CN101280278A - Separation method and application of algicadal bacteria - Google Patents

Separation method and application of algicadal bacteria Download PDF

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Publication number
CN101280278A
CN101280278A CNA2008100584163A CN200810058416A CN101280278A CN 101280278 A CN101280278 A CN 101280278A CN A2008100584163 A CNA2008100584163 A CN A2008100584163A CN 200810058416 A CN200810058416 A CN 200810058416A CN 101280278 A CN101280278 A CN 101280278A
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algae
separation method
separation
molten phycomycete
bacterial strain
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赖泳红
崔晓龙
文孟良
王海玉
李一青
赵江源
彭谦
李欣毅
葛娟
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Yunnan University YNU
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Abstract

The invention relates to an algae-lysing bacteria separation method and the application thereof. The separation method adopts that firstly, the solid separation culture medium of algae-lysing bacteria is configured; secondly, a group of environmental samples containing the algae-lysing bacteria are diluted to one tenth to form concentration gradient, 0.1-0.2ul of each concentration of the samples is respectively taken and coated on the petri dish of the solid culture medium, to culture to form colonies, and then single colony is selected to perform separation and purification to obtain the purified bacterial strain; thirdly, the liquid cultural medium is prepared; fourthly, the purified bacterial strain is inoculated in the liquid cultural medium according to 2 to 30 percent inoculum size to enlarge the culture to obtain the algae-lysing bacteria liquid. The algae-lysing bacteria liquid is applied in the water body polluted by the blue algae to be used for treating the blue algae plankton blooms. The algae-lysing bacteria separation method can ensure that the algae-lysing bacteria with better algae-lysing effect can be effectively obtained, the new method of obtaining algae-lysing bacteria is provided, and the usage of algae-lysing bacteria used for treating the blue algae plankton blooms is also provided.

Description

The separation method of molten phycomycete and application
Technical field
The invention belongs to the new purposes of the method for screening and separating and the molten phycomycete of microbe to screen method and applied technical field, particularly molten phycomycete.
Background technology
Molten phycomycete (algicidal bacteria) typically refers to can cracking or suppress the mushrooms such as bacterium, actinomycetes and fungi of algal tufa.The molten algae of taking from the molten phycomycete of inhomogeneity of varying environment mainly has following characteristic: the molten phycomycete of the first kind is the cracking blue-green algae fully, the molten phycomycete of second class can kill blue-green algae but can not degrade, there are nutrient competition in molten phycomycete of the 3rd class and blue-green algae, the ability that bacterium obtains nutrition is better than algae, thereby suppress the breeding of blue-green algae, can not cracking or the degraded blue-green algae, so algae still exists, just quantity does not increase.
Molten phycomycete in environment quantity seldom, the method for screening and separating of existing molten phycomycete carries out enrichment culture normally 1. with wawter bloom and blue-green algae solution co-cultivation to molten algae bacterium; 2. with 10 times of dilution-plate methods molten algae liquid is coated on the beef extract-peptone solid plate substratum again, carries out bacterium and separate; 3. get single bacterium colony line purifying respectively; 4. get the good single bacterium colony shake-flask culture of purifying, get the bacterium culture and carry out the experiment of molten algae, can molten algae be molten phycomycete.Adopt this method to screen molten phycomycete, it is many to require efforts, and the time is long, and is difficult for obtaining molten algae effect bacterial classification preferably.
At present, blue-green alga bloom causes serious environmental to pollute to the whole world.And the method for existing improvement blue-green alga bloom mainly is physics, chemistry and biological method.Physical method mainly comprises methods such as mechanical removal method, ultraviolet removing method, ultrasonic wave removing method and ozone removing method.Chemical process is employing multiple organic acid, aldehyde, ketone compounds suppresses or directly kill frustule, comprises that mainly copper agent is removed method, herbicide spraying is removed method, and adsorbs condense water body ammonia nitrogen, H with mineral substance such as zeolite powder, salt, high ferro, Gao Mei 2S, NO 2Reach materials such as bacterial pathogens, with improvement substrate and water body.Biological method mainly is to utilize in the ecosystem restriction or checking relation in five elements relation of mutual promotion between the biology to control or suppress the method for algal grown, and conventional biological method has stocked carp, plants higher plant, puts hydrophyta natantia etc.Physical method efficient is low, time and effort consuming, cost height.Chemical process often causes secondary pollution of water.Biological method treatment cost height, the required cycle is long, and floor space is big.
