CN106929422B - A kind of method of chlorella and yeast co-cultivation purification yeast wastewater - Google Patents
A kind of method of chlorella and yeast co-cultivation purification yeast wastewater Download PDFInfo
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- CN106929422B CN106929422B CN201710156087.5A CN201710156087A CN106929422B CN 106929422 B CN106929422 B CN 106929422B CN 201710156087 A CN201710156087 A CN 201710156087A CN 106929422 B CN106929422 B CN 106929422B
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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
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/32—Biological treatment of water, waste water, or sewage characterised by the animals or plants used, e.g. algae
- C02F3/322—Biological treatment of water, waste water, or sewage characterised by the animals or plants used, e.g. algae use of algae
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
- C02F3/347—Use of yeasts or fungi
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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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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2209/00—Controlling or monitoring parameters in water treatment
- C02F2209/06—Controlling or monitoring parameters in water treatment pH
Abstract
The present invention provides the method for a kind of chlorella and yeast co-cultivation purification yeast wastewater, includes the following steps: 1) chlorella and yeast cells activation culture, obtains chlorella seed liquor I and yeast starter liquid I；2) chlorella seed liquor I is inoculated in improvement basal medium, yeast starter liquid I is inoculated in culture medium, is placed in outdoor carry out seed liquor culture, obtains chlorella seed liquor II and yeast starter liquid II；3) chlorella seed liquor II rhodotorula glutinis seed liquor II is inoculated in the yeast wastewater that need to be handled, forms co-culture system, carry out outdoor co-cultivation.The energy consumption and production cost of chlorella and yeast co-cultivation can not only be effectively reduced using the luminous energy under natural conditions by three easy steps, using a small amount of raw material in the present invention, and realizes and utilize microbial co culture large scale processing yeast wastewater.
Description
Technical field
The invention belongs to cell culture technologies, are related to microalgae and yeast Coculture techniques, relate generally to one kind and pass through bead
The method that algae and yeast co-culture purification yeast wastewater.
Background technique
Yeast wastewater is a kind of high concentrated organic wastewater difficult, coloration is deep generated in Yeast Fermentation Process, tool
There are high-content COD, BOD5, total nitrogen, total phosphorus, not biodegradable organic pollutant and the features such as high chroma.As biology is made
The yeast fermentation waste water amount of pharmaceutical factory popularization, generation increases, and causes very big pollution to environment.Study the processing of high-efficiency environment friendly
Method is most important to the sustainable development of yeast industry.
Chlorella is the widely used algae in municipal wastewater processing, has that growth rate is fast, tolerance is strong, albumen
The advantages that matter or high fat content, can synthesize own cells component using carbon, nitrogen, the phosphorus in waste water, effectively remove nitrogen in waste water
The nutriments such as phosphorus.Yeast can then effectively remove the organic substance in waste water, and COD removal rate is up to 68%-86%, and yeast is raw
Long very fast, cultivation cycle is short.Some researches show that, microalgae and yeast in same cultivating system there are the relationship of mutualism, micro-
Algae growth generates O2It can promote yeast growth；Yeast can secrete powerful extracellular enzyme small point of the organic inaction of macromolecular of degradation
Son breathes the CO of generation2It can be used in micro algae growth, so that the shadow of the variations such as pH and dissolved oxygen in incubation be effectively relieved
It rings.Microalgae and yeast co-culture the advantage that can make full use of two kinds of microorganisms in yeast wastewater, harvest valuable biology
Matter, while waste water is purified, turn waste into wealth.
Currently, the research in terms of although microalgae and yeast co-culture accumulation grease and handle waste water has received significant attention,
But utilize the research of microbial co culture technology scaleization processing waste water relatively fewer.For example, Publication No. CN102080119
Chinese patent application, disclosing using industrial wastewater is culture medium, is mixed yeast and algae, produces microbial oil
Method；Miao Jin is prosperous etc., rhodotorula glutinis and blunt top spirulina mixed culture production grease in gourmet powder waste water (Beijing University of Chemical Technology's report,
The supplementary issue II of volume 34,2007).But, the studies above belongs in microbial room co-culture, and handles waste water on a small scale.
Therefore, develop it is a kind of using outside microbial room co-culture scale processing yeast wastewater method be necessary.
Summary of the invention
In order to overcome the shortcomings in the prior art, the present invention provides a kind of utilize and co-cultures mould outside chlorella and yeast room
Formula, the method for large scale processing yeast wastewater；And this method is relatively easy, cultivates under outdoor natural conditions, can effectively drop
Low energy consumption and production cost；Microbial co culture is being utilized, there is important practical value in terms of improving yeast wastewater clean-up effect.
To solve the above problems, the technical solution adopted is as follows:
A kind of method of chlorella and yeast co-cultivation purification waste water, includes the following steps:
1) chlorella and yeast cells activation culture obtain chlorella seed liquor I and yeast starter liquid I；
2) chlorella seed liquor I is inoculated in improvement basal medium, yeast starter liquid I is inoculated in culture medium, is placed in room
Outside, seed liquor culture is carried out, chlorella seed liquor II and yeast starter liquid II are obtained；
3) chlorella seed liquor II and yeast starter liquid II are inoculated in yeast wastewater, form co-culture system, carried out
Outdoor co-cultivation.
