CN106929422A - A kind of method that chlorella and yeast co-culture purification yeast wastewater - Google Patents

Abstract

The invention provides a method for purifying yeast wastewater by co-cultivating chlorella and yeast, comprising the following steps: 1) activating and culturing chlorella and yeast cells to obtain chlorella seed liquid I and yeast seed liquid I; Chlorella seed liquid I was inoculated in the improved basal medium, yeast seed liquid I was inoculated in the culture medium, and placed outdoors for seed liquid cultivation to obtain Chlorella seed liquid II and Yeast seed liquid II; 3) Chlorella seed liquid II Rhodotorula viscosum seed solution II is inoculated in the yeast wastewater to be treated to form a co-cultivation system for outdoor co-cultivation. Through three simple steps, the present invention uses a small amount of raw materials and utilizes light energy under natural conditions, not only can effectively reduce the energy consumption and production cost of the co-cultivation of chlorella and yeast, but also realizes the large-scale treatment of yeast by co-cultivation of microorganisms waste water.

Description

A method for co-culturing chlorella and yeast to purify yeast wastewater

technical field

The invention belongs to cell culture technology, relates to microalgae and yeast co-cultivation technology, and mainly relates to a method for purifying yeast wastewater by co-culturing chlorella and yeast.

Background technique

Yeast wastewater is a kind of difficult-to-treat, dark-colored, high-concentration organic wastewater produced in the yeast production process, with high content of COD, BOD ₅ , total nitrogen, total phosphorus, non-biodegradable organic pollutants and high color, etc. features. With the expansion of the scale of biopharmaceutical factories, the amount of yeast fermentation wastewater produced has increased, causing great pollution to the environment. Research on efficient and environmentally friendly treatment methods is crucial to the sustainable development of the yeast industry.

Chlorella is an algal species widely used in urban wastewater treatment. It has the advantages of fast growth rate, strong tolerance, and high protein or oil content. It can use carbon, nitrogen, and phosphorus in wastewater to synthesize its own cell components and effectively remove Nutrients such as nitrogen and phosphorus in wastewater. Yeast can effectively remove organic substances in wastewater, and the COD removal rate is as high as 68%-86%. Yeast grows faster and the culture period is short. Studies have shown that there is a mutualistic symbiosis between microalgae and yeast in the same culture system. O ₂ produced by microalgae growth can promote the growth of yeast; yeast can secrete powerful extracellular enzymes to degrade macromolecules, organic compounds and small molecules. _CO2 can be used for the growth of microalgae, thus effectively mitigating the impact of changes in pH and dissolved oxygen during the cultivation process. