CN101139140A

CN101139140A - Aerugo microcapsule algae degradation method using animalcule

Info

Publication number: CN101139140A
Application number: CNA2007100451951A
Authority: CN
Inventors: 樊正球; 母锐敏; 王祥荣
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2007-08-23
Filing date: 2007-08-23
Publication date: 2008-03-12

Abstract

The present invention belongs to the technical field of environment protection, in particular to a method to degrade Microcystic aeruginosa through microorganisms. The method is that firstly an algae-lysing bacterial strain Brevibacillus spp.FDK2 can be achieved by separating from and sublimating deep eutrophication urban lake water body;the algae-lysing bacterial strain Brevibacillus spp.FDK2 is cultured by a culture medium to acquire bacterial liquid with a certain concentration. The bacterial liquid is added into a water sample containing Microcystic aeruginosa. Under a certain condition, the bacterial strain has a good degrading effect on the Microcystic aeruginosa. The degrading removing rate is more than 90 percent.

Description

A kind of method of utilizing the microbiological deterioration microcystic aeruginosa

Technical field

The invention belongs to environmental technology field, be specifically related to a kind of method of utilizing the microbiological deterioration microcystic aeruginosa.

Background technology

In recent years, along with developing rapidly of industrial or agricultural, especially the phosphorus-containing detergent consumption increases greatly, causes nutritive substance, as a large amount of dischargings such as nitrogen, phosphorus, causes algal bloom, is gathered into large stretch of wawter bloom (lake) and red tide (ocean) on the water surface.According to investigations, the lake of China more than 90% reached middle nutritional status or eutrophic state.Eutrophication can cause microorganism such as algae to breed in a large number, at first makes the water body top layer painted, causes look to pollute, particularly algae during the high-incidence season water surface be yellow-green colour, influence the photosynthesis of benthos.Algal bloom also can cause oxygen in water to reduce, fish kills, the eubiosis of destruction water body.In addition, eutrophication can cause negative impact to coming aquatic production art and pipe network and ductwork water quality.

Along with the aggravation of global body eutrophication, the outburst of harmful algae wawter bloom is increased, and environment that it causes and economic problems paid more and more attention are sought effective wawter bloom and the red-tide control approach is imperative.The main method of algae pollution improvement at present mainly contains micro-filtration, physical methods such as air supporting, and the chemical process and the biology that add oxygenant and algicide contact pretreated microbial method except that methods such as algaes.There is inefficiency in physical method, the shortcoming that cost is high; Though the chemical process instant effect often causes secondary pollution; And conventional Biological Pretreatment algae-removing method such as biological contact oxidation process construction investment are big, and operation is difficulty, and floor space is big, and it is also not satisfactory to remove the algae effect.Utilizing physics, chemistry and conventional biological method to administer under the not satisfactory situation of wawter bloom, it is imperative to seek a kind of effective more wawter bloom controlling way.Utilizing under physics, chemistry and the very unfavorable situation of other biological method improvement wawter bloom, molten algae bacterium, biology as wawter bloom and red-tide control, cause more and more people's concern, utilized the algae in the molten algae removal of bacteria eutrophication water to become a kind of new approaches that biological process removes algae.Therefore, separation and purification goes out to remove the molten algae bacterium of microcystic aeruginosa (Microcystis) (in the wawter bloom common reason algae) from natural water, has important more practical value for safe, fast and efficient improvement wawter bloom problem.

Summary of the invention

The purpose of this invention is to provide a kind of method of utilizing removal of microorganisms microcystic aeruginosa (in the wawter bloom common algae), to eliminate the problem of environmental pollutions that a large amount of breedings of microcystic aeruginosa bring in the current wawter bloom.

The present invention screens and obtains a strain has good molten algae effect to microcystic aeruginosa bacterium Brevibacillus spp.FDK2 from the natural lake water body of eutrophication, be kept at China Committee for Culture Collection of Microorganisms common micro-organisms center, No. 11, BeiYiTiao, ZhongGuanCun, Haidian District, BeiJing City, preservation date: on August 18th, 2007, deposit number: CGMCCNo.XXXXXX).Under temperature 25-35 ℃, pH 7.0-8.1 and 18h condition, this bacterial strain has higher degradation capability to microcystic aeruginosa.

The inventive method comprises the steps:

(1) screening of microorganism strains

As molten algae bacterium separation source, adopt spread plate and sectional streak method through purifies and separates repeatedly with degree of depth eutrophication city water body in lake, finishing screen is selected the bacterial strain with higher molten algae usefulness, numbering FDK2; It is inserted slant medium, in 4 ℃ of preservations of refrigerator; Bacteria culture medium adopts beef-protein medium.

