CN110343623A - A kind of degraded cellulose, the penicillium oxalicum bacterial strain for adsorbing cadmium and its application - Google Patents
A kind of degraded cellulose, the penicillium oxalicum bacterial strain for adsorbing cadmium and its application Download PDFInfo
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Abstract
The present invention provides a kind of degraded cellulose, the penicillium oxalicum bacterial strain of absorption cadmium, classification naming is penicillium oxalicum (Penicilliumoxalicum) WAF6, and deposit number is ACCC 32731.The concentration of cadmium ions tolerance range of the penicillium oxalicum bacterial strain is 0-150mg/L；Its energy cellulase-producing, and have good degradation effect to rice straw, it is applied in the degradation of rice straw, was degraded substantially at the 15th day；Meanwhile the bacterial strain have adsorbing heavy metal cadmium ability, applied cadmium pollution soil improvement in, the 30th day penicillium oxalicum bacterial strain to every adsorption rate be more than 76%.The bacterial strain is suitable for the biological prosthetic of cadmium pollution rice field, while degrading rice straw, can also achieve the effect that rehabilitating soil metal cadmium pollution.
Description
Technical field
The present invention relates to technical field of environmental management, in particular to a kind of degraded cellulose, the penicillium oxalicum for adsorbing cadmium
Strain and its application.
Background technique
China is one of stalk resource country the most abundant.It is reported that the annual output of China's agricultural crop straw is 6~7
Hundred million tons, account for about the 1/3 of world's stalk total output, stalk resource can be larger using space, and therefore, stalk resource recycles pair
Improve agroecological environment, reduces environmental pressure and be of great significance.Currently, the method for being directed to straw degradative, mainly has directly
Returning to the field, mechanical degradation, chemical degradation and biodegrade etc., compared to other methods, biological degradation method have processing mode it is mild,
Low energy consumption and it is without secondary pollution to environment the advantages that and be concerned.
Cadmium is classified as the food contaminant of primary study by the World Health Organization (WHO)；International cancer research institution (IARC)
Cadmium is regarded as into I class carcinogenic substance.Cadmium is accumulated to a certain degree in vivo, can damage urinary system, make body connective tissue damage,
Reproductive system dysfunction, teratogenesis, and breast cancer can be caused.In the method administered for cadmium pollution in current environment, biology is repaired
Multiple method has research advantage.
Chinese patent CN 105296363A discloses a kind of penicillium oxalicum NJDL-03, which can significantly improve hydroxyl
The solubility of base apatite, while the phosphate anion discharged can precipitate the lead ion in environment reduces lead in the environment
Validity；Chinese patent CN 106047730A discloses a kind of penicillium oxalicum bacterial strain EU2101, and the bacterium is after solid fermentation culture
Leaching liquor have cellulase activity, can effectively hydrolyzing oxygenation pretreatment manioc waste；Currently, temporarily having no related has while dropping
Solve cellulose, adsorbing heavy metal cadmium ability penicillium oxalicum report.
Summary of the invention
The present invention provides a kind of degraded cellulose, adsorb cadmium penicillium oxalicum bacterial strain and its application, its purpose is to
So that penicillium oxalicum bacterial strain is suitable for the biological prosthetic of cadmium pollution rice field, while degrading rice straw, can also reach a reparation huge sum of money
Belong to the effect of contaminated soil.
In order to achieve the above object, the invention provides the following technical scheme:
A kind of degraded cellulose, the penicillium oxalicum bacterial strain for adsorbing cadmium, the classification naming of the penicillium oxalicum bacterial strain are oxalic acid
Mould (Penicilliumoxalicum) WAF6, deposit number are ACCC 32731, biological nature are as follows: colony edge is in white
Color, velvet shape；Surface raw dirty-green spore, the spore it is easy to fall off；Most suitable culture pH is 6.5, and most suitable cultivation temperature is 28
℃；Spore can be produced by cultivating 3~5 days under conditions of 25~28 DEG C of solid fermentation；It can be micro- with glucose, sucrose, wheat bran or stalk
Biological carbon source is grown；It can be grown by microorganism nitrogen source of peptone, yeast extract or corn flour.
