CN105985917A - Method for improving biomass of chlorella in swine wastewater - Google Patents
Method for improving biomass of chlorella in swine wastewater Download PDFInfo
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- CN105985917A CN105985917A CN201510063872.7A CN201510063872A CN105985917A CN 105985917 A CN105985917 A CN 105985917A CN 201510063872 A CN201510063872 A CN 201510063872A CN 105985917 A CN105985917 A CN 105985917A
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Abstract
The invention provides a method for improving the biomass of chlorella in swine wastewater, belonging to the field of microbe application. Chlorella assigned with the accession number of CGMCC NO.9225 and paenibacillus elgii assigned with the accession number of CGMCC NO.7724 are co-cultured in the swine wastewater, the result shows that paenibacillus elgii can obviously improve the biomass of chlorella in the swine wastewater, and the promotion effect achieves 1.6 times. With the adoption of the method, the treatment problem of the swine wastewater is solved, and the environment pollution is reduced; due to the addition of paenibacillus elgii, the water body microenvironment in the swine wastewater is improved, the biomass of chlorella is obviously improved, further, the production cost of microalgae biodiesel is reduced, thus good economic benefit is obtained, and therefore, the method has a good market application prospect.
Description
Technical field
The invention belongs to microorganism utilization and bio-oil preparation field, specifically, relate to
A kind of improve chlorella method of Biomass in Pig raising wastewater.
Background technology
One of Biodiesel of most future that oil-rich microalgae industry has become as, and standby
Concerned.Microalgae has multiple advantage compared with other energy crops, such as low input high yield
Amount, does not strive ground, does not strives fertilizer with agriculture with agriculture, available waste gas, waste water, salt water growth, no
Need herbicide, insecticide, microalgae cell residue can be used for agricultural fertilizer or animal feed or
Continue to produce biogas or ethanol etc..
At present, animal excrements pollute further severe, not only result in body eutrophication,
Agricultural production is caused harm, and the living environment to people also creates negative effect.With
The day by day serious of environmental problem and energy problem, microdisk electrode combines with sewage disposal
Through becoming a kind of new trend.Pig raising wastewater (Liquid swine manure, LSM) contains
A large amount of pig excrements, rich in organic carbon source and such as other nutritional labelings such as nitrogen, phosphorus, are regarded
For high-concentration sewage, direct discharge can cause body eutrophication, polluted source.Additionally,
Pig raising wastewater carries substantial amounts of pathogen, distributes the denseest stink, day by day causes environment sternly
Heavily contaminated.Microalgae then has and utilizes the organic and inorganic pollution in sewage and converted
Ability for cellular component (such as lipid and carbohydrate).Microalgae is to produce biodiesel
Quality raw materials.The oil productivity of microalgae is 30-100 times of oil crop such as Semen sojae atricolor, for producing
Alternative fuel oil is laid a good foundation.Algae, using luminous energy as the energy, utilizes nitrogen, phosphorus etc. to seek
Support the organic matter that material synthesis is complicated.Therefore, algae can reduce the nitrogen in sewage, phosphorus contains
Amount.Nitrogen, the removal of phosphorus in sewage are served preferable effect.
But, the selection of algae kind is most important, and only specific algae kind can be at Pig raising wastewater
Middle growth.This is mainly due to ammonia nitrogen (NH in Pig raising wastewater3-N) excessive concentration and containing
Other noxious substances, and major part microalgae is the most sensitive to it, thus limit microalgae and exist
Growth in waste water.Research before shows the most a fraction of chlorella Chlorella
Sp., scenedesmus Scenedesmus sp. and green Euglena Euglena sp. can be applied to sewage disposal
In.Although it addition, some algae kinds can grow in Pig raising wastewater, but its Biomass is low
And fat content is low, relative to the acquisition of biodiesel uneconomical.It can be said that in order to allow
Microalgae produces more biodiesel in sewage need to 2 breakthroughs, and one i.e. selects properly
Algae kind and cultural method increase the Biomass of microalgae, it is second for increasing in microalgae cell
Oil content.
There are some researches show, microalgae can be the oxygen required for heterotrophic bacteria provides and Organic substance,
Carbon dioxide and nutrient (little dinitrogen, phosphorus) that meanwhile heterotrophic bacteria discharges are permissible
Utilize for micro algae growth.