This bacterial strain is aerobic, Gram-negative product genus bacillus, size is 0.8-1.0 * 2.8-4.0 μ m, gemma is oval, mostly central authorities give birth to bacterium colony and are nearly almond yellow, have simultaneously that sporangiocyst expands, the whole pH of peritrichous, hydrogen peroxide enzyme positive, V.P (acetyl methyl carbinol generations) feminine gender, V.P culture is 7.5, do not produce H ₂S and indoles, can gelatin hydrolysate, growth such as is suppressed at characteristics in 5%NaCl, can utilize glucose and N.F,USP MANNITOL to produce acid simultaneously, but not utilize pectinose and wood sugar to produce acid.

Use primer 2 7F (5 '-AGA GTT TGA TCM TGG CTC AG-3 ') and 1512R (5 '-ACG GCT ACCTTG TTA CGA CT-3 '), and use round pcr, amplification 16S rDNA fragment from total DNA of this bacterial strain.Detect through agarose gel electrophoresis, find respectively have a PCR characteristic band that is about the 1.5kb size, this theoretical estimated value with the 16S rDNA of this bacterial strain conforms to.Further utilize two-way primer order-checking, the sequence that finally obtains the 16S rDNA of bacterial strain is SEQ.ID.No.1

Through identifying that this bacterial strain is bacillus brevis Brevibacillus spp., called after Brevibacillus spp.FDK2.

(2) utilize bacterial strain Brevibacillus spp.FDK2 degrading to microcystic aeruginosa

To be inoculated into behind the storage medium actication of culture in the aseptic triangular flask of the 500mL that substratum is housed, after 12h was cultivated in concussion under 35 ℃, 200rpm condition, bacterium entered increased logarithmic phase, and (concentration is 3.0 * 10 with 50ml bacterium liquid ⁸Individual/as mL) to pour in the water sample that 200ml contains the verdigris Microcystis aeruginosa, under temperature 25-35 ℃, pH7.0-8.1 condition, bacterial strain Brevibacillusspp.FDK2 removes ability (is metric with the chlorophyll a clearance) to the higher degraded of having of microcystic aeruginosa, and has feature over time.The result shows that bacterial strain Brevibacillus spp.FDK2 has degradation effect preferably to microcystic aeruginosa, increases clearance with the reaction times and progressively increases, bacterial strain reaches the highest to the degradation efficiency of microcystic aeruginosa during to 18h, be 93%, after this then no longer change, maintain this level substantially.General degradation efficiency reaches more than 90%.

Brevibacillus spp.FDK2 to remove algae effect this variation characteristic in time relevant with the biological activity of molten algae bacterium.According to the growth curve of bacterium as can be known, this bacterium enters logarithmic phase about 10h, add the bacterium liquid that is tried algae liquid and be the bacterium liquid that is in increased logarithmic phase, this moment, the bacterial cell metabolic activity was the strongest, synthesize the fastest of novel substance, bacterial growth is vigorous, and cell quantity not only increases with geometricprogression, and the timed interval that the cell per minute splits once is the shortest.Bacterium is strong to the resistibility of poor environment.Therefore the removal speed of chlorophyll a is very fast.Thereafter bacterium is through entering decline phase stationary phase, and at this moment the bacterium mortality ratio increases, and viable count reduces, even death toll is greater than new aerobic plate count.The bacterium of decline phase breeds less or does not breed, or self-dissolving, and bacterial activity is low, so the clearance of chlorophyll a tends towards stability gradually.

Bacterial strain Brevibacillus spp.FDK2 is further studied, find this bacterial strain after membrane filtration, centrifugal settling, high-temperature inactivation and ultrasonication, Brevibacillus spp.FDK2 is still to the microcystic aeruginosa had strong inhibitory effects of having grown.Infer that thus Brevibacillus spp.FDK2 plays molten algae effect by secreting the outer material of a kind of special born of the same parents, and high temperature resistant, the high pressure of this secretory product, is non-protein matter.

Description of drawings

Fig. 1 is that chlorophyll-a concentration is over time after adding molten algae bacterium Brevibacillus spp.FDK2 in the algae-containing water.

Embodiment

Embodiment 1

Present embodiment is washed yarn lake degree of depth eutrophication water as molten algae bacterium separation source with Huang Xing park, Shanghai, adopts spread plate and sectional streak method through purifies and separates repeatedly, places 200mL liquid nutrient medium, 200rmin respectively with separating the 18 strain bacteriums that obtain ^-1, cultivate 2d down for 35 ℃, add 100mL then respectively to contain in the water sample of verdigris Microcystis aeruginosa, to investigate, compare the molten algae effect of each bacterium, finishing screen is selected the bacterial strain FD01 with higher molten algae usefulness.Through being accredited as bacillus brevis Brevibacillus spp., called after Brevibacillus spp.FDK2 inserts slant medium with it, in 4 ℃ of preservations of refrigerator.

