CN108624506A - The method of microalgae and yeast mixed culture purification biogas slurry coproduction microbial biomass - Google Patents
The method of microalgae and yeast mixed culture purification biogas slurry coproduction microbial biomass Download PDFInfo
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- CN108624506A CN108624506A CN201810469213.7A CN201810469213A CN108624506A CN 108624506 A CN108624506 A CN 108624506A CN 201810469213 A CN201810469213 A CN 201810469213A CN 108624506 A CN108624506 A CN 108624506A
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- microalgae
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/12—Unicellular algae; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/30—Fuel from waste, e.g. synthetic alcohol or diesel
Abstract
The present invention relates to a kind of methods of microalgae and yeast mixed culture purification biogas slurry coproduction microbial biomass, include the following steps：1) pure culture of microalgae and yeast cells respectively obtains microalgae seed liquor and yeast starter liquid；2) supernatant that biogas slurry pretreatment obtains is diluted, allocated, obtain the marsh liquid culture medium of mixed culture；3) microalgae seed liquor and the seed liquor of yeast are inoculated in marsh liquid culture medium in proportion, establish co-culture system；4) biogas slurry after discharge is purified harvests microbial biomass.While this method handles biogas slurry, the recycling of high-protein biological matter is realized, reduce the discharge of waste to greatest extent, achieve the purpose that resource reclaim and conversion, energy saving, emission reduction, synergy, realize circular economy, turn waste into wealth, green production.
Description
Technical field
The invention belongs to field of microbial fermentation, are related to microalgae and yeast mixed culture technique, relate generally to one kind and pass through
The method of microalgae and yeast mixed culture purification biogas slurry coproduction microbial biomass.
Background technology
Biogas slurry is the by-product of biogas production, processing and the main bottleneck for recycling always biogas industrial development.For
Evading biogas slurry is directly used in the problem that ecological risk and treating capacity are limited existing for agricultural irrigation, often utilizes Aerobic biological process
The ingredients such as nitrogen, phosphorus, organic matter in method reduction biogas slurry, but a large amount of activated sludge and CO that this method generates2Deng it is difficult to sharp again
With causing secondary pollution and carbon emission, not meeting the requirement that resource recycling utilizes.
Biogas slurry nitrogen and phosphorus is sufficient, can meet the nitrogen phosphorus demand of micro algae growth, and micro algae growth can make biogas slurry reach reason
The nitrogen phosphorus ligands effect thought.The utilizable inorganic carbon source of microalgae and simple organic carbon source (acetate, glycerine are added into system
Deng) growth and the nitrogen phosphorus ligands effect of microalgae can be promoted.In addition, by with can utilize organic matter, release CO2The micro- life of heterotrophism
Object co-cultures the supply of the further degradation and carbon source that can strengthen organic pollution, such as certain saccharomyces oleaginosus (such as Yarrowia
Lipolytic) in addition to can using system in other than original organic matter grown, cheap organic carbon source can also be utilized (such as
The crude glycerin byproduct that production of biodiesel generates) it carries out the production of microbial grease and discharges CO2.Microalgae-saccharomyces oleaginosus is mixed
Culture is closed compared to single cultivating system, there is apparent advantage in terms of pollutant removal and microbial biomass accumulation.
Currently, the research of microalgae and yeast mixed culture is widely paid close attention to：Such as Publication No.
The Chinese patent application of CN200910237936.5 discloses a kind of side using mixed culture yeast and algae production grease
Method；The Chinese patent application of Publication No. CN03105314.9 discloses a kind of algae and yeast mixed culture fermenting and producing shrimp
The method of green element.Application of the studies above in terms of illustrating grease and production of astaxanthin, but so far there are no using microalgae and
Yeast mixed culture technique handles the research of biogas slurry.Therefore, a kind of biogas slurry treatment being mixed based on microalgae and yeast is developed
The method of coproduction microbial biomass is to solving the problems, such as that the recycling of biogas slurry is necessary.