Outdoor seed liquor culture carries out extensive, further activation culture to seed liquor using the luminous energy of nature,
A large amount of high density seed liquors can be obtained in a short time.
Chlorella and yeast, which co-culture, carries out extensive wastewater treatment, a large amount of high density seed liquors is needed, if cannot provide
Sufficient seed liquid, inoculum density is too low, is unfavorable for purification of waste water.Present inventor's discovery, directly progress chlorella and ferment
Mother, which co-cultures, cannot obtain highdensity seed liquor, so at the beginning not by chlorella and yeast Co-culture；And it will be small
Ball algae and yeast carry out activation and seed liquor culture with different culture mediums respectively, can obtain a large amount of high density in a short time
Seed liquor.It is substantially the same to cultivate seed liquor I and seed liquor II, is all to provide high density seed for outdoor large-scale culture
Liquid.Seed liquor I is and to be difficult meet the needs of extensive seed liquor in laboratory in laboratory cultures.For quick expanding species, mention
For largely stable high density seed liquor, first in laboratory activation culture, expanded culture later in outdoor big shaking table.
The chlorella includes one of chlorella pyrenoidosa, chlorella vulgaris, chlorella ellipsoidea, Chlorella protothecoides；
The yeast includes rhodotorula glutinis, Si Shi saccharomyces oleaginosus, Saccharomycopsis lipolytica, rhodothece rubra, red phaffia rhodozyma, the false silk in the torrid zone
One of yeast, Cryptococcus laurentii, circle rhodosporidium toruloides.
Preferably, chlorella is chlorella pyrenoidosa, and yeast is rhodotorula glutinis.Through screening matched pair study, the present application
It is best that people has found that chlorella pyrenoidosa and rhodotorula glutinis co-culture processing yeast wastewater effect.
The formula of the improvement basal medium is NaNO33450-4050mg/L, KH2PO41150-1350mg/L,
MgSO4·7H2O 900-1100mg/L, EDTA 450-650mg/L, H3BO3105-125mg/L, CaCl2·2H2O 101-
121mg/L, FeSO4·7H2O 45-55mg/L, ZnSO4·7H2O 80-96mg/L, MnCl2.4H2O 13-15mg/L,
Na2MoO4·2H2O 11-13mg/L, CuSO4·5H2O14.5-16.5mg/L, Co (NO3)2·6H2O 4.5-5.5mg/L,
C6H12O645-55g/L；PH6.10 ± 0.2 of the improvement basal medium.
Preferably, the formula of the improvement basal medium is NaNO33750mg/L, KH2PO41250mg/L, MgSO4·
7H2O 1000mg/L, EDTA 500mg/L, H3BO3114.2mg/L, CaCl2·2H2O 111mg/L, FeSO4·7H2O
49.8mg/L ZnSO4·7H2O 88.2mg/L, MnCl2·4H2O 14.2mg/L, Na2M0O4·2H2O 11.92mg/L,
CuSO4·5H2O 15.7mg/L, Co (NO3)2·6H2O 4.9mg/L, C6H12O650g/L；The improvement basal medium
pH6.10。
Improve NaNO in basal medium3Dosage be 3 times of ordinary culture medium, while adding glucose as carbon source.
When concentration of glucose is 50g/L, sodium nitrate concentration is 3.75g/L, Heterotrophic culture 4 days, glucose can all exhaust, at this time
The biomass concentration of chlorella reaches highest, and biomass concentration 21.31g/L is significantly higher than sodium nitrate concentration 1.25g/L when institute
Attainable maximum biomass concentration 13.64g/L.As it can be seen that the growth of chlorella can be promoted using improvement basal medium, significantly
The biomass concentration for improving chlorella, realizes quick expanding species, provides stable high density seed for the scale processing of outdoor waste water
Liquid.
As a kind of specific embodiment, yeast starter liquid I is inoculated in culture medium in step 2), and the culture medium is YM
One of culture medium, malt extract medium.
Preferably, yeast starter liquid I is inoculated in culture medium in step 2), and the culture medium is YM culture medium.
The formula of the YM culture medium is casein peptone 5.0g/L, fructus hordei germinatus leaching powder 3.0g/L, glucose 10.0g/L, yeast
Soak powder 3.0g/L, the pH value 6.2 ± 0.2 (25 DEG C) of the YM culture medium.Yeast is grown comparatively fast in YM culture medium, can be in short-term
Interior acquisition high density seed liquor, dosage are considerably less than malt extract medium.In view of extensive seed liquor demand and at
This, YM culture medium is more suitable for.
The following are the preferred embodiments of step 1):
Chlorella is inoculated in improvement basal medium, yeast cells is inoculated in YM culture medium, carries out cell activation culture.
The condition of the cell activation culture are as follows: cultivation temperature is 27-29 DEG C of constant temperature, revolving speed 140-160r/m, and intensity of illumination is
3500-4500lux, cultivation cycle are 5-7 days.
It is particularly preferred, in step 1), the condition of the cell activation culture are as follows: cultivation temperature is 28 DEG C of constant temperature, revolving speed
For 150r/m, intensity of illumination 4000lux, cultivation cycle is 6 days.