Preferably, the concentration of cadmium ions tolerance range of the penicillium oxalicum bacterial strain is 0~150mg/L.
The present invention also provides a kind of application of above-mentioned penicillium oxalicum bacterial strain in rice straw degradation.
Preferably, the application includes the following steps:
(1) rice straw fluid nutrient medium is prepared；
(2) the penicillium oxalicum bacterial strain is prepared into seed liquor, is connected to rice straw Liquid Culture by 10% inoculum concentration
In base, shaken cultivation 5~30 days under 28 DEG C, the revolving speed of 120r/min.
Preferably, the rice straw fluid nutrient medium is by CMC-Na, KH2PO4、MgSO4·7H2O, NaCl, peptone,
Yeast extract, distilled water and rice straw composition.
It is highly preferred that the rice straw fluid nutrient medium includes following parts by weight of component: 10 parts of CMC-Na, KH2PO4 1
Part, MgSO4·7H2After 0.5 part of O, 0.5 part of NaCl, 2 parts of peptone, 0.5 part of yeast extract, 1000 parts of distilled water, and shearing
10 parts of rice straw.
Preferably, evaluation penicillium oxalicum bacterial strain includes by the rice after step (2) are cultivated to rice straw degradation effect
Straw solution is centrifuged, is cleaned and drying and processing, and rice straw weight-loss ratio is calculated.
The present invention also provides a kind of application of above-mentioned penicillium oxalicum bacterial strain in cadmium pollution soil improvement.
Preferably, the application includes the following steps:
(1) the penicillium oxalicum bacterial strain is obtained into conidia powder after solid fermentation, drying and crushing；
(2) spare after drying, clean by cadmium pollution soil, pulverizing, being sieved；
(3) conidia powder obtained by step (1) is added step (2) treated in cadmium pollution soil, contain soil miospore
Amount is 1 × 106~1 × 107CFU/g is cultivated 10~50 days naturally at room temperature after adding water and stirring.
Preferably, after step (3) is cultivated 30 days naturally the penicillium oxalicum bacterial strain to every adsorption rate be more than 76%.
Above scheme of the invention have it is following the utility model has the advantages that
Penicillium oxalicum bacterial strain WAF6 provided by the invention, classification naming are penicillium oxalicum
(Penicilliumoxalicum) WAF6 is preserved in Chinese agriculture Microbiological Culture Collection administrative center, deposit number are as follows:
ACCC 32731.It can cellulase-producing, and have good degradation effect to rice straw；It is applied in rice straw
In degradation, rice straw was degraded substantially at the 15th day；Meanwhile the bacterial strain has the ability of adsorbing heavy metal cadmium, can reduce
The biological effectiveness of heavy metal cadmium in contaminated soil and in the soil have good colonization ability, applied in cadmium pollution
In soil remediation, after the 30th day, penicillium oxalicum bacterial strain to every adsorption rate be more than 76%.The bacterial strain is suitable for cadmium pollution rice field
It is biological prosthetic, while degrading rice straw, can also achieve the effect that repairing heavy metal in soil pollutes.
Detailed description of the invention
Fig. 1 is the strain culturing 25 days disintegration effects to rice straw of the invention；
Fig. 2 is the degradation effect of different strains of the invention to rice straw；
Fig. 3 is the adsorption effect of bacterial strain WAF6 of the invention in cadmium pollution soil.
Specific embodiment
To keep the technical problem to be solved in the present invention, technical solution and advantage clearer, below in conjunction with specific implementation
Example is described in detail.