Invention content
In order to solve the above-mentioned problems in the prior art, at a kind of microalgae and yeast mixed culture
Reason biogas slurry and the method for generating microbial biomass realize the recycling of biomass, reduce nitrogen to greatest extent while handling biogas slurry
The discharge of phosphorus.
The technical purpose of the present invention is achieved through the following technical solutions：
The method of microalgae of the present invention and yeast mixed culture purification biogas slurry coproduction microbial biomass, including walk as follows
Suddenly：
1) pure culture of microalgae and yeast cells respectively obtains microalgae seed liquor and yeast starter liquid；
2) supernatant that biogas slurry pretreatment obtains is diluted, allocated, obtain the marsh liquid culture medium of mixed culture；
3) microalgae seed liquor and the seed liquor of yeast are inoculated in marsh liquid culture medium in proportion, establish co-culture system；
4) biogas slurry after discharge is purified harvests microbial biomass.
Preferably, microalgae described in step 1) of the present invention is the green algas such as chlorella, scenedesmus or glueballs algae；Preferably, described
Microalgae is photoautotrophy or simultaneous foster chlorella bacterial strain, can be selected from chlorella vulgaris, chlorella pyrenoidosa, chlorella ellipsoidea, original
One kind in shell chlorella；The yeast cells is selected from using glycerine as the aerobic bacterial strain of carbon source, be can be selected from sub- sieve solution fat yeast, is glued
One kind in rhodotorula, this formula saccharomyces oleaginosus, rhodothece rubra, hair husband rhodotorula, candida tropicalis.Chlorella of the present invention
Preferably chlorella vulgaris or chlorella pyrenoidosa, the yeast are selected from sub- sieve solution fat yeast.
It is highly preferred that pure culture condition is in step 1)：25-30 DEG C of temperature, 40-80 μm of ol photons/ of intensity of illumination
m2/ s, 150-200 revs/min of rotating speed, cultivated days 5-8 days.
Specifically, the pretreatment of biogas slurry described in step 2) of the present invention includes the following steps：Natural subsidence simultaneously centrifuges, and is placed in
Disinfection in bioreactor.
More specifically, the total nitrogen content in biogas slurry supernatant described in step 2) of the present invention is in 436mg/L or more, ammonia nitrogen
Content is 385mg/L or more, and total phosphorus content is 58mg/L or more, and COD contents are 1278mg/L or more, pH value 7.3-8.5.
Preferably, described in step 2) of the present invention dilution refer to using tap water, natural water body water or incubation return water into
Row dilution, dilution ratio 1:1 to 1:Between 5；The allotment refers to is adjusted to 7.0 ± 0.5 by the pH value of biogas slurry after dilution, and mends
Fill carbon source.
Preferably, carbon source of the present invention refers to pure glycerin or crude glycerine (biological diesel oil byproduct), and final concentration
For 1-5g/L.
Preferably, condition is mixed described in step 3) of the present invention is：25-30 DEG C of temperature, 40-80 μm of ol of intensity of illumination
photons/m2/ s, 150-200 revs/min of rotating speed, cultivated days 5-8 days.
Preferably, inoculative proportion described in step 3) of the present invention refers to microalgae and yeast count ratio (3-5):1, it is described
The initial concentration of yeast cells is 0.1-1 × 107Cell/mL.
Preferably, microbial biomass described in step 4) of the present invention is to obtain as follows：Harvest algae solution, drying
Obtain biomass dry powder；Harvesting culture solution trigger condition be：Work as NH3When-N (mg/L) and TP (mg/L) content are less than 5mg/L.
Beneficial effects of the present invention：
Compared with prior art, the method tool of the microalgae and yeast mixed culture purification biogas slurry coproduction microbial biomass
There is following advantage：
1), the inactive sludge of this method generates, and the glycerine (or crude glycerine) of introducing can be converted into microbial biomass, without residual
It stays；
2) recycling that biomass, is realized while handling biogas slurry, reduces nitrogen discharge and phosphorus discharge, reaches resource reclaim to greatest extent
With the purpose of conversion, emission reduction, synergy, realizes circular economy, turns waste into wealth, green production.
