetry; The centrifugal nutrient solution that goes out and the ultrapure water that cleans this mycoplasma are mixed the back adopt the atom fluorimetry total arsenic content as supernatant liquor;
Total arsenic content in the mycoplasma is as the biological accumulation amount of penicillium janthinellum to arsenic, and penicillium janthinellum is to the total arsenic content in the total arsenic content-supernatant liquor in the total arsenic content-mycoplasma of the volatile quantity=configuration of arsenic.
Preferably,
With reference at total arsenic content being the processing that inserts penicillium janthinellum in the PGP substratum of 50mg/L, respectively with at described total arsenic content be do not insert in the PGP substratum of 50mg/L that penicillium janthinellum is handled and in the PGP substratum, insert penicillium janthinellum and the processing of not disposing arsenic content in contrast, every processing is all repeated repeatedly.
Preferably, under indoor conditions, be determined at the variation of penicillium janthinellum biomass under the culture condition of the solution that contains different concns arsenic, specifically comprise:
Preparation total arsenic content scope is the PGP substratum of 0～200mg/L, after sterilizing 14～16 minutes under 120～122 ℃ of temperature, 8～10mm penicillium janthinellum bacterium piece that growth velocity is identical adds respectively in the above processing, temperature is 23～27 ℃ on shaking table, and rotating speed is 138～142rpm shaking culture; Cultivate after 5 days, nutrient solution centrifugal 8～12 minutes with 3800～4200rpm, and adopt the spore number that dilutes in the gradient method counting bacteria suspension, promptly express the penicillium janthinellum biomass;
Clean mycoplasma repeatedly 4 times with ultrapure water, wash residual substratum off after, this mycoplasma is dried to constant weight under 48～52 ℃, then this mycoplasma weight of weighing, i.e. penicillium janthinellum biomass weight.
Preferably,
The corresponding processing of different total arsenic contents all repeats repeatedly.
Preferably, this method also comprises:
Observation is along with the variation tendency of the biomass of the increase penicillium janthinellum of total arsenic content in the culture solution, and observe the highest total arsenic content of biomass of penicillium janthinellum, it is stronger to arsenic biological accumulation and evaporable total arsenic content scope to determine that thus described penicillium janthinellum shows.
In order to solve the problems of the technologies described above, to the invention provides a kind of penicillium janthinellum and administer and the application aspect the accumulation in crop and agricultural-food of reduction arsenic at As polluted soil.
By the screening of the present invention to penicillium janthinellum, can make corresponding microorganism preparation for repairing, after being applied in the microorganism preparation for repairing that makes in contaminated soil or the environment, by enrichment and the volatilization of this penicillium janthinellum to arsenic, reduce the content of arsenic in the soil to a certain extent, particularly reduce the content of effective arsenic in the soil, thereby guarantee the crop normal growth, and make the content of arsenic in crop and the agricultural-food reach the non-polluted farm product requirement.
Description of drawings
Penicillium janthinellum the colony growth situation under the filter paper effect that be soaked with different concns arsenic of Fig. 1 for screening and turn out with method of the present invention;
Fig. 2 is the growth curve chart of penicillium janthinellum under different arsenic concentration levels.
Embodiment
The screening of penicillium janthinellum provided by the invention and cultural method comprise: separate fungi from the soil of arsenic contamination, observe isolated fungi and coerce the upgrowth situation of bacterium colony down at different concns arsenic, filter out penicillium janthinellum thus; Under the indoor cultivation condition, observe accumulation and the volatilization ability of isolated penicillium janthinellum to arsenic.
The penicillium janthinellum of cultivating, filtering out is inoculated in different concns arsenic coerces in the solid-state PGP flat board down, the colony growth situation of observation penicillium janthinellum under different concns arsenic is coerced.Compare with other fungi, penicillium janthinellum is still to have good upgrowth situation under the condition of 10000mg/L at arsenic content, shows that this fungi has good patience to arsenic.The penicillium janthinellum that carries out shows the volatile quantity determination experiment of arsenic subsequently, and when being 5 days to this penicillium janthinellum incubation time, and the arsenic concentration that adds in substratum is when being 50mg/L, and this penicillium janthinellum reaches 142.195 μ g to the biological volatile quantity of arsenic.This penicillium janthinellum of description of test has stronger conversion and volatilization ability to arsenic, arsenate can be converted into gasiform arsenide, can be applied in the biological restoration process of As polluted soil, thereby reduce the accumulation of arsenic in crop and agricultural-food.