Fixed nitrogen series bacillus belongs to azotobacter, and it is that a class can produce spore, anti-
Inverse property is strong, and the time-to-live is long, strong adaptability, widely distributed, it is possible to the microorganism of fixed nitrogen.
While fixed nitrogen series bacillus has nitrogen fixation, also can produce some and promote plant growing
Material and some antibiotic substance, can effectively improve Soil Micro-environment, promote plant growing
And the ability of enhancing Genes For Plant Tolerance disease, the many bacterial strains in this genus are promoted as plant rhizosphere
Raw bacterium and widely studied, but be all only limitted to macrophyte, the most not fixed nitrogen class spore bar
Bacterium is as the example of Promoting bacteria in microdisk electrode.
Summary of the invention
It is an object of the invention to provide and a kind of improve chlorella side of Biomass in Pig raising wastewater
Method.
Present invention firstly provides a kind of method improving chlorella (Chlorella sp.) Biomass,
It is in the culture medium cultivating chlorella, add series bacillus (Paenibacillus sp.).
The present invention provides a kind of and improves chlorella (Chlorella sp.) Biomass in Pig raising wastewater
Method, cultivate chlorella Pig raising wastewater in add series bacillus (Paenibacillus
sp.)。
In the said method of the present invention, described chlorella is 34-2, and its deposit number is
CGMCC NO.9225, this bacterial strain is deposited in China Microbiological on May 15th, 2014
CGMCC, address: Beijing (are called for short in culture presevation administration committee common micro-organisms center
North Star West Road, Chaoyang District 1 institute 3, Institute of Microorganism, Academia Sinica, postcode
100101), Classification And Nomenclature is chlorella Chlorella sp..
Described series bacillus is 18WLY, and its deposit number is CGMCC NO.7724,
This bacterial strain is deposited in Chinese microorganism strain preservation conservator on June 19th, 2013
(CGMCC, address: BeiChen West Road, Chaoyang District, BeiJing City 1 can be called for short in common micro-organisms center
Number institute 3, Institute of Microorganism, Academia Sinica, postcode 100101) preservation, classification life
Entitled Paenibacillus sp..
Described Pig raising wastewater be pig farm raise pigs produce waste water through dilution 3-6 times of volume after
The waste water arrived.Preferably, for the waste water obtained after dilution 5 times.Diluent is selected from tap water
Or deionized water.The purpose of dilution is to provide more preferable water body microenvironment for chlorella and make it
More fully accept illumination and carry out photosynthesis.
Raising chlorella (Chlorella sp.) Biomass in Pig raising wastewater that the present invention provides
In method, the initial inoculum of chlorella is 3 × 108-5×108Individual/ml Pig raising wastewater;Class
The initial inoculum of bacillus cereus is 1 × 10-4-2×10-4G/ml Pig raising wastewater.
Preferably, the initial inoculum of chlorella is 4.3 × 108Individual/ml Pig raising wastewater;Class bud
The initial inoculum of spore bacillus is 1.26 × 10-4G/ml Pig raising wastewater.
Further, in the inventive method, inoculate the dry weight ratio of series bacillus and micrococcus
For 15-20:1.Preferably, the dry weight of series bacillus and micrococcus is than for 18:1.
The condition of culture that series bacillus and micrococcus are co-cultured in Pig raising wastewater by the present invention
For 20-35 DEG C, in one day, illumination and interlunation are than for 14-18:6-10, cultivate 3-8 days.
Preferably, cultivating 3 days for 28 DEG C, in one day, ratio is for 16:8 illumination and dark time.Illumination
Optional natural light or artificial light source, when intensity of illumination is 5000lux, growth-promoting effect is obvious.
The said method of the present invention can be applied in preparing bio-oil.
Present invention also offers the series bacillus that deposit number is CGMCC NO.7724
(Paenibacillus sp.) and the microbial inoculum containing it are improving chlorella (Chlorella sp.) life
Application in thing amount.
The invention provides the series bacillus that deposit number is CGMCC NO.7724
(Paenibacillus sp.) and the microbial inoculum containing it are reducing answering during animal farm wastewater pollutes
With.