The following are the preferred embodiments of step 2):
Chlorella seed liquor I is inoculated in improvement basal medium, yeast starter liquid I is inoculated in YM culture medium, is placed in room
Outer progress seed liquor culture, obtains chlorella seed liquor II and yeast starter liquid II；The condition of the seed liquor culture are as follows: culture
Temperature is 27-29 DEG C, and chlorella seed liquor culture revolving speed is 150-210r/m, and yeast starter liquid culture revolving speed is 90-150r/m,
Intensity of illumination is 1500-3400lux, and cultivation cycle is 6-8 days.
Particularly preferred, in step 2), the condition of the seed liquor culture is that cultivation temperature is 28 DEG C, chlorella seed liquor
Culture revolving speed is 180r/m, and yeast starter liquid culture revolving speed is 120r/m, intensity of illumination 1500-3400lux, and cultivation cycle is
6-8 days.
Chlorella seed liquor culture rotating ratio yeast starter liquid culture revolving speed is high, is because chlorella is close in outdoor shaking flask
Degree is higher, if revolving speed is too low, might have cell settlement, is unfavorable for its growth；In outside scenery yeast, yeast growth compared with
Fastly, density is high, since, containing compared with polyprotein, yeast growth generates CO in culture medium2If revolving speed is excessively high, had in shaking flask
A large amount of foams generate, and are unfavorable for the growth of yeast, therefore revolving speed is unsuitable excessively high.Seed liquor culture revolving speed can according to physical condition and
What cell growth condition was adjusted.
The following are the preferred embodiments of step 3):
Scheme one:
First stage: taking the 25%-35% (volume) of yeast wastewater total amount, and the pH value for adjusting yeast wastewater is 5.8-6.3,
And it is inoculated with chlorella seed liquor II and yeast starter liquid II thereto, co-culture system is formed, cultivation cycle is 4-6 days；
Second stage: adding the 25%-35% (volume) into yeast wastewater total amount, and cultivation cycle is 2-4 days；
Phase III: it is added again into remaining yeast wastewater, cultivation cycle is 2-4 days.
From first stage to phase III, cultivation temperature is controlled at 26-35 DEG C；The pH value of culture is controlled in 6.0-7.0；
Phosphate in waste water and total phosphorus content are measured by sampling daily, when cultivating system total phosphorus content is few less than 40mg/L or phosphate content
When 10mg/L, according to remaining phosphate and total phosphorus content, 20-80mg/L PO is added4 3-The phosphate of-P, phosphate are phosphoric acid hydrogen
At least one of dipotassium, potassium dihydrogen phosphate, sodium dihydrogen phosphate.
Phosphorus is chlorella and the indispensable element of yeast growth, and phosphorus lacks or is unfavorable for cell growth when insufficient, in turn
Influence purification of waste water effect.Phosphate content is not high in yeast wastewater, in order to guarantee cell normal growth, needs to add phosphoric acid
Salt.
Preferably, it is 2-8: 1 that the chlorella seed liquor II and yeast starter liquid II, which is inoculated in the ratio in yeast wastewater,.
Particularly preferred, the chlorella seed liquor II and yeast starter liquid II are inoculated in the ratio in yeast wastewater as 3-
5∶1。
Wherein, it is cell number ratio that the chlorella seed liquor II and yeast starter liquid II, which is inoculated in the ratio in yeast wastewater,
Example.
Using preferred chlorella seed liquor II and yeast starter liquid II inoculative proportion, Initial stage of culture pH reduction pair can be relieved
The influence of chlorella growth；More useful chlorellas and yeast can be not only obtained, but also it is useless preferably to remove yeast
COD, NH in water3- N, TN, TP and PO4 3-Equal substances.
Scheme two:
First stage: yeast starter liquid II is inoculated in yeast wastewater, and the amount of yeast wastewater is yeast wastewater total amount
25%-35% (volume).Culture to pH value rises to 4.7-5.7, accesses chlorella seed liquor II, forms co-culture system,
Cultivation cycle is 3-5 days；
Second stage: adding the 18%-28% (volume) into yeast wastewater total amount, and cultivation cycle is 2-3 days；
Phase III: it is added again into remaining yeast wastewater, cultivation cycle is 5-6 days.
From first stage to phase III, cultivation temperature is controlled at 26-35 DEG C；The pH value of culture is controlled in 6.0-7.0；
Phosphate in waste water and total phosphorus content are measured by sampling daily, when cultivating system total phosphorus content is less than 20-40mg/L or phosphate radical contains
When amount is less than 10mg/L, according to remaining phosphate and total phosphorus content, 20-80mg/L PO is added4 3-The phosphate of-P, phosphate are phosphorus
At least one of sour hydrogen dipotassium, potassium dihydrogen phosphate, sodium dihydrogen phosphate.
Preferably, the yeast wastewater is useless by the pretreated molasses yeast of sand filtration, active carbon filtering, ultrafiltration three-level
Water.High organic content, waste water color value are high in false yeasts waste water；If directly cultivating chlorella in raw wastewater, due to translucency
It is very poor, photosynthetic efficiency is low, chlorella growth is slow, cell density is low.By pretreatment, the organic matter that can reduce yeast wastewater is dense
Degree and coloration improve and co-culture effect, while improving purification of waste water ability.Pretreated yeast wastewater: pH value 3.0-
5.5；COD, NH in yeast wastewater3- N, TN and TP content are respectively 10000-15000,300-1000,450-1800 and 50-
200mg/L.But since every batch of waste strength may be different, range fluctuation may be bigger.