Below in conjunction with accompanying drawing and preferred embodiment technical scheme of the present invention is at length set forth.Following examples only are used for description and interpretation the present invention, and do not constitute the restriction to technical solution of the present invention.
Below among all embodiment, arsenic is all with Na
3AsO
412H
2The form of O adds in the substratum.
Separation, screening and the cultivation of embodiment 1 penicillium janthinellum
Gather the contaminated soil sample from the arsenic contamination district, install and transport back the laboratory, go up at arsenical solid-state PGP substratum (potato-glucose-protein culture medium) and from this pedotheque, isolate fungi, filter out penicillium janthinellum thus with plastics bag.
Specifically comprise step:
Above-mentioned As polluted soil sample is near slag accumulation place the arsenic disulfide of shimen Hunan area, after transporting this pedotheque back laboratory, through gradient dilution and coat on Martin's substratum that arsenic content is 400～600mg/L and isolate fungi, the composition of this Martin's substratum comprises: glucose, peptone, KH
2PO
4, MgSO
47H
2O, agar and water, its weight ratio are 24: 8: 2: 1: 60: 4000.
Embodiment 2 different concns arsenic are coerced down the colony growth situation of little purple mould
With isolated fungi through purifying repeatedly, cultivate after, cut-off directly is that the circular bacterium piece of 8～10mm or square bacterium piece that the length of side is 8～10mm place on the PGP substratum, 1000,3000,5000,10000mg/L be that the circular filter paper sheet that the diameter that is soaked with the different concns arsenic solution is 2～4mm is placed at 1～3cm place respectively apart from this bacterium piece, arsenic concentration is set to respectively:.The PGP cultivation after 5 days, is observed the growing state of bacterial strain based on 24～26 ℃ of following cultivations.The PGP medium component comprises: potato, glucose, peptone and water, the weight ratio of each composition are that weight ratio is 60: 8: 1: 400.
Penicillium janthinellum is compared with isolating other fungi, is still to have good upgrowth situation under the condition of 10000mg/L at arsenic content.As shown in Figure 1, A is isolated penicillium janthinellum in Fig. 1, and it is that the filter paper of 10000mg/L arsenic covers fully that its mycelia will be soaked with concentration; B, C, D are respectively the colony growth situation of contrast fungi under the filter paper effect that is soaked with different concns arsenic that is numbered SM-5F7, SM-5F9, SM-5F1.
Embodiment 3 penicillium janthinellums are to the determination experiment of the volatilization function of arsenic
The preparation total arsenic content is the PGP substratum of 50mg/L, and sterilization is after 14～17 minutes down at 120～125 ℃, and inserting 0.1ml penicillium janthinellum content is 10
4The bacteria suspension of cfu/ml, culture temperature is that 24～27 ℃, rotating speed are to cultivate under 138～142rpm condition after 5 days, rotating speed with 3800～4200rpm carried out centrifugal treating 8～12 minutes, clean mycoplasma repeatedly 4 times with ultrapure water, wash residual substratum off, this mycoplasma being dried to constant weight under 48～52 ℃, then this mycoplasma is weighed, is 4～6: 1 HNO with 11～13ml volume ratio
3, HClO
4Mixed solution disappears this mycoplasma under 158～162 ℃ of conditions and boils 10 hours, adopts the total arsenic content in this mycoplasma of atom fluorimetry; The centrifugal nutrient solution that goes out and the ultrapure water that cleans this mycoplasma are mixed the back also adopt atom fluorimetry total arsenic content in it as supernatant liquor.
As the biological accumulation amount of penicillium janthinellum to arsenic, calculate the volatile quantity of penicillium janthinellum to arsenic with following formula: penicillium janthinellum is to total arsenic content in the total arsenic content-supernatant liquor in the total arsenic content-mycoplasma of the volatile quantity=configuration of arsenic with the total arsenic content in the mycoplasma.
This experiment does not add penicillium janthinellum to add 50mg/L arsenic, adds the processing that penicillium janthinellum does not add arsenic, contrasts above-mentioned adding 50mg/L arsenic and adds the processing of penicillium janthinellum, and each handles counterpoise multiple 3 times.