Series bacillus (Paenibacillus sp.) 18WLY (deposit number CGMCC
NO.7724) it is that applicant is at a strain Azobacter bacillus cereus, tool obtained by early stage screening
There is strong adaptability, the feature that nitrogen fixing capacity is strong, the product of the crops such as Semen Maydis can be effectively facilitated
Amount improves.Applicants have unexpectedly found that, this bacterium is trained altogether with chlorella Chlorella sp.34-2
Support after Pig raising wastewater, find that Paenibacillus sp.18WLY is at of short duration suppression Chlorella
After the growth of sp.34-2, the Biomass to it serves significant facilitation, and facilitation is high
Reach 1.6 times.This result and application thereof is that those skilled in the art lead at the microalgae biomass energy
Territory is unpredictable to be arrived.
The present invention also provides for a kind of method producing bio-oil, by chlorella (Chlorella
Sp.) 34-2 and series bacillus (Paenibacillus sp.) 18WLY co-cultures useless in pig farm
In water.
The chlorella Chlorella sp.34-2 of the present invention picks up from Xuancheng City of Anhui Province, and it has
The feature mushroomed out in unsterilised Pig raising wastewater (is cultivated 10 days, algae in Pig raising wastewater
The balanced growth speed of dry cell weight is: 165mg L-1·d-1), and can be dirty by carbon source in sewage
Dye thing changes into the bio-oil of up to 30%, and its settleability is good, it is easy to collect.This
Outward, this chlorella is to nutrient removal efficiency such as the carbon in Pig raising wastewater, nitrogen, phosphorus relatively
Height, after Pig raising wastewater is processed 7 days, nitrogen, tp removal rate are respectively 60% and 85%.Again
Person, this algae environmental suitability is very strong, all can be good under 20-40 DEG C and pH 3-11 scope environment
Growth, has bigger production application potentiality.The present invention is first by chlorella and class spore bar
Bacterium (Paenibacillus sp.) co-cultures in piggery wastewater, finds that class spore is felt bead
The raising of algae biomass creates the effect of remarkably promoting, and the Biomass of its stable phase chlorella reaches
1.80×1010Individual/ml, biological for not inoculating chlorella in Paenibacillus sp.18WLY bottle
Amount (1.15 × 1010Individual/ml) 1.6 times, growth-promoting functions is notable.The inventive method solves
Pig raising wastewater processes problem, reduces environmental pollution, and the addition of series bacillus improves raises pigs
Water body microenvironment in waste water, significantly improves chlorella Biomass, reduces microalgae biodiesel
Production cost, obtain preferable economic benefit, there is good market application foreground and guarantor
The effect of probationer nurse state.
Accompanying drawing explanation
Fig. 1 is the growth curve chart of chlorella 34-2.
Detailed description of the invention
Following example are used for illustrating the present invention, but are not limited to the scope of the present invention.If
Not specializing, technological means used in embodiment is well known to those skilled in the art
Conventional means, raw materials used is commercial goods.
Embodiment 1 Pig raising wastewater composition measurement and the preparation of dilution Pig raising wastewater
In the present embodiment, Pig raising wastewater is from Changping County, Beijing Cui Cun Hong Yun plant.Pig raising wastewater
The assay method of total nitrogen (total nitrogen, TN) be that alkaline chitinase is cleared up ultraviolet and divided
Light photometry, according to People's Republic of China (PRC) state environment protecting standard HJ 636-2012;And
The assay method of total phosphorus (total phosphorus, TP) is ammonium molybdate spectrophotometric method, foundation
National Standard of the People's Republic of China GB118WLY93-93.Ammonia nitrogen (NH3-N) and
The mensuration of COD is then according to even China water quality rapid detection apparatus 5B-6C.Through said determination method
Measure TN, NH of Pig raising wastewater3-N, TP and COD composition, measurement result is listed in table
1:
Table 1 Pig raising wastewater composition measurement
Take above-mentioned Pig raising wastewater and add after tap water is diluted to former supernatant volume 5 times, subpackage
In 250ml cleaning conical flask, every bottle of 100ml.Sewage composition after adding tap water dilution
It is calculated as: TN is about 121mg L-1, NH3-N is about 104mg ml-1, TP is about
16.4mg·L-1, COD is about 332mg L-1。
Embodiment 2 improves chlorella method of Biomass in Pig raising wastewater
1, chlorella and the isolated and purified and cultivation of azotobacter
(1) the isolated and purified and cultivation of chlorella: solid plate TAP culture medium: agar
Powder 1.5%;Chlorella Chlorella sp.34-2 monoclonal on picking flat board is by line side
Method is seeded on flat board preservation and purification algae kind, and subculture is once weekly.