Embodiment 1: the culture medium used is invented
(1) isolation medium: CMC culture medium: sodium carboxymethylcellulose (CMC-Na) 10.0g, KH2PO4 0.25g, potato
Juice 100mL, agar powder 18.0g, distilled water 1000mL.
(2) purifying, Storaged media: PDA culture medium: potato 200g, glucose 20.0g, agar powder 18.0g, distillation
Water 1000mL.
(3) culture medium containing cadmium ion: potato 200g, glucose 20.0g, agar powder 18.0g, distilled water 1000mL, root
Various concentration concentration of cadmium ions solid medium is configured to according to needing to add mother liquor containing cadmium ion (20mg/mL).
(4) screening and culturing medium:
A. Congo red culture medium: CMC-Na 2.0g, KH2PO40.5g, MgSO4·7H2O 0.25g, (NH4)2SO4
1.0g, Congo red 0.1g, agar powder 18.0g, distilled water 1000mL；
B. secondary screening culture medium: CMC-Na 5.0g, KH2PO41.0g, MgSO4·7H2O 0.5g, NaNO33.0g, KCl
0.5g, FeCl3·6H2O 0.01g, distilled water 1000mL；
(5) rice straw fluid nutrient medium (by weight): 10 parts of CMC-Na, KH2PO41 part, MgSO4·7H2O 0.5
10 parts of rice straw after part, 0.5 part of NaCl, 2 parts of peptone, 0.5 part of yeast extract, 1000 parts of distilled water, and shearing.
Embodiment 2: bacterial strain isolates and purifies
Strain isolation purifying method particularly includes: use dilution plate rubbing method, pedotheque is passed through into gradient dilution
10 are made with sterile water-3-10-5Bacteria suspension, and be coated on isolation medium, it is placed in 28 DEG C of constant incubators and cultivates 2-3
The fungi being separated to, is carried out scribing line purifying and preservation by it in PDA culture medium, and purified strain is put in 4 DEG C of refrigerators and is protected
Hiding.
The classification naming of the penicillium oxalicum bacterial strain is penicillium oxalicum (Penicilliumoxalicum) WAF6, preservation
Number is ACCC 32731, biological nature are as follows: colony edge is white, velvet shape；Surface raw dirty-green spore, the spore
It is easy to fall off；Most suitable culture pH is 6.5, and most suitable cultivation temperature is 28 DEG C；It is cultivated 3~5 days under conditions of 25~28 DEG C of solid fermentation
Spore can be produced；It can be grown using glucose, sucrose, wheat bran or stalk as microbe carbon source；It can be with peptone, yeast extract or jade
Rice flour is that microorganism nitrogen source is grown.
Embodiment 3: the cadmium ion tolerance evaluation of bacterial strain WAF6
The cadmium ion tolerance evaluation method of bacterial strain WAF6 of the present invention, specifically includes the following steps:
(1) prepare the PDA culture medium containing different concentration of cadmium ions, concentration is set as 0,10,50,80,100,150,
200mg/L；
(2) bacteria cake is made with 5mm punch in bacterial strain, is aseptically inoculated in step (1) described plating medium,
It is cultivated 3-5 days in 26-28 DEG C of constant incubator, observes the growing state of bacterial strain.
Bacterial strain WAF6 growing state on the plate for the concentration of cadmium ions that concentration of cadmium ions is 0~150mg/L is good, and
It grows on the plate of 200mg/L and is suppressed completely, therefore the concentration of cadmium ions tolerance range of the bacterial strain is 0-150mg/L.