Studies show that molten phycomycete is the important component part of biotic population 26S Proteasome Structure and Function in the aquatic ecosystem, can regulate the primary productivity in the aquatic ecosystem, it is the important factor that influences occurring in nature algae population checker, have vital role to keeping algae bio amount balance, also disappearing rapidly of harmful algae wawter bloom played an important role.The report of present molten phycomycete is less, and the research of administering blue-green alga bloom is still at the experimental stage.
Summary of the invention
The objective of the invention is to solve the deficiency that above-mentioned prior art exists and a kind of molten algae effect novel method of molten phycomycete preferably that can efficiently obtain is provided, the present invention also provides the purposes of molten phycomycete.
The objective of the invention is to be achieved through the following technical solutions.
A kind of separation method of molten phycomycete, separate according to the following steps:
1. prepare the solid separation culture medium of molten phycomycete by following proportioning: (NH 4) 2SO 40.1-2.5g, yeast powder 1-5g, pectin powder 0.5-20g, K 2PO 41-5g, MgSO 40.5-2.5g, MnSO 4H 2O 0.01-0.035g, FeSO 4.7H 2O 0.0015-0.01g, NaCl 0.3-3g, agar 15-20g, H 2O800-1200ml;
One group of environmental sample that 2. will contain molten phycomycete forms concentration gradient with 10 times of dilutions, and the sample 0.1-0.2ul that gets each concentration respectively is coated on the solid medium plate, cultivates to form bacterium colony, chooses single bacterium colony separation and purification then and obtains the purifying bacterial strain;
3. obtaining liq substratum;
4. enlarged culturing: the inoculum size of the purifying bacterial strain being pressed 2-30% inserts liquid nutrient medium, and the pH=value of liquid nutrient medium is 6.5-8.5, and culture temperature is 25-50 ℃, and the incubation time bacterium is 24-72hr, and fungi is 48-96hr, and actinomycetes are 5-8 days.
The present invention can carry out culture presevation with standby with the purifying bacterial strain.
The purifying bacterial strain that separation and purification of the present invention obtains is bacterium or fungi or actinomycetes.
The inhomogeneous purifying bacterial strain that correspondence obtains, liquid nutrient medium of the present invention are bacteria culture medium or fungi culture medium or actinomycetes substratum; Bacteria culture medium is prepared by following proportioning: extractum carnis 2-4g, peptone 5-10g, yeast powder 0.5-1g, sugared 9-11g, agar powder 15～20g, H 2O 800-1200ml.Fungi culture medium is by following proportioning preparation: K 2HPO 41-2g, KH 2PO 41-5g, MgSO 40.5-1.5g, peptone 4-6g, glucose 9-11g, agar 15-20g, H 2O 800-1200ml.The actinomycetes substratum is prepared by following proportioning: Zulkovsky starch 15-25g, NaCl0.5-1g, KNO 31-1.5g K 2HPO 4.3H 2O 1-3g, MgSO 4.7H 2O 0.5-1g, FeSO 4.7H 2O 0.005-0.1g, agar 15-25g, H 2O 800-1200ml.
The present invention is applied to administer blue-green alga bloom with molten phycomycete.
Compared with prior art the present invention has following distinguishing feature: can obtain a large amount of molten phycomycetes in the short period of time.Can obtain dissimilar molten phycomycetes such as fungi, actinomycetes and bacterium, the bacterial strain that separation obtains all has molten algae or effect of algae restraint in various degree more than 90%.The inventive method is not only applicable to the screening of the molten phycomycete of fresh water, is applicable to the screening of the molten phycomycete in ocean yet.