The clean-up effect of the yeast wastewater with higher of preferred embodiment one of step 3), COD, NH in yeast wastewater3-N、
TN、TP、PO4 3-And BOD5Total removal rate can respectively be up to 80.98%, 78.54%, 83.21%, 100%, 100% and
87.76%.
The step of the step of preferred embodiment two of step 3), is more simple, simplifies the pH value for first adjusting yeast wastewater；And
Using first inoculation yeast seed liquor II, degraded larger molecular organics (especially residual protein) using the growth of yeast, only ammonia
Base nitrogen content increases, and improves the pH value of yeast wastewater；Inoculate chlorella.Not only step is simple, reduces cost, is more suitable for work
Industry application, and the clean-up effect of good yeast wastewater can be reached, COD, NH in yeast wastewater3- N, TN, TP and PO4 3-
Total removal rate can respectively be up to 81.57%, 67.27%, 76.94%, 100% and 100%.In addition, the preferred side of step 3)
Case one, two is all made of the method for gradually adding yeast wastewater.Yeast wastewater is gradually added to be equivalent to first with partial yeast waste water
Chlorella and yeast starter liquid culture are carried out, high density co-cultured cell is obtained in the short time, and the cell is useless by yeast
The cell of water domestication, is more suitable in yeast wastewater and grows.This not only shortens the cultivation cycle of outdoor co-cultivation, also improves
Yeast wastewater clean-up effect.On the other hand, with the extension of incubation time, in yeast wastewater, the cell number of chlorella and yeast is not
It is disconnected to increase, need the nutriments such as more nitrogen phosphorus, by gradually add yeast wastewater can be provided for cell growth it is more sufficient
Nutriment, also improve wastewater treatment capacity.
After outdoor co-cultivation, pipeline bioreactor is coupled by hyperfiltration membrane assembly and realizes chlorella and yeast
Online concentration and Sewage treatment.Chlorella and yeast concentrate can be used as seed liquor and continue on for wastewater treatment, can also be into
One step, which is concentrated and dried, is used for other purposes；Waste water can appropriately processed rear discharge.
The invention has the advantages that and the utility model has the advantages that
1, the step of technical solution of the present invention is simple, and seed liquor under indoor seed liquor culture and outdoor natural conditions is taken to train
It supports and co-cultures, using a small amount of raw material, using the luminous energy under natural conditions, chlorella can not only be effectively reduced and yeast is total
The energy consumption and production cost of culture, and realize and utilize microbial co culture large scale processing yeast wastewater.
2, compared with chlorella or yeast individually cultivate processing yeast wastewater, co-cultivation can make full use of two kinds of microorganisms
Mutualism the characteristics of, issuable unfavorable factor in incubation is effectively relieved, especially in pH and dissolved oxygen adjusting side
Face.Chlorella growth generates oxygen, and the dissolved oxygen of excessive concentrations can bring oxygen injury to chlorella；Yeast growth generates CO2, together
When can generate the small organic molecules such as organic acid.The oxygen that chlorella photosynthesis discharges in co-cultivation can be by yeast cells benefit
With the CO that yeast respiration generates2And the small organic molecule etc. that growth generates can be used as carbon source promotion chlorella
Growth, thus can be very good the influence of dissolved oxygen and pH variation in balance incubation, co-culture system can be made certain
It is maintained in time in metastable environment.
3, chlorella and yeast co-cultivation also significantly increase the clean-up effect of yeast wastewater, can not only be to a certain degree
The pollutants such as COD, BOD, nitrogen, phosphorus, heavy metal in upper removal yeast wastewater, COD, NH in yeast wastewater3-N、TN、TP、PO4 3-
It can be up to 80.98%, 78.54%, 83.21%, 100%, 100% and 87.76% respectively with the total removal rate of BOD；And it can
With fresh-water-saving resource, production cost is substantially reduced, while the chlorella with comprehensive utilization value and yeast life can also be harvested
Object amount.As it can be seen that the present invention is the effective way of yeast wastewater resource utilization of reducing environmental pollution, realize.
Detailed description of the invention
Fig. 1: the biomass dry weight concentration of chlorella pyrenoidosa and rhodotorula glutinis when being inoculated with different proportion in yeast wastewater
Variation；
Fig. 2: pH value variation in yeast wastewater when inoculation different proportion chlorella pyrenoidosa and rhodotorula glutinis；
Fig. 3 (A-E): chlorella pyrenoidosa in chlorella pyrenoidosa seed liquor and rhodotorula glutinis seed liquor and yeast wastewater
Displaing micro picture (the A: chlorella pyrenoidosa seed liquor co-cultured with rhodotorula glutinis；B: rhodotorula glutinis seed liquor；C, D, E are yeast
Chlorella pyrenoidosa and rhodotorula glutinis co-culture in waste water).
Specific embodiment
Technical scheme is described further with reference to the accompanying drawing.