Embodiment 4: the degraded cellulose merit rating of bacterial strain WAF6
Specifically includes the following steps:
(1) primary dcreening operation of bacterial strain: the fungal bacterial strain spore suspension isolated and purified is coated on PDA, in 28 DEG C of perseverances
It is cultivated 2-3 days in warm incubator, uniformly to the growth of media surface bacterium colony, bacteria cake is made with 5mm punch and is inoculated in the Congo
It on red culture medium, is placed in 28 DEG C of constant incubators and cultivates 3-5 days, whether have hydrolysis, and measure if observing around bacteria cake
Colony diameter (d) and hydrolytic circle (D)；
(2) measurement of carboxymethylcelluloenzyme enzyme (CMCase) living:
1) Specification Curve of Increasing: drying to constant weight under the conditions of 80 DEG C for DEXTROSE ANHYDROUS, and being prepared into concentration is 1.0mg/
The glucose standard of mL is separately added into glucose standard 0,0.2,0.4,0.6,0.8,1.0mL in 6 clean tubes, mends
Distilled water is added boiling water bath 5min after 1.5mLDNS reagent, is settled to 25mL after cooling, measures OD under wavelength 540nm to 2.0mL
Value draws standard curve using Excel.
2) crude enzyme liquid preparation and enzyme activity determination: seed liquor is prepared using the fungal bacterial strain that primary dcreening operation obtains, by 10% inoculation
Amount is connected in secondary screening culture medium, and liquid amount is 90mL/250mL triangular flask, 28 DEG C of constant-temperature shaking cultures, respectively 3,5,7,9,11
It takes fermentation liquid 1.5mL in centrifuge tube, and 10000r/min centrifugation 10min obtains crude enzyme liquid, measures bacterial strain in different fermentations
Between under carboxymethylcelluloenzyme enzyme (CMCase) vigor (enzyme activity determination method reference literature " Li Jing, Zhang Hanneng, the high such as Zhao's Chong
Imitate cellulose-degrading bacteria separation screening, composite microbial system building and straw degradative effect analysis apply with environmental organism journal,
2016,22(4):0689-0696.).Measure the time of occurrence of post analysis record highest enzyme activity.
As shown in table 1, in order to illustrate the effect of bacterial strain more of the present invention, table 1 provides the degraded cellulose of different strains
Indexes of capability evaluation.The ratio size of hydrolytic circle (D) and colony diameter (d) can only qualitative performance's bacterial strain there is CMC-Na
Capacity of decomposition, and enzyme activity size is then the quantitative target for determining strains for degrading cellulose ability；As shown in Table, bacterial strain WAF6
Highest enzyme activity value is maximum, and the required time for reaching highest enzyme activity is most short, i.e., higher with respect to other bacterial strain decomposition efficiencies.
Each strains for degrading cellulose merit rating of table 1.
Embodiment 5: application of the bacterial strain WAF6 in rice straw degradation
Specifically includes the following steps:
(1) disintegration effect of the bacterial strain to rice straw: rice straw is cut into about 0.5cm*3cm strip with scissors, by water
Rice straw substitutes carbon source (CMC-Na) in secondary screening culture medium, and seed liquor is connected to by 0.5g rice straw/bottle by 10% inoculum concentration
Liquid amount is in 45mL/250mL triangular flask, and 28 DEG C, constant-temperature shaking culture under the conditions of 120r/min, routine observation rice straw collapse
Solution situation simultaneously records.
(2) degradation effect of the bacterial strain to rice straw: crossing 40 meshes after rice straw is crushed, rice straw is substituted multiple
Carbon source (CMC-Na) in culture medium is sieved, 2.0g rice straw/bottle, seed liquor, which is connected to liquid amount, by 10% inoculum concentration is
In 90mL/250mL triangular flask, to access 10mL distilled water as control, 28 DEG C, constant-temperature shaking culture under the conditions of 120r/min, 25
After it, culture 5000r/min is centrifuged 10min, supernatant is abandoned, is cleaned 3-5 times repeatedly with distilled water, 80 DEG C are dried to perseverance
Weight calculates weight-loss ratio.