Embodiment
Below in conjunction with embodiment the present invention is specifically described.
Embodiment 1: divide the exsolution phycomycete according to the following steps:
1. prepare the solid separation culture medium of molten phycomycete by following proportioning: (NH 4) 2SO 40.1g, yeast powder 1g, pectin powder 0.5g, K 2PO 41g, MgSO 40.5g, MnSO 4H 2O0.01g, FeSO 4.7H 2O 0.0015g, NaCl 0.3g, agar 15g, H 2O 800ml.
One group of environmental sample that 2. will contain molten phycomycete forms concentration gradient with 10 times of dilution-plate methods, and the sample 0.1ul that gets each concentration respectively is coated on the solid medium plate, cultivates and forms bacterium colony, chooses single bacterium colony separation and purification then and obtains the purifying bacterial strain.During the selection environment sample, selection contains the sample of the molten phycomycete of a class of cleavable blue-green algae.
3. prepare inhomogeneous liquid nutrient medium, liquid nutrient medium can adopt microbial technology field liquid nutrient medium commonly used, also can be respectively applied for culturing bacterium, fungi or actinomycetic bacteria culture medium, fungi culture medium or actinomycetes substratum by following proportioning preparation:
The proportioning of bacteria culture medium: extractum carnis 2g, peptone 5g, yeast powder 0.5g, sugared 9g, agar powder 15g, H 2O800ml.
The proportioning of fungi culture medium: K 2HPO 41g, KH 2PO 41g, MgSO 40.5g, peptone 4g, glucose 9g, agar 15g, H 2O 800ml.
The proportioning of actinomycetes substratum: Zulkovsky starch 15g, NaCl 0.5g, KNO 31g K 2HPO 4.3H 2O 1g, MgSO 4.7H 2O 0.5g, FeSO 4.7H 2O 0.005g, agar 15g, H 2O 800ml.
4. enlarged culturing: the purifying bacterial strain is inserted liquid nutrient medium by 2% inoculum size.When bacterial strain is bacterium, insert bacteria culture medium; When bacterial strain is fungi, insert fungi culture medium; When bacterial strain is actinomycetes, insert the actinomycetes substratum.The pH=value of liquid nutrient medium is 6.5, and culture temperature is 25 ℃, and the incubation time bacterium is 24hr, and fungi is 48hr, and actinomycetes are 5 days.
As required, separate the purifying bacterial strain that obtains and directly to insert liquid nutrient medium, also can carry out culture presevation with standby.
The molten phycomycete liquid that above-mentioned enlarged culturing is obtained is applied in the water body that is subjected to the blue-green algae pollution, with the cracking blue-green alga bloom, reaches the effect of administering blue-green algae.
Embodiment 2: divide the exsolution phycomycete according to the following steps:
1. prepare the solid separation culture medium of molten phycomycete by following proportioning: (NH 4) 2SO 42.5g, yeast powder 5g, pectin powder 20g, K 2PO 45g, MgSO 42.5g, MnSO 4H 2O 0.035g, FeSO 4.7H 2O 0.01g, NaCl 3.0g, agar 20g, H 2O 1200ml;
One group of environmental sample that 2. will contain molten phycomycete forms concentration gradient with 10 times of dilution-plate methods, and the sample 0.2ul that gets each concentration respectively is coated on the solid medium plate, cultivates and forms bacterium colony, chooses single bacterium colony separation and purification then and obtains the purifying bacterial strain;
3. press bacteria culture medium or the fungi culture medium or the actinomycetes substratum of following proportioning obtaining liq:
Bacteria culture medium: extractum carnis 4g, peptone 10g, yeast powder 1g, sugared 11g, agar powder 20g, H 2O 1200ml.
Fungi culture medium: K 2HPO 42g, KH 2PO 45g, MgSO 41.5g, peptone 6g, glucose 11g, agar 20g, H 2O 1200ml.