Present inventor when developing a kind of chlorella and yeast co-cultures the method for purification yeast wastewater, carried out as
Lower research work:
Embodiment 1: the inoculative proportion of chlorella pyrenoidosa and rhodotorula glutinis is investigated
Experimental procedure:
1) chlorella pyrenoidosa and rhodotorula glutinis cell are inoculated in respectively equipped with improvement basal medium and YM culture medium
250ml triangular flask in, be placed in constant-temperature table, 28 DEG C, 150r/m, light intensity 4000lux, continuous culture 6 days obtains albumen
Core chlorella seed liquor I and rhodotorula glutinis seed liquor I.
Improve basal medium: NaNO33750mg/L, KH2PO41250mg/L, MgSO4·7H2O 1000mg/L, EDTA
500mg/L, H3BO3114.2mg/L, CaCl2·2H2O 111mg/L, FeSO4·7H2O49.8mg/L, ZnSO4·7H2O
88.2mg/L MnCl2·4H2O 14.2mg/L, Na2M0O4·2H2O 11.92mg/L, CuSO4·5H2O 15.7mg/L, Co
(NO3)2·6H2O 4.9mg/L, C6H12O650g/L.Adjust pH6.10.
YM culture medium: casein peptone 5.0g/L, fructus hordei germinatus leaching powder 3.0g/L, glucose 10.0g/L, yeast extract 3.0g/L,
PH value 6.2 ± 0.2 (25 DEG C).
2) yeast raw wastewater pH value is 5.21, COD, NH3- N, TN and TP content be respectively 11240 ± 40,356.50 ±
2.83,625 ± 7.07 and 48.20 ± 0.69mg/L.The pH value of yeast wastewater is adjusted to 6.10, then by rhodotorula glutinis and pyrenoids
Chlorella is inoculated into the 250ml triangular flask equipped with yeast wastewater (pH value 6.10) by 1: 0,0: 1,1: 1,1: 2,1: 3 respectively,
Yeast wastewater liquid amount is 50ml, is placed in 28 DEG C, 150r/m, light intensity is continuously to cultivate 6 in the constant-temperature table of 8000lux or so
It.
In incubation, sample daily, with micro- sem observation cellular morphology and growth conditions；Flow cytometer measures cell
Number；Measure biomass dry weight；Measure COD, NH in waste water3-N、TN、PO4 3-, the water quality indicators such as TP, for assessing cell growth shape
Condition and yeast wastewater clean-up effect.
Experimental result is referring to Fig. 1, Fig. 2 and table 1.
Fig. 1 is the change of biomass dry weight concentration in yeast wastewater when being inoculated with different proportion chlorella pyrenoidosa and rhodotorula glutinis
Change.As shown in Figure 1, rhodotorula glutinis growth rate is individually cultivated in yeast wastewater is significantly greater than individually culture chlorella pyrenoidosa；
The inoculative proportion of rhodotorula glutinis and chlorella pyrenoidosa is 1: 2 and 1: 3, and after culture 6 days, biomass dry weight can be respectively reached
5.38g/L and 5.23g/L, be significantly higher than rhodotorula glutinis and chlorella pyrenoidosa individually cultivate, rhodotorula glutinis and pyrenoids bead
Culture gained maximum biomass dry weight when the inoculative proportion of algae is 1: 1.
Fig. 2 is the variation of pH value in yeast wastewater when being inoculated with different proportion chlorella pyrenoidosa and rhodotorula glutinis.It can by Fig. 2
Know, rhodotorula glutinis individually cultivates, rhodotorula glutinis: chlorella pyrenoidosa=1: under 1 inoculation condition, Initial stage of culture, pH have it is obvious under
Drop, is gradually increasing later；Rhodotorula glutinis and chlorella pyrenoidosa inoculative proportion are 1: 2 and 1: 3, and pH variation is individually trained with chlorella
Change when feeding it is almost the same, illustrate increase yeast and chlorella inoculative proportion, carry out co-culture can be effectively relieved culture just
The reduction of phase pH may be to the influence of chlorella growth.
Table 1: inoculation different proportion rhodotorula glutinis and chlorella pyrenoidosa co-culture lower yeast wastewater clean-up effect
As can be seen from Table 1, when rhodotorula glutinis is individually cultivated, pyrenoids is significantly higher than to COD removal rate in yeast wastewater
Chlorella is individually cultivated, but to yeast wastewater TN, TP and NH3The removal rate of-N is individually cultivated far away from chlorella pyrenoidosa.It is viscous
When rhodotorula and chlorella pyrenoidosa co-culture, NH in yeast wastewater3- N and TN removal rate are obviously higher than rhodotorula glutinis or egg
White nucleus chlorella is individually cultivated.
Embodiment 2: step 3) is using preferred embodiment one, chlorella pyrenoidosa and rhodotorula glutinis in outdoor 700L pipeline photoproduction
Processing yeast wastewater is co-cultured in object reactor
Experimental procedure:
1) chlorella pyrenoidosa and rhodotorula glutinis cell are inoculated in respectively equipped with improvement basal medium and YM culture medium
250ml triangular flask in, be placed in constant-temperature table, 28 DEG C, 150r/m, light intensity 4000lux, continuous culture 6 days obtains albumen
Core chlorella seed liquor I and rhodotorula glutinis seed liquor I.