Description of the drawings
Fig. 1 is biomass variety accumulation figure in microalgae of the present invention and the culture of yeast list and the lower biogas slurry of mixed culture.
Specific implementation mode
The present invention is also applied for all types of biogas slurries by taking the biogas slurry of cattle farm as an example.The present invention implements specific steps such as
Under：
(1) cattle farm biogas slurry is pre-processed：Through natural subsidence and centrifugation, to remove the solid content in biogas slurry, to
The permeability for reducing light, is conducive to the mixture growth of microalgae.Be subsequently placed in bioreactor (including shaking flask, column photoproduction
Object reactor, Flat photobioreactor, pipeline bioreactor and open pond), carry out disinfection sterilization processing.
Preprocessed obtained supernatant, total nitrogen content is in 436mg/L or more, and ammonia-nitrogen content is 385mg/L or more, always
Phosphorus content is 58mg/L or more, and COD contents are 1278mg/L or more, pH value 7.3-8.5.
To treated, biogas slurry is diluted, allocates, to meet the growth demand of microalgae and yeast.It is diluted to using originally
Water or natural water body water or and incubation return water the supernatant of preprocessed acquisition is diluted, dilution ratio 1:1
To 1:Between 5；The allotment refers to adjusts pH value to 7.0 ± 0.5 by the HCl solution that 1N is added, and final concentration is added
It is 1-5g/L glycerine or crude glycerine as carbon source.
(2) in the bioreactor equipped with pretreated cattle farm biogas slurry inoculation in logarithmic growth microalgae and
Yeast cells is mixed.Microalgae need to meet specific inoculative proportion with yeast：Chlorella and yeast count concentration ratio
Example (3-5):1, yeast cells initial concentration is 0.1-1 × 107Cell/mL.
Used yeast bacterial strain is aerobic bacterial strain (sub- sieve solution fat yeast, rhodotorula glutinis, this formula that can be carbon source using glycerine
One kind in saccharomyces oleaginosus, rhodothece rubra, hair husband rhodotorula, candida tropicalis), chlorella is photoautotrophy or and bacteria strain
(one kind in chlorella vulgaris, chlorella pyrenoidosa, chlorella ellipsoidea, Chlorella protothecoides).Preferably, chlorella vulgaris/egg
The effect of white nucleus chlorella and Ya Luo solution fat yeast mixed culture processing biogas slurries is ideal.
Mixed culture is：25-30 DEG C of temperature, 40-80 μm of ol photons/m of intensity of illumination2/ s, 150-200 turns of rotating speed/
Point, it cultivates 4-8 days.Work as NH3(mg/L) it can be harvested when and TP (mg/L) content is less than 5mg/L.
(3) after cultivating, frustule is harvested by ultrafiltration apparatus and prepares concentrate, centrifuges and obtains wet mud, and dry
To biomass dry powder.This technique can by the nutriments such as nitrogen phosphorus in biogas slurry together be added system low value glycerine be converted into micro- life
Object biomass obtains microbial biomass and contains albumen, grease, carbohydrate, can be used as single cell protein, Unicell Oils and Fats
And the raw material of chemical products, realize the recycling of the emission reduction of nitrogen phosphorus, biogas slurry purification and waste water.
With reference to embodiment, the present invention is described in further detail, and embodiments of the present invention are not limited thereto.
Embodiment 1
Microalgae-yeast, which is carried out, using cattle farm biogas slurry is mixed (glycerol concentration 2g/L)
1.1 algaes activate and prepared by seed liquor
It is single from one chlorella vulgaris of picking (Chlorella vulgaris) on the BG11 culture medium solid plates of activation
Algae falls, and is inoculated into the 250mL triangular flasks equipped with BG11 culture mediums, liquid amount 100mL.It is 28 DEG C to be placed in temperature, and light intensity is
40μmol photons/m2It is cultivated 6 days in/s, 150 revs/min of constant-temperature table, 2% sterile CO is passed through in incubation2.From
Sub- sieve solution fat Asia Lip river yeast (Yarrowia lipolytica) single bacterium colony of picking one on the YPD culture medium solid plates of activation,
It is inoculated into the 250mL triangular flasks equipped with YPD culture mediums, liquid amount 100mL.It is 28 DEG C to be placed in temperature, 150 revs/min of perseverance
It is cultivated 24 hours in warm shaking table.