Actinomycetes substratum: Zulkovsky starch 25g, NaCl 1g, KNO 31.5g K 2HPO 4.3H 2O 3g, MgSO 4.7H 2O 1g, FeSO 4.7H 2O 0.01g, agar 25g, H 2O 1200ml.
4. enlarged culturing: the purifying bacterial strain is inserted liquid nutrient medium by 30% inoculum size, and the pH=value of liquid nutrient medium is 8.5, and culture temperature is 50 ℃, and the incubation time bacterium is 72hr, and fungi is 96hr, and actinomycetes are 8 days.
The molten phycomycete liquid that above-mentioned enlarged culturing is obtained is applied in the water body that is subjected to the blue-green algae pollution, with the cracking blue-green alga bloom, reaches the effect of administering blue-green algae.
Embodiment 3: divide the exsolution phycomycete according to the following steps:
1. prepare the solid separation culture medium of molten phycomycete by following proportioning: (NH 4) 2SO 41g, yeast powder 3g, pectin powder 10g, K 2PO 44g, MgSO 41g, MnSO 4H 2O 0.02g, FeSO 4.7H 2O0.05g, NaCl 2g, agar 18g, H 2O 1000ml;
One group of environmental sample that 2. will contain molten phycomycete forms concentration gradient with 10 times of dilution-plate methods, and the sample 0.15ul that gets each concentration respectively is coated on the solid medium plate, cultivates and forms bacterium colony, chooses single bacterium colony separation and purification then and obtains the purifying bacterial strain;
3. press bacteria culture medium or the fungi culture medium or the actinomycetes substratum of following proportioning obtaining liq:
Bacteria culture medium: extractum carnis 3g, peptone 8g, yeast powder 0.8g, sugared 10g, agar powder 18g, H 2O1000ml.
Fungi culture medium: K 2HPO 41.5g, KH 2PO 43g, MgSO 41g, peptone 5g, glucose 10g, agar 18g, H 2O 1000ml.
Actinomycetes substratum: Zulkovsky starch 20g, NaCl 0.8g, KNO 31.2g K 2HPO 4.3H 2O 2g, MgSO 4.7H 2O 0.8g, FeSO 4.7H 2O 0.03g, agar 20g, H 2O 1000ml.
4. enlarged culturing: the purifying bacterial strain of preservation is inserted liquid nutrient medium by 10% inoculum size, and the pH=value of liquid nutrient medium is 7, and culture temperature is 30 ℃, and the incubation time bacterium is 48hr, and fungi is 70hr, and actinomycetes are 6 days.
The molten phycomycete liquid that above-mentioned enlarged culturing is obtained is applied in the water body that is subjected to the blue-green algae pollution, with the cracking blue-green alga bloom, reaches the effect of administering blue-green algae.
Embodiment 4: divide the exsolution phycomycete according to the following steps:
1. prepare the solid separation culture medium of molten phycomycete by following proportioning: (NH 4) 2SO 42g, yeast powder 3g, pectin powder 15g, K 2PO 41.0g, MgSO 42g, MnSO 4H 2O 0.035g, FeSO 4.7H 2O 0.008g, NaCl 0.8g, agar 15g, H 2O 1200ml;
One group of environmental sample that 2. will contain molten phycomycete forms concentration gradient with 10 times of dilution-plate methods, and the sample 0.2ul that gets each concentration respectively is coated on the solid medium plate, cultivates and forms bacterium colony, chooses single bacterium colony separation and purification then and obtains the purifying bacterial strain;
3. press bacteria culture medium or the fungi culture medium or the actinomycetes substratum of following proportioning obtaining liq:
Bacteria culture medium: extractum carnis 4g, peptone 8g, yeast powder 0.5g, sugared 9g, agar powder 20g, H 2O1000ml.
Fungi culture medium: K 2HPO 42g, KH 2PO 44g, MgSO 41.2g, peptone 4g, glucose 10g, agar 15g, H 2O 800ml.