This penicillium janthinellum has shown stronger anti-arsenic performance, and when incubation time was 5 days, its biological volatile quantity to arsenic is 142.195 μ g, and was as shown in table 1.
Table 1 penicillium janthinellum is to the biological volatile quantity of arsenic (mean value ± S.D)
| Penicillium janthinellum | Mycoplasma dry weight (g) | Biological volatile quantity (μ g) |
| P.janthinellum | 0.255±0.034 | 142.195±15.294 |
At present domestic also not about arsenic being had the report of the fungi of volatilization ability, the report that some this respects are abroad arranged, but be based on mostly in theory and breadboard research, the existing penicillium janthinellum that the present invention is separated, filters out is compared with the anti-arsenic fungi of abroad reporting, and is as shown in table 2.
Table 2 the present invention compares with the situation of the anti-arsenic fungi of external report
The above is preferred embodiment of the present invention only, is not to be used to limit the scope that comprises of the present invention.All any modifications of being done within the spirit and principles in the present invention, be equal to alternative, improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. the screening method of a penicillium janthinellum comprises:
Gather the As polluted soil sample, in the laboratory, from described pedotheque, isolate fungi, on solid-state PGP substratum,, observe the colony growth situation of fungi under different concns arsenic is coerced, filter out penicillium janthinellum thus isolated fungi and the filter paper effect that is soaked with different concns arsenic.
2. in accordance with the method for claim 1, it is characterized in that, specifically comprise:
Described pedotheque is isolated fungi through coating behind the gradient dilution on Martin's substratum that arsenic content is 400～600mg/L;
With isolated fungi through purifying repeatedly, cultivate after, cut-off directly is that the circular fungi bacterium piece of 8～10mm or square fungi bacterium piece that the length of side is 8～10mm are put on the described PGP substratum, be that the circular filter paper sheet that the diameter that is soaked with the different concns arsenic solution is 2～4mm is placed at 1～3cm place respectively apart from described bacterium piece, the scope of described arsenic concentration is set to 1000～10000mg/L; Described PGP cultivation after 5 days, is filtered out described penicillium janthinellum by the growing state of observing bacterial strain based on 24～26 ℃ of following cultivations.
3. in accordance with the method for claim 2, it is characterized in that the composition of described Martin's substratum comprises: glucose, peptone, KH
2PO
4, MgSO
47H
2O, agar and water, the weight ratio of each composition are 24: 8: 2: 1: 60: 4000;
The composition of described PGP substratum comprises: potato, glucose, peptone and water, the weight ratio of each composition are 60: 8: 1: 400.
4. a penicillium janthinellum comprises the authentication method of the resistance of arsenic:
Under the indoor cultivation condition, measure biological accumulation and the volatilization ability of penicillium janthinellum to arsenic, and the variation of penicillium janthinellum biomass under the culture condition of the solution that contains different concns arsenic, the resistance of described penicillium janthinellum identified thus to arsenic.
5. in accordance with the method for claim 4, it is characterized in that described biological accumulation and the volatilization ability of penicillium janthinellum to arsenic of measuring specifically comprises under the indoor cultivation condition:
The preparation total arsenic content is the PGP substratum of 50mg/L, and sterilization is after 14～17 minutes down at 120～125 ℃, and inserting 0.1ml penicillium janthinellum biomass is 10
4The bacteria suspension of cfu/ml, culture temperature is 24～27 ℃, rotating speed is 138～142rpm, cultivate after 5 days, carried out centrifugal treating 8～12 minutes, clean mycoplasma repeatedly 4 times with ultrapure water with 3800～4200rpm rotating speed, wash residual substratum off, described mycoplasma being dried to constant weight under 48～52 ℃, then described mycoplasma is weighed, is 4～6: 1 HNO with 11～13ml volume ratio
3, HClO
4Mixed solution disappears described mycoplasma under 158～162 ℃ of conditions and boils 10 hours, adopts the total arsenic content in the described mycoplasma of atom fluorimetry; The centrifugal nutrient solution that goes out and the ultrapure water that cleans described mycoplasma are mixed the back adopt described atom fluorimetry total arsenic content as supernatant liquor;
As the biological accumulation amount of penicillium janthinellum to arsenic, calculate the volatile quantity of penicillium janthinellum to arsenic with following formula: penicillium janthinellum is to the total arsenic content in the total arsenic content-described supernatant liquor in the total arsenic content-described mycoplasma of the volatile quantity=configuration of arsenic with the total arsenic content in the described mycoplasma.