The pretreatment and inoculation of 1.2 biogas slurries
By cattle farm biogas slurry after settling, centrifuging pretreatment, the dilution of isometric tap water is added, extremely with the salt acid for adjusting pH of 1N
7.04, after 0.22 μm of sterilised membrane filter filters after, be fitted into 250mL triangular flasks, then liquid amount 100mL is added sterile
Glycerine makes its final concentration of 2g/L.Chlorella vulgaris seed liquor and Ya Luo are solved into the seed liquor kind of fat yeast to cultivating system
In, make the frustule a concentration of 24.5 × 10 of culture starting6Cell/mL, yeast are 8.05 × 106Cell/mL.It is cultivated when initial
Liquid index is：TN 218mg/L、NH3-N 192.5mg/L、TP 29mg/L。
1.3 cultural method
Mixed culture is：27 DEG C of temperature, 40 μm of ol photons/m of intensity of illumination2/ s, 170 revs/min of rotating speed are cultivated 6 days.
1.4 test method
1.3.1 TN, NH in waste water3- N, TP assays
Use the dedicated kit of HACH companies of the U.S., TN, NH3- N, TP measurement range be respectively 0-150mg/L, 0-
50mg/L、0-3.5mg/L.According to kit standard operating procedure.Each sample is averaged after being repeated three times, and is measured and is read
Number is multiplied by sample extension rate, TN, NH in water sample as to be measured3- N, TP contents.
1.5 biomass estimation
Work as NH3When-N (mg/L) and TP (mg/L) content are less than 5mg/L, by culture solution through centrifugation, the wet mud of gained spend from
Sub- water is resuspended, and repeated centrifugation obtains biomass.Biomass is transferred in pre-weighed 1.5mL centrifuge tubes, high speed centrifugation
(12000 revs/min centrifuge 5 minutes), removes supernatant, is put into 60 DEG C of baking ovens and dries and weigh.
1.6 interpretation of result
With TN, NH3- N, TP removal rates are index, reflect the purge cases of waste water.As shown in table 1, the front and back mixing training of culture
Foster TP removal rates can reach 100%, TN, NH3- N removal rates are respectively 88.26%, 99.74%.
Table 1：Microalgae and the culture of yeast list and the lower biogas slurry clean-up effect comparison of mixed culture
Table 2：The biomass comparison that microalgae and the culture of yeast list and mixed culture obtain
| Biomass yield (g/L/d) | Maximum biomass concentration (g/L) | |
| Single algae culture | 0.13±0.010 | 0.85±0.098 |
| Single Yeast Cultivation | 0.09±0.003 | 0.92±0.005 |
| Mixed culture | 0.21±0.026 | 1.62±0.196 |
Table 3：The biomass nutrient comparison that microalgae and the culture of yeast list and mixed culture obtain
The changing rule of biomass and ultimate yield situation such as Fig. 1 and table 2 in single culture and co-culture system.Analysis can
Know and cultivated by 144h, be mixed the maximum biomass concentration (1.62g/L) of acquisition, is more than single algae cultivating system (0.85g/
L) it is more than single algae with single Yeast Cultivation system (0.92g/L), the biomass yield (0.21g/L/d) of co-culture system and cultivates
(0.13g/L/d) and single yeast (0.09g/L/d) cultivating system.
The trophic analysis for the biomass that single culture and co-culture system obtain and output situation such as table 3, analysis is it is found that mixed
The yield (0.31g/L and 0.51g/L) that culture obtains grease and albumen is closed, single algae cultivating system (0.28g/L and 0.22g/ are more than
) and single Yeast Cultivation system (0.04g/L and 0.18g/L) L.Consider removal, biomass output, grease and the albumen of nitrogen phosphorus
Output, co-culture system have a clear superiority compared to single cultivating system.