Actinomycetes substratum: Zulkovsky starch 15g, NaCl 0.5g, KNO 31.5g K 2HPO 4.3H 2O 2g, MgSO 4.7H 2O 1g, FeSO 4.7H 2O 0.05g, agar 25g, H 2O 1000ml.
4. enlarged culturing: the purifying bacterial strain of preservation is inserted liquid nutrient medium by 18% inoculum size, and the pH=value of liquid nutrient medium is 7.5, and culture temperature is 50 ℃, and the incubation time bacterium is 50hr, and fungi is 70hr, and actinomycetes are 6 days.
The molten phycomycete liquid that above-mentioned enlarged culturing is obtained is applied in the water body that is subjected to the blue-green algae pollution, with the cracking blue-green alga bloom, reaches the effect of administering blue-green algae.
Embodiment 5: divide the exsolution phycomycete according to the following steps:
1. prepare the solid separation culture medium of molten phycomycete by following proportioning: (NH 4) 2SO 41.5g, yeast powder 2g, pectin powder 12g, K 2PO 43.5g, MgSO 42g, MnSO 4H 2O 0.001g, FeSO 4.7H 2O 0.005g, NaCl1g, agar 17g, H 2O 1000ml;
One group of environmental sample that 2. will contain molten phycomycete forms concentration gradient with 10 times of dilution-plate methods, and the sample 0.12ul that gets each concentration respectively is coated on the solid medium plate, cultivates and forms bacterium colony, chooses single bacterium colony separation and purification then and obtains the purifying bacterial strain;
3. by following proportioning obtaining liq substratum, liquid nutrient medium is bacteria culture medium or fungi culture medium or actinomycetes substratum:
Bacteria culture medium: extractum carnis 3.5g, peptone 9g, yeast powder 0.7g, sugared 11g, agar powder 15g, H 2O900ml.
Fungi culture medium: K 2HPO 41.8g, KH 2PO 42g, MgSO 41g, peptone 5g, glucose 9g, agar 16g, H 2O 900ml.
Actinomycetes substratum: Zulkovsky starch 22g, NaCl 1g, KNO 31.5g K 2HPO 4.3H 2O 2g, MgSO 4.7H 2O 0.5g, FeSO 4.7H 2O 0.08g, agar 20g, H 2O 900ml.
4. enlarged culturing: the purifying bacterial strain of preservation is inserted liquid nutrient medium by 15% inoculum size, and the pH=value of liquid nutrient medium is 8, and culture temperature is 30 ℃, and the incubation time bacterium is 65hr, and fungi is 80hr, and actinomycetes are 7 days.
The molten phycomycete liquid that above-mentioned enlarged culturing is obtained is applied in the water body that is subjected to the blue-green algae pollution, with the cracking blue-green alga bloom, reaches the effect of administering blue-green algae.
Above embodiment, with the algae liquid of solubilization phycomycete liquid not in contrast.Observe algae liquid colour-change every day, whether yellow of frond, whether aggegation.Through carrying out molten algae effect detection, the bacterial strain that separation obtains all has molten algae effect in various degree more than 90%.Detect by an unaided eye, also do not observe the chlorophyll of frustule, examining under a microscope frustule quantity is 0 substantially, places and does not also see frustule breeding reproduction in 2 months.

Claims (7)

1, a kind of separation method of molten phycomycete is characterized in that, separates according to the following steps:
1. prepare the solid separation culture medium of molten phycomycete by following proportioning: (NH 4) 2SO 40.1-2.5g, yeast powder 1-5g, pectin powder 0.5-20g, K 2PO 41-5g, MgSO 40.5-2.5g, MnSO 4H 2O 0.01-0.035g, FeSO 4.7H 2O 0.005-0.1g, NaCl 0.3-3g, agar 15-20g, H 2O800-1200ml;
One group of environmental sample that 2. will contain molten phycomycete forms concentration gradient with 10 times of dilutions, and the sample 0.1-0.2ul that gets each concentration respectively is coated on the solid medium plate, cultivates to form bacterium colony, chooses single bacterium colony separation and purification then and obtains the purifying bacterial strain;
3. obtaining liq substratum;
4. enlarged culturing: the inoculum size of the purifying bacterial strain being pressed 2-30% inserts liquid nutrient medium, and the pH=value of liquid nutrient medium is 6.5-8.5, and culture temperature is 25-50 ℃, and the incubation time bacterium is 24-72hr, and fungi is 48-96hr, and actinomycetes are 5-8 days.