Improve basal medium: NaNO33750mg/L, KH2PO41250mg/L, MgSO4·7H2O 1000mg/L, EDTA
500mg/L, H3BO3114.2mg/L, CaCl2·2H2O 111mg/L, FeSO4·7H2O 49.8mg/L, ZnSO4·7H2O
88.2mg/L MnCl2·4H2O 14.2mg/L, Na2MoO4·2H2O 11.92mg/L, CuSO4·5H2O 15.7mg/L, Co
(NO3)2·6H2O 4.9mg/L, C6H12O650g/L.Adjust pH6.10.
YM culture medium: casein peptone 5.0g/L, fructus hordei germinatus leaching powder 3.0g/L, glucose 10.0g/L, yeast extract 3.0g/L,
PH value 6.2 ± 0.2 (25 DEG C).
2) chlorella pyrenoidosa seed liquor I and rhodotorula glutinis seed liquor I are inoculated into respectively respectively equipped with the training of improvement basis
It in the big triangular flask of 2L for supporting base and YM culture medium, is placed in outdoor big shaking table, temperature control is at 28 DEG C or so, intensity of illumination
1500-3400lux, the revolving speed 180r/m of chlorella pyrenoidosa seed liquor.The revolving speed 120r/m of rhodotorula glutinis seed liquor, culture 7
It, obtains chlorella pyrenoidosa seed liquor II and rhodotorula glutinis seed liquor II.
It 3) is yeast wastewater by the pretreated molasses yeast wastewater of sand filtration, active carbon filtering, ultrafiltration three-level, yeast is useless
Water pH value is 5.21, COD, NH in yeast wastewater3-N、TN、PO4 3-Be respectively 10910 ± 70 with TP content, 307.0 ± 0.5,
490 ± 10,35 ± 0 and 82.0 ± 0.4mg/L.First stage: the pH value of yeast wastewater is adjusted to 6.08；In outdoor 700L pipeline
In bioreactor, 200L yeast wastewater (pH value 6.08) first is added, by chlorella pyrenoidosa seed liquor II and glues red ferment
Female seed liquor II is inoculated in yeast wastewater according to 4.6: 1 ratios, forms co-culture system, is cultivated 5 days；Wherein, pyrenoids is small
Ball algae and rhodotorula glutinis initial cell density are respectively 4.2 × 106Cells/ml and 9.0 × 105cells/ml.Second stage: add
Enter 200L yeast wastewater (pH value 6.08), cultivates 3 days.Phase III: yeast wastewater (pH value 6.08) is added, until pipeline
The capacity 700L of bioreactor is cultivated 3 days.Entire cultivation cycle is 11 days.Spray system is whole system cooling, temperature
Control is between 27.1-32.7 DEG C；Carbon-pH feed back control system in situ of mending is that whole system adjusts pH, and late stage of culture pH exists
6.0-7.0 or so passes through supplement CO when pH is excessively high2It is adjusted；When co-culture system lacks phosphorus source, (total phosphorus content is less than
40mg/L or phosphate content are less than 10mg/L), according to remaining phosphate and total phosphorus content, add 20-80mg/L PO4 3-The phosphorus of-P
Sour hydrogen dipotassium, potassium dihydrogen phosphate, sodium dihydrogen phosphate.
In incubation, sample daily, with micro- sem observation cellular morphology and growth conditions；Flow cytometer measures cell
Number；Measure biomass dry weight；Measure COD, BOD, NH in waste water3-N、TN、PO4 3-With the water quality indicators such as TP, for assessing cell
Upgrowth situation and yeast wastewater clean-up effect.
Experimental result is referring to table 2.
2 step 3) of table is anti-in outdoor 700L pipeline photo-biological using preferred embodiment one, chlorella pyrenoidosa and rhodotorula glutinis
Answer co-cultivation purification of waste water effect in device
| Removal rate (%) | COD | NH3-N | TN | BOD |
| Pretreatment | 22.29±0.08 | 30.66±0.67 | 21.59±0.40 | 53.06 |
| First stage (200L) | 69.84±0.12 | 58.39±0.48 | 74.50±0.71 | 52.17 |
| Second stage (400L) | 48.82±0.08 | 48.59±0.16 | 54.01±0.23 | - |
| Phase III (700L) | 22.16±0.41 | 52.56±0.24 | 55.34±1.67 | - |
| Entire incubation | 75.53±0.09 | 69.06±0.04 | 78.58±0.82 | 73.91 |
| Overall process | 80.98±0.05 | 78.54±0.18 | 83.21±0.56 | 87.76 |
As shown in Table 2, false yeasts waste water is by pretreatment, the pyrenoids bead in outdoor 700L pipeline bioreactor
The amplification step by step of algae and rhodotorula glutinis co-cultures, COD, NH in overall process yeast wastewater3- N, TN and BOD5Total removal rate can
Respectively reach 80.98%, 78.54%, 83.21% and 87.76%.In addition, during the cultivation process, co-culture system lacks
Phosphorus also needs supplement phosphate, therefore TP and PO in overall process yeast wastewater4 3-Total removal rate be 100%.
Embodiment 3: step 3) is using preferred embodiment two, chlorella pyrenoidosa and rhodotorula glutinis in outdoor 1300L pipeline light
Processing yeast wastewater is co-cultured in bioreactor
Experimental procedure:
1) chlorella pyrenoidosa and rhodotorula glutinis cell are inoculated in respectively equipped with improvement basal medium and YM culture medium
250ml triangular flask in, be placed in constant-temperature table, 28 DEG C, 150r/m, light intensity 4000lux, continuous culture 6 days obtains albumen
Core chlorella seed liquor I and rhodotorula glutinis seed liquor I.