Microbial culture adopts beef-protein medium.Wherein be constructed as follows in the 1000mL liquid nutrient medium: 1) extractum carnis 3.0g; 2) peptone 10.0g; 3) NaCl 5.0g; 4) H ₂O 1000ml; 5) pH 7.4-7.6.The 1000ml slant medium is constructed as follows: 1) extractum carnis 3.0g; 2) peptone 10.0g; 3) NaCl 5.0g; 3) H ₂O 1000ml; 4) agar 18.0g; 5) pH7.4-7.6.

With bacterial strain Brevibacillus spp.FDK2 in 35 ℃, cultivate 12h under the 200rpm condition after, by the volume ratio of 20-30% bacterium liquid is inoculated into and cultivates 25-35 days tried in the algae liquid (microcystic aeruginosa), bacterial concentration is 3.0 * 10 ₈Individual/ml, chlorophyll-a concentration is 396.52 μ g/L, establishes blank.In temperature is 35 ℃, and rotating speed is under the condition of 200rpm, measures the content of the chlorophyll a tried algae liquid, continuous monitoring 24h every 3h.Chlorophyll a (the μ g/L of unit) adopts the acetone extraction spectrophotometry, at 750nm, and 663nm, 645nm under the 630nm wavelength, by measuring the absorbancy of water sample, measures the content of its chlorophyll a.The result shows that chlorophyll a content is the fastest at preceding 9h fall off rate, and clearance is near 70%, and clearance reaches maximum value behind the 18h, is 93%, after this is stabilized in this level and no longer changes.

Sequence SEQ.ID.No.1:

ACTTATGCTAATACCGGGATAGGTTTTGGATTGCATGATCCGAAAAGAAAAGATGGCT

TCGGCTATCACTGGGAGATGGGCCTGCGGCGCATTAGCTAGTTGGTGGGGTAACGGC

CTACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGACCGGCCACACTGGGACT

GAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATTTTCCACAATGGAC

GAAAGTCTGATGGAGCAACGCCGCGTGAACGATGAAGGTCTTCGGATTGTAAAGTTC

TGTTGTCAGGGACGAACACGTGCCGTTCGAATAGGGCGGTACCTTGACGGTACCTGA

CGAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAG

CGTTGTCCGGATTTATTGGGCGTAAAGCGCGCGCAGGCGGCTATGTAAGTCTGGTGTT

AAAGCCCGGAGCTCAACTCCGGTTCGCATCGGAAACTGTGTAGCTTGAGTGCAGAA

GAGGAAAGCGGTATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACAC

CAGTGGCGAAGGCGGCTTTCTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGG

GAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAGGTG

TTGGGGGTTTCAATACCCTCAGTGCCGCAGCTAACGCAATAAGCACTCCGCCTGGGG

AGTACGCTCGCAAGAGTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTG

GAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCCG

CTGACCGCTCTGGAGACAGAGCTTCCCTTCGGGGCAGCGGTGACAGGTGGTGCATG

GTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCC

TTATCTTTAGTTGCCAGCATTCAGTTGGGCACTCTAGAGAGACTGCCGTCGACAAGAC

GGAGGAAGGCGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACAC

GTGCTACAATGGTTGGTACAACGGGATGCTACCTCGCGAGGGGACGCCAATCTCTGA

AAACCAATCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCT

AGTAAT

Claims

1. method of utilizing the microbiological deterioration microcystic aeruginosa is characterized in that concrete steps are as follows:

(1) screening microorganism strains

As molten algae bacterium separation source, adopt spread plate and sectional streak method through purifies and separates repeatedly with degree of depth eutrophication city water body in lake, finishing screen is selected the bacterial strain with higher molten algae usefulness, numbering FDK2; It is inserted slant medium, in 4 ℃ of preservations of refrigerator; Bacteria culture medium adopts beef-protein medium;

Through identifying that this bacterial strain is bacillus brevis Brevibacillus spp., called after Brevibacillus spp.FDK2; Microbial preservation is numbered CGMCC NO.XXXXX, and preservation date is on August 18th, 2007;

To be inoculated into behind the storage medium actication of culture in the aseptic triangular flask that substratum is housed, after 12h was cultivated in concussion under 35 ℃, 200rpm condition, bacterium entered increased logarithmic phase, is 3.0 * 10 with 40-60 ml concentration ⁸The bacterium liquid of individual/mL is poured in the water sample that 200ml contains the verdigris Microcystis aeruginosa, and temperature is 25-35 ℃, and the pH value is 7.0-8.1, and bacterial strain Brevibacillus spp.FDK2 has higher degraded to microcystic aeruginosa and removes ability, and has feature over time; This bacterial strain reaches more than 90% the degradation efficiency of microcystic aeruginosa.