On the TAP flat board of chlorella Chlorella sp.34-2, picking monoclonal is inoculated in
In 20ml TAP fluid medium, then take 1ml culture fluid and be inoculated in 100ml TAP liquid
In culture medium, it is placed in quiescent culture in 25 DEG C of illumination boxs.
(2) separation of series bacillus 18WLY, purification and cultivation: solid LB cultivates
Base: agar powder 1.5%;Picking passes through line from Paenibacillus sp.18WLY monoclonal
Method is seeded on flat board preservation and purification strain, and every three days subcultures are once
Picking monoclonal on the LB flat board of series bacillus Paenibacillus sp.18WLY
It is inoculated in 20ml LB culture medium, then takes 1ml culture fluid in 100ml LB liquid culture
In base, it is placed in 28 DEG C of shaking tables concussion and cultivates, horizontal shaker rotating speed 150rpm.
2, series bacillus 18WLY co-cultures with chlorella 34-2
The pure series bacillus Paenibacillus sp.18WLY training of logarithmic (log) phase will be in
Nutrient solution and pure chlorella Chlorella sp.34-2 culture fluid are respectively at 4 DEG C of 4000rpm
Under be centrifuged 10min, obtain cell precipitation.Cell precipitation is clear with sterilizing deionized water respectively
Wash 3 times, remove wherein remaining medium component.
Amount according to cell precipitation adds appropriate aquesterilisa and suspends, with vortex oscillation instrument vortex
Mixing is prepared as concentrated solution, inoculates chlorella Chlorella sp. respectively in each bottle according to table 2
And series bacillus Paenibacillus sp.18WLY is dilute in the 100ml sterilizing of embodiment 1
Release in Pig raising wastewater, add aquesterilisa and ensure that in all bottles, liquid final volume is identical.Utilize micro-
Mirror and red blood cell count plate control frustule inoculum concentration, make the chlorella Chlorella of inoculation
Sp.34-2 is 4.3 × 108Individual/ml, 7 × 10-6G/ml (dry weight);Series bacillus
Paenibacillus sp.18WLY is 1.26 × 10-4G/ml (dry weight).Inoculated class spore bar
Bacterium and inoculated chlorella dry weight are than for 18:1.
2 each bottles of initial inoculums of table
Being put in 25 DEG C of illumination boxs by above-mentioned each bottle and cultivate, its day mode is
16h under 5000lux, night pattern 8h.Just it is observed every 24h, contrast its growth
The difference of situation, and utilize blood counting chamber that microalgae cell number in each group is counted.
Fermentation is terminated after co-culturing 6 days.
Cultivate it has been observed that blank group and only inoculate the bacterium of Paenibacillus sp.18WLY
In group, bottle does not all have obvious color to change, the most unchanged green, this is because wherein do not inoculate little
Ball algae Chlorella sp.34-2 and cause.And only inoculate chlorella Chlorella sp.34-2
Negative control group (i.e. the 3rd group) with simultaneously inoculation Paenibacillus sp.18WLY and
The positive controls (i.e. the 4th group) of chlorella Chlorella sp.34-2 all has green product
Raw.And from postvaccinal 3rd day, positive controls compared to negative control group and shows
Deeper green, show that the quantity of wherein microalgae cell is more compared with the latter.