Obviously, the above embodiment of the present invention be only to clearly illustrate example of the present invention, and not be pair
The restriction of embodiments of the present invention.For those of ordinary skill in the art, may be used also on the basis of the above description
To make other variations or changes in different ways.Here all embodiments can not be exhaustive.It is every to belong to this hair
Row of the obvious changes or variations that bright technical solution is extended out still in protection scope of the present invention.
Claims (10)
1. a kind of method of microalgae and yeast mixed culture purification biogas slurry coproduction microbial biomass, which is characterized in that including such as
Lower step：
1) pure culture of microalgae and yeast cells respectively obtains microalgae seed liquor and yeast starter liquid；
2) supernatant that biogas slurry pretreatment obtains is diluted, allocated, obtain the marsh liquid culture medium of mixed culture；
3) microalgae seed liquor and the seed liquor of yeast are inoculated in marsh liquid culture medium in proportion, establish co-culture system；
4) biogas slurry after discharge is purified harvests microbial biomass.
2. according to the method described in claim 1, it is characterized in that, microalgae described in step 1) is chlorella, scenedesmus or glueballs
Algae；Preferably, the microalgae be photoautotrophy or and foster chlorella bacterial strain, selected from chlorella vulgaris, chlorella pyrenoidosa, ellipse
One kind in circle chlorella, Chlorella protothecoides；The yeast cells is selected from using glycerine as the aerobic bacterial strain of carbon source, selected from sub- sieve solution
One kind in fat yeast, rhodotorula glutinis, this formula saccharomyces oleaginosus, rhodothece rubra, hair husband rhodotorula, candida tropicalis.
3. according to the method described in claim 1, in step 1), pure culture condition is：25-30 DEG C of temperature, intensity of illumination 40-80
μmol photons/m2/ s, 150-200 revs/min of rotating speed, cultivated days 5-8 days.
4. according to the method described in claim 1, it is characterized in that, the pretreatment of biogas slurry described in step 2) includes following step
Suddenly：
Natural subsidence simultaneously centrifuges, and is placed in disinfection in bioreactor.
5. according to the method described in claim 1, it is characterized in that, the total nitrogen content in biogas slurry supernatant described in step 2) exists
436mg/L or more, ammonia-nitrogen content are 385mg/L or more, and total phosphorus content is 58mg/L or more, and COD contents are 1278mg/L or more,
PH value is 7.3-8.5.
6. according to the method described in claim 1, it is characterized in that, dilution described in step 2) refers to using tap water, natural water
Body water or incubation return water are diluted, dilution ratio 1:1 to 1:Between 5；The allotment refers to the pH of biogas slurry after dilution
Value is adjusted to 7.0 ± 0.5, and supplementary carbon source.
7. according to the method described in claim 6, it is characterized in that, the carbon source refers to pure glycerin or crude glycerine, and final
A concentration of 1-5g/L.
8. according to the method described in claim 1, it is characterized in that, mixed culture condition described in step 3) is：Temperature 25-30
DEG C, 40-80 μm of ol photons/m of intensity of illumination2/ s, 150-200 revs/min of rotating speed, cultivated days 5-8 days.
9. according to the method described in claim 1, it is characterized in that, inoculative proportion described in step 3) refers to that microalgae and yeast are thin
Born of the same parents' number ratio (3-5):1, the initial concentration of the yeast cells is 0.1-1 × 107Cell/mL.
10. according to the method described in claim 1, it is characterized in that, microbial biomass described in step 4) is by as follows
Step obtains：Harvesting algae solution is dried to obtain biomass dry powder；Harvesting culture solution trigger condition is：Work as NH3- N (mg/L) and TP
(mg/L) when content is less than 5mg/L.
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| CN112299562A (en) * | 2019-07-29 | 2021-02-02 | 仲恺农业工程学院 | Method for promoting microalgae to degrade carbon, nitrogen and phosphorus by using yeast secretion |
| CN115594310A (en) * | 2022-10-17 | 2023-01-13 | 上海碳迹生物科技有限公司（Cn） | Method for producing single-cell protein for feed from livestock and poultry manure biogas slurry |
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