If strains tested is as shown in table 2 to rice straw disintegration effect, strains tested is inoculated in the training of rice straw liquid
After supporting base culture 20 days, bacterial strain WF5 and F19 are respectively at first 9 days, 7 days, and rice straw is without apparent metamorphosis；And from the 5th
It starts, and rice straw is gradually degraded under bacterial strain WAF6 effect, and in 15 days, can degrade most stalks.Different bacterium
To the disintegration effect of rice straw as shown in Figure 1, under bacterial strain WAF6 effect, rice straw is substantially degraded for strain.
2. rice straw of table is disintegrated degree
Note :+: stalk is degraded less；++: stalk is degraded half；+++: stalk is degraded substantially；++++: stalk is complete
Degradable
As shown in Fig. 2, 3 kinds of bacterial strains for examination have certain degradation to rice straw, the present embodiment bacterial strain is utilized
Obtain to the calculation method in the degradation effect of rice straw about weight-loss ratio: the degradation rate highest of bacterial strain WAF6 is
45.72%, the degradation rate of bacterial strain WF5, F19 are respectively 18.55%, 13.58%.
Embodiment 6: bacterial strain WAF6 is in the application in cadmium pollution soil is administered
Specifically includes the following steps:
1) by different strains testeds by solid fermentation, drying, crushing and etc. obtain spore pulvis, and detection level；
2) it will be air-dried for examination soil natural, remove the sundries such as plant roots and stems, it is spare to pulverize 80 meshes of sieving；
3) it is dispensed for examination soil, dress 100g cadmium pollution soil in 250mL beaker adds conidia powder by different disposal, and adjust
Inoculum concentration makes each fungal spore content 1 × 10 in soil6~1 × 107CFU/g, while control group is set；
4) soil incubation, each processing addition 100mL water are simultaneously stirred evenly with glass bar, put at room temperature cultivation 10 naturally~
50 days；
5) the 0th, 10,20,30d be measured by sampling effective cadmium content of soil, after every sub-sampling, be added equivalent amount of water with
Ensure soil moisture regime；
As shown in figure 3, with the extension of soil incubation time, effective cadmium content in soil was gradually reduced, at the 30th day
Afterwards, compared to initial effective cadmium content, the adsorption rate of bacterial strain has reached 76.26%, illustrates bacterial strain in the present invention in the soil
With good colonization ability, and to provide available microbial resources in heavy metal pollution repairing and treating.
The above is a preferred embodiment of the present invention, it is noted that for those skilled in the art
For, without departing from the principles of the present invention, it can also make several improvements and retouch, these improvements and modifications
It should be regarded as protection scope of the present invention.
Claims (10)
1. a kind of degraded cellulose, the penicillium oxalicum bacterial strain for adsorbing cadmium, which is characterized in that the classification of the penicillium oxalicum bacterial strain is ordered
Entitled penicillium oxalicum (Penicilliumoxalicum) WAF6, deposit number are ACCC 32731, biological nature are as follows: bacterium colony
Edge is white, velvet shape；Surface raw dirty-green spore, the spore it is easy to fall off；Most suitable culture pH is 6.5, most suitable culture
Temperature is 28 DEG C；Spore can be produced by cultivating 3~5 days under conditions of 25~28 DEG C of solid fermentation；Can with glucose, sucrose, wheat bran or
Stalk is that microbe carbon source is grown；It can be grown by microorganism nitrogen source of peptone, yeast extract or corn flour.
2. penicillium oxalicum bacterial strain according to claim 1, which is characterized in that the concentration of cadmium ions of the penicillium oxalicum bacterial strain is resistance to
It is 0~150mg/L by range.
3. a kind of application of penicillium oxalicum bacterial strain as claimed in claim 1 or 2 in rice straw degradation.
4. application according to claim 3, which comprises the steps of:
(1) rice straw fluid nutrient medium is prepared；
(2) the penicillium oxalicum bacterial strain is prepared into seed liquor, is connected in rice straw fluid nutrient medium by 10% inoculum concentration,
Shaken cultivation 5~30 days under 28 DEG C, the revolving speed of 120r/min.