Improve basal medium: NaNO33750mg/L, KH2PO41250mg/L, MgSO4·7H2O 1000mg/L, EDTA
500mg/L, H3BO3114.2mg/L, CaCl2·2H2O 111mg/L, FeSO4·7H2O 49.8mg/L, ZnSO4·7H2O
88.2mg/L MnCl2·4H2O 14.2mg/L, Na2MoO4·2H2O 11.92mg/L, CuSO4·5H2O 15.7mg/L, Co
(NO3)2·6H2O 4.9mg/L, C6H12O650g/L.Adjust pH6.10.
YM culture medium: casein peptone 5.0g/L, fructus hordei germinatus leaching powder 3.0g/L, glucose 10.0g/L, yeast extract 3.0g/L,
PH value 6.2 ± 0.2 (25 DEG C).
2) chlorella pyrenoidosa seed liquor I and rhodotorula glutinis seed liquor I are inoculated into respectively respectively equipped with the training of improvement basis
It in the big triangular flask of 2L for supporting base and YM culture medium, is placed in outdoor big shaking table, temperature control is at 28 DEG C or so, intensity of illumination
1500-3400lux, the revolving speed 180r/m of chlorella pyrenoidosa seed liquor.The revolving speed 120r/m of rhodotorula glutinis seed liquor, culture 7
It, obtains chlorella pyrenoidosa seed liquor II and rhodotorula glutinis seed liquor II.
It 3) is yeast wastewater by the pretreated molasses yeast wastewater of sand filtration, active carbon filtering, ultrafiltration three-level, yeast is useless
Water pH value is 3.64, COD, NH in yeast wastewater3-N、TN、PO4 3-It is respectively 13460 ± 40,847 ± 13,1480 with TP content
± 20,0 and 54.6 ± 1.4mg/L.First stage: in outdoor 700L pipeline bioreactor (organic glass pipe outside diameter 5cm)
In, 400L yeast wastewater (pH value 3.64) first is added, red ferment seed liquor II will be glued and be inoculated in yeast wastewater, rises to pH value
When to 5.2, chlorella pyrenoidosa seed liquor II is accessed, forms co-culture system, is cultivated 4 days；Wherein, rhodotorula glutinis kind is first accessed
Sub- liquid, initial cell density are 1.7 × 105Cells/ml accesses chlorella pyrenoidosa when pH value rises to 5.2, and starting is thin
Born of the same parents' density is 7.5 × 105cells/ml.Second stage: 300L yeast wastewater is added, until the capacity of pipeline bioreactor
700L is cultivated 2 days.Phase III: all cultures are transferred to 1300L pipeline photo-biological from 700L pipeline bioreactor
In reactor (organic glass pipe outside diameter 10cm), 600L yeast wastewater is added, until the capacity of pipeline bioreactor
1300L carries out outdoor co-cultivation, cultivates 6 days.Entire cultivation cycle is 12 days altogether.Spray system is whole system cooling, temperature
Control is between 29.0-33.6 DEG C；Carbon-pH feed back control system in situ of mending is that whole system adjusts pH, and late stage of culture pH exists
6.0-7.0 or so passes through supplement CO when pH is excessively high2It is adjusted；When co-culture system lacks phosphorus source, (total phosphorus content is less than
40mg/L or phosphate content are less than 10mg/L), according to remaining phosphate and total phosphorus content, add 20-80mg/L PO4 3-The phosphorus of-p
Sour hydrogen dipotassium, potassium dihydrogen phosphate, sodium dihydrogen phosphate.
In incubation, sample daily, with micro- sem observation cellular morphology and growth conditions；Flow cytometer measures cell
Number；Measure biomass dry weight；Measure COD, NH in waste water3-N、TN、PO4 3-, the water quality indicators such as TP, for assessing cell growth shape
Condition and yeast wastewater clean-up effect.
Experimental result is referring to table 3.
3 step 3) of table is anti-in outdoor 1300L pipeline photo-biological using preferred embodiment two, chlorella pyrenoidosa and rhodotorula glutinis
Answer co-cultivation purification of waste water effect in device
| Removal rate (%) | COD | NH3-N | TN |
| Pretreatment | 24.13±0.04 | 19.87±1.42 | 17.78±0.28 |
| First stage (400L) | 47.55±0.01 | 18.70±1.35 | 42.57±0.14 |
| Second stage (expands 300L) | 25.58±0.09 | 16.56±3.43 | 25.00±2.72 |
| Phase III (is expanded to 1300L) | 52.26±0.50 | 34.53±0.46 | 51.73±0.77 |
| Entire incubation | 75.71±0.01 | 59.15±0.14 | 71.96±0.06 |
| Overall process | 81.57±0.01 | 67.27±0.47 | 76.94±0.03 |
As shown in Table 3, false yeasts waste water carries out albumen in outdoor 1300L pipeline bioreactor by pretreatment
The amplification step by step of core chlorella and rhodotorula glutinis co-cultures, COD, NH in overall process yeast wastewater3The total removal rate of-N and TN can
Respectively reach 81.57%, 67.27% and 76.94%.In addition, during the cultivation process, co-culture system occurs lacking phosphorus, also need to mend
Phosphate is filled, therefore TP and PO in overall process yeast wastewater4 3-Total removal rate be 100%.