6. in accordance with the method for claim 5, it is characterized in that,
With reference at described total arsenic content being the processing that inserts penicillium janthinellum in the PGP substratum of 50mg/L, respectively with at described total arsenic content be do not insert in the PGP substratum of 50mg/L that penicillium janthinellum is handled and in the PGP substratum, insert penicillium janthinellum and the processing of not disposing arsenic content in contrast, every processing is all repeated repeatedly.
7. in accordance with the method for claim 4, it is characterized in that, under indoor conditions, be determined at the variation of penicillium janthinellum biomass under the culture condition of the solution that contains different concns arsenic, specifically comprise:
Preparation total arsenic content scope is the PGP substratum of 0～200mg/L, after sterilizing 14～16 minutes under 120～122 ℃ of temperature, 8～10mm penicillium janthinellum bacterium piece that growth velocity is identical adds respectively in the above processing, temperature is 23～27 ℃ on shaking table, and rotating speed is 138～142rpm shaking culture; Cultivate after 5 days, nutrient solution centrifugal 8～12 minutes with 3800～4200rpm, and adopt the spore number that dilutes in the gradient method counting bacteria suspension, promptly express described penicillium janthinellum biomass;
Clean mycoplasma repeatedly 4 times with ultrapure water, wash residual substratum off after, described mycoplasma is dried to constant weight the described mycoplasma weight of weighing then, promptly described penicillium janthinellum biomass weight under 48～52 ℃.
8. in accordance with the method for claim 7, it is characterized in that,
The corresponding processing of different total arsenic contents all repeats repeatedly.
9. in accordance with the method for claim 8, it is characterized in that, also comprise:
Observation is along with the variation tendency of the biomass of the described penicillium janthinellum of increase of total arsenic content in the culture solution, and the total arsenic content of the biomass of observing described penicillium janthinellum when the highest, it is stronger to arsenic biological accumulation and evaporable total arsenic content scope to determine that thus described penicillium janthinellum shows.
10. a penicillium janthinellum is administered and the application aspect the accumulation in crop and agricultural-food of reduction arsenic at As polluted soil.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104556399A (en) * | 2014-11-14 | 2015-04-29 | 山东省农业科学院农产品研究所 | Method for purifying heavy metal ions |
| CN104673678A (en) * | 2014-11-14 | 2015-06-03 | 山东省农业科学院农产品研究所 | Penicillium subjanthinellum HG2014 |
| CN105112307A (en) * | 2015-09-15 | 2015-12-02 | 沈阳农业大学 | Fungus strain with induced resistance as well as culture method and application of fungus strain |
| CN105647817A (en) * | 2016-01-26 | 2016-06-08 | 华中农业大学 | Penicillium janthinellum for decomposing hard-soluble aluminum phosphate in acid soil and application thereof |
-
2010
- 2010-03-11 CN CN 201010122178 patent/CN102191181A/en not_active Withdrawn
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104556399A (en) * | 2014-11-14 | 2015-04-29 | 山东省农业科学院农产品研究所 | Method for purifying heavy metal ions |
| CN104673678A (en) * | 2014-11-14 | 2015-06-03 | 山东省农业科学院农产品研究所 | Penicillium subjanthinellum HG2014 |
| CN104556399B (en) * | 2014-11-14 | 2016-06-08 | 山东省农业科学院农产品研究所 | A kind of method of purification of heavy metal ion |
| CN104673678B (en) * | 2014-11-14 | 2017-11-21 | 山东省农业科学院农产品研究所 | One species penicillium janthinellum |
| CN105112307A (en) * | 2015-09-15 | 2015-12-02 | 沈阳农业大学 | Fungus strain with induced resistance as well as culture method and application of fungus strain |
| CN105112307B (en) * | 2015-09-15 | 2018-07-20 | 沈阳农业大学 | A kind of fungal bacterial strain and its cultural method and application with induction of resistance |
| CN105647817A (en) * | 2016-01-26 | 2016-06-08 | 华中农业大学 | Penicillium janthinellum for decomposing hard-soluble aluminum phosphate in acid soil and application thereof |
| CN105647817B (en) * | 2016-01-26 | 2019-04-26 | 华中农业大学 | The penicillium janthinellum of one plant of soil slightly solubility aluminum phosphate that reduces sourness and its application |
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