In 24h uses microscope and red blood cell count plate to each bottle, microalgae cell is carried out
Counting.Count results is carried out counting statistics and draws growth curve and compare, its result
See Fig. 1.As it is shown in figure 1, in positive controls, although have during chlorella growth
Longer lag phase, but its logarithmic (log) phase is shorter, and logarithmic (log) phase Biomass increases apparently higher than not
The negative control group of inoculation Paenibacillus sp.18WLY, and when stable phase, positive
In matched group, the Biomass of chlorella is 1.80 × 1010Individual/ml, for bead in negative control group
Algae biomass (1.15 × 1010Individual/ml) 1.6 times, growth-promoting functions is notable.
Result above shows, the class spore bar with Paenibacillus sp.18WLY as representative
Bacterium can remarkably promote the chlorella with Chlorella sp.34-2 as representative in Pig raising wastewater
Biomass, the reason of this result may improve water body residing for microalgae with series bacillus
Microenvironment relevant.
Although, used general explanation, detailed description of the invention and test, to this
Detailed description has been made in invention, but on the basis of the present invention, can to works some revise or
Improving, this will be apparent to those skilled in the art.Therefore, without departing from this
These modifications or improvements on the basis of spirit, belong to claimed
Scope.
Claims (10)
1. the method improving chlorella (Chlorella sp.) Biomass, its feature exists
In, the culture medium cultivating chlorella is added series bacillus (Paenibacillus sp.).
2. improve chlorella (Chlorella sp.) method for Biomass in Pig raising wastewater,
It is characterized in that, the Pig raising wastewater cultivating chlorella adds series bacillus
(Paenibacillus sp.)。
3. method as claimed in claim 2, it is characterised in that described chlorella is 34-
2, its deposit number is CGMCC NO.9225, and described series bacillus is 18WLY,
Its deposit number is CGMCC NO.7724.
4. method as claimed in claim 2, it is characterised in that described Pig raising wastewater is pig
Raise pigs the waste water that obtains after wastewater dilution 3-6 times produced in field.
5. the method as described in claim 2-4 is arbitrary, it is characterised in that at the beginning of chlorella
Beginning inoculum concentration is 3 × 108-5×108Individual/ml Pig raising wastewater;The initial inoculation of series bacillus
Amount is 1 × 10-4-2×10-4G/ml Pig raising wastewater.
6. the method as described in claim 2-4 is arbitrary, it is characterised in that inoculation class spore
The dry weight of bacillus and micrococcus is than for 15-20:1.
7. the method as described in claim 2-4 is arbitrary, it is characterised in that condition of culture is
20-35 DEG C, in one day, illumination and interlunation are than for 14-18:6-10, cultivate 3-8 days.
8. claim 1-7 arbitrary described method application in preparing bio-oil.
9. deposit number is the series bacillus (Paenibacillus of CGMCC NO.7724
Sp.) and containing its microbial inoculum in the application improved in chlorella (Chlorella sp.) Biomass.
10. deposit number is the series bacillus (Paenibacillus of CGMCC NO.7724
Sp.) and containing its microbial inoculum reduce animal farm wastewater pollute in application.
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CN108085283B (en) * | 2018-01-19 | 2019-12-06 | 杭州渔森农业技术开发有限公司 | method for culturing high-density algae through symbiosis of bacteria and algae |
CN109609382A (en) * | 2018-12-07 | 2019-04-12 | 中国石油大学(华东) | A kind of method that phycomycete co-cultures promotion chlorella growth and oil and fat accumulation |
CN109609382B (en) * | 2018-12-07 | 2022-03-22 | 中国石油大学(华东) | Method for promoting growth of chlorella and oil accumulation by algal-bacteria co-culture |
CN109402025A (en) * | 2018-12-12 | 2019-03-01 | 河西学院 | A kind of both culturing microalgae prevention and cure of pollution method |
CN110699258A (en) * | 2019-11-18 | 2020-01-17 | 江苏师范大学 | Culture method for improving chlorella cell biomass |
CN110699258B (en) * | 2019-11-18 | 2021-09-24 | 江苏师范大学 | Culture method for improving chlorella cell biomass |
CN110982859A (en) * | 2019-12-23 | 2020-04-10 | 哈尔滨工业大学 | Method for producing carbohydrate by using pig-raising sewage |
CN110982859B (en) * | 2019-12-23 | 2023-05-05 | 哈尔滨工业大学 | Method for producing carbohydrate by utilizing pig raising sewage |
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