2, the separation method of a kind of molten phycomycete according to claim 1 is characterized in that, the purifying bacterial strain is carried out culture presevation.
3, the separation method of a kind of molten phycomycete according to claim 1 is characterized in that, the purifying bacterial strain that separation and purification obtains is bacterium or fungi or actinomycetes.
4, the separation method of a kind of molten phycomycete according to claim 1 is characterized in that, described liquid nutrient medium is a bacteria culture medium, prepares by following proportioning: extractum carnis 2-4g, peptone 5-10g, yeast powder 0.5-1g, sugared 9-11g, agar powder 15～20g, H 2O 800-1200ml.
5, the separation method of a kind of molten phycomycete according to claim 1 is characterized in that, described liquid nutrient medium is a fungi culture medium, by following proportioning preparation: K 2HPO 41-2g, KH 2PO 41-5g, MgSO 40.5-1.5g, peptone 4-6g, glucose 9-11g, agar 15-20g, H 2O 800-1200ml.
6, the separation method of a kind of molten phycomycete according to claim 1 is characterized in that, described liquid nutrient medium is the actinomycetes substratum, prepares by following proportioning: Zulkovsky starch 15-25g, NaCl 0.5-1g, KNO 31-1.5g K 2HPO 4.3H 2O 1-3g, MgSO 4.7H 2O 0.5-1g, FeSO 4.7H 2O 0.005-0.1g, agar 15-25g, H 2O 800-1200ml.
7, molten phycomycete is used to administer the application of blue-green alga bloom.
CNA2008100584163A 2008-05-20 2008-05-20 Separation method and application of algicadal bacteria Pending CN101280278A (en)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101955904A (en) * 2010-10-25 2011-01-26 北京大学 Algicidal bacteria separation method in natural water environment
CN102660460A (en) * 2012-05-10 2012-09-12 同济大学 Method for screening non-photosynthetic high-efficiency carbon immobilization microorganism under aerobic conditions
CN103695342A (en) * 2013-12-12 2014-04-02 上海交通大学 Bacillus having alga-lysing activity and application thereof
CN109279700A (en) * 2018-09-06 2019-01-29 中海新世纪量子生物科技（深圳）有限责任公司 Administer leavening, composite bacteria agent, the Preparation method and use of blue algae polluted water body
CN112689669A (en) * 2018-09-14 2021-04-20 克亚诺斯生物技术公司 Method for cultivating a microorganism of interest and related apparatus
CN114698652A (en) * 2022-01-17 2022-07-05 中国科学院生态环境研究中心 Application of composition in preparation of algaecide, algaecide and in-situ algaecide method

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101955904A (en) * 2010-10-25 2011-01-26 北京大学 Algicidal bacteria separation method in natural water environment
CN102660460A (en) * 2012-05-10 2012-09-12 同济大学 Method for screening non-photosynthetic high-efficiency carbon immobilization microorganism under aerobic conditions
CN103695342A (en) * 2013-12-12 2014-04-02 上海交通大学 Bacillus having alga-lysing activity and application thereof
CN109279700A (en) * 2018-09-06 2019-01-29 中海新世纪量子生物科技（深圳）有限责任公司 Administer leavening, composite bacteria agent, the Preparation method and use of blue algae polluted water body
CN112689669A (en) * 2018-09-14 2021-04-20 克亚诺斯生物技术公司 Method for cultivating a microorganism of interest and related apparatus
CN114698652A (en) * 2022-01-17 2022-07-05 中国科学院生态环境研究中心 Application of composition in preparation of algaecide, algaecide and in-situ algaecide method

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