5. application according to claim 4, which is characterized in that the rice straw fluid nutrient medium by CMC-Na,
KH2PO4、MgSO4·7H2O, NaCl, peptone, yeast extract, distilled water and rice straw composition.
6. application according to claim 4, which is characterized in that the rice straw fluid nutrient medium includes following parts by weight
Component: 10 parts of CMC-Na, KH2PO41 part, MgSO4·7H20.5 part of O, 0.5 part of NaCl, 2 parts of peptone, yeast extract 0.5
10 parts of rice straw after part, 1000 parts of distilled water, and shearing.
7. application according to claim 3, which is characterized in that evaluation penicillium oxalicum bacterial strain is to rice straw degradation effect packet
It includes and the rice straw solution after step (2) culture is centrifuged, is cleaned and drying and processing, calculate rice straw weight-loss ratio.
8. a kind of application of penicillium oxalicum bacterial strain as claimed in claim 1 or 2 in cadmium pollution soil improvement.
9. application according to claim 8, which comprises the steps of:
(1) the penicillium oxalicum bacterial strain is obtained into conidia powder after solid fermentation, drying and crushing；
(2) spare after drying, clean by cadmium pollution soil, pulverizing, being sieved；
(3) conidia powder obtained by step (1) is added step (2) treated in cadmium pollution soil, make soil miospore content 1
×106~1 × 107CFU/g is cultivated 10~50 days naturally at room temperature after adding water and stirring.
10. application according to claim 9, which is characterized in that the penicillium oxalicum bacterial strain after step (3) is cultivated 30 days naturally
To every adsorption rate be more than 76%.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111534439A (en) * | 2020-04-03 | 2020-08-14 | 河北科技大学 | Penicillium oxalicum SDF-25 strain and application thereof |
| CN113333459A (en) * | 2021-06-08 | 2021-09-03 | 湘潭大学 | Solid fermentation of penicillium oxalicum and method for efficiently dissolving phosphorus and removing lead by using same |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103667069A (en) * | 2013-07-11 | 2014-03-26 | 南京农业大学 | Agricultural straw degrading fungus penicillium oxalicum NJGZ-2 and fungal agent thereof |
| CN105296363A (en) * | 2015-10-28 | 2016-02-03 | 南京农业大学 | Oxalic acid penicillium strain NJDL-03 and application thereof |
| CN106434386A (en) * | 2016-11-24 | 2017-02-22 | 郑州大学 | Microorganism and microbial inoculum for degrading straws as well as preparation method and application of microbial inoculum |
-
2019
- 2019-08-05 CN CN201910717927.XA patent/CN110343623A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103667069A (en) * | 2013-07-11 | 2014-03-26 | 南京农业大学 | Agricultural straw degrading fungus penicillium oxalicum NJGZ-2 and fungal agent thereof |
| CN105296363A (en) * | 2015-10-28 | 2016-02-03 | 南京农业大学 | Oxalic acid penicillium strain NJDL-03 and application thereof |
| CN106434386A (en) * | 2016-11-24 | 2017-02-22 | 郑州大学 | Microorganism and microbial inoculum for degrading straws as well as preparation method and application of microbial inoculum |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111534439A (en) * | 2020-04-03 | 2020-08-14 | 河北科技大学 | Penicillium oxalicum SDF-25 strain and application thereof |
| CN111534439B (en) * | 2020-04-03 | 2022-05-17 | 河北科技大学 | Penicillium oxalicum SDF-25 strain and application thereof |
| CN113333459A (en) * | 2021-06-08 | 2021-09-03 | 湘潭大学 | Solid fermentation of penicillium oxalicum and method for efficiently dissolving phosphorus and removing lead by using same |
| CN113333459B (en) * | 2021-06-08 | 2022-06-03 | 湘潭大学 | Solid fermentation of penicillium oxalicum and method for efficiently dissolving phosphorus and removing lead by using same |
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