The detection method used in the embodiment of the present invention can refer to progress as described below:
(1) the following method of the measurement of microbes biomass dry weight concentrations:
Appropriate microbial culture medium is drawn, 8000r/m is centrifuged 10 minutes, is washed repeatedly with sterile water 3 times, 60 DEG C are dried to
After constant weight, with electronic balance weighing and calculating difference, 3 Duplicate Samples of each sample are indicated with mean+SD.
(2) yeast wastewater BOD5, COD, total nitrogen, total phosphorus concentration measurement with the following method:
(1) yeast wastewater BOD5Measurement:
According to the number and wastewater source of content of organics in water sample, suitable extension rate is selected.Measurement is at 20 ± 1 DEG C
At a temperature of cultivate 5 days front and back solution in dissolved oxygen difference.With BOD5Form indicates.
(2) measurement of yeast wastewater COD, total nitrogen, total phosphorus concentration:
Using the dedicated kit of HACH company, according to kit operating procedure, sample is diluted to measurement range, and examination is added
It is cleared up at different temperatures on digestion device DRB200 after agent, resolution is completely and after cooling down, in DR2700 spectrophotometer
Middle reading.
(3) yeast wastewater NH3-N、PO4 3-The measurement of concentration is with the following method:
It is measured using the multiparameter water quality analyzer of Italian Kim Yong-jin.Illustrate according to instrumentation, sample is dilute
Measurement range is released, product and corresponding reagent is loaded in cuvette, is placed in instrument and is measured and reads.
The above described is only a preferred embodiment of the present invention, be not intended to limit the present invention in any form, therefore
Without departing from the technical solutions of the present invention, to the above embodiments according to the technical essence of the invention any simply to repair
Change, equivalent variations and modification, all of which are still within the scope of the technical scheme of the invention.
Claims (4)
1. a kind of method that chlorella and yeast co-culture purification yeast wastewater, which comprises the steps of:
1) chlorella pyrenoidosa and rhodotorula glutinis cell activation culture, obtain chlorella seed liquor I and yeast starter liquid I；
2) chlorella seed liquor I being inoculated in improvement basal medium, yeast starter liquid I is inoculated in culture medium, is placed in outdoor, into
Row seed liquor culture, obtains chlorella seed liquor II and yeast starter liquid II；
3) chlorella seed liquor II and yeast starter liquid II are inoculated in the yeast wastewater that need to be handled, form co-culture system,
Carry out outdoor co-cultivation；
The formula of the improvement basal medium includes following component: NaNO3 3750mg/L, KH2PO4 1250mg/L, MgSO4·
7H2O 1000mg/L, EDTA 500mg/L, H3BO3 114.2mg/L, CaCl2·2H2O 111mg/L, FeSO4·7H2O
49.8mg/L ZnSO4·7H2O 88.2mg/L, MnCl2·4H2O 14.2mg/L, Na2MoO4·2H2O 11.92mg/L,
CuSO4·5H2O 15.7mg/L, Co (NO3)2·6H2O 4.9mg/L, C6H12O650g/L；The pH of the improvement basal medium
It is 6.10 ± 0.2；
The condition of the culture of the sub- liquid I of chlorella vulgaris and yeast starter liquid I in step 2 are as follows: cultivation temperature is 28 DEG C, intensity of illumination
For 1500-3400lux, cultivation cycle is 6-8 days；The culture revolving speed of chlorella seed liquor I is 180r/m, yeast starter liquid I
Culture revolving speed is 120r/m；
Step 3) carries out as follows:
First stage: the pH value for adjusting yeast wastewater is 5.8-6.3, takes the 25%-35% of yeast wastewater total amount, and be inoculated with thereto
The inoculative proportion of chlorella seed liquor II and yeast starter liquid II, the chlorella seed liquor II and the yeast starter liquid II is
4.6:1, forms co-culture system, and cultivation cycle is 4-6 days；
Second stage: adding the 25%-35% into yeast wastewater total amount, and cultivation cycle is 2-4 days；
Phase III: it is added again into remaining yeast wastewater, cultivation cycle is 2-4 days；
Or step 3) carries out as follows:
First stage: yeast starter liquid II is inoculated in yeast wastewater, and the amount of yeast wastewater is yeast wastewater total amount
25%-35%, the pH value of the yeast wastewater to be seeded for having yeast starter liquid II rise to 4.7-5.7, access chlorella seed liquor
II, co-culture system is formed, cultivation cycle is 3-5 days；
Second stage: adding the 18%-28% into yeast wastewater total amount, and cultivation cycle is 2-3 days；
Phase III: it is added again into remaining yeast wastewater, cultivation cycle is 5-6 days.
2. the method according to claim 1, wherein cultivation temperature is 26-35 DEG C in step 3).
3. the method according to claim 1, wherein in step 3), when the total phosphorus content of the co-culture system
Less than 40mg/L or when phosphate content is less than 10mg/L, phosphate is added.
4. described the method according to claim 1, wherein yeast starter liquid I is inoculated in culture medium in step 2
Culture medium be one of YM culture medium, malt extract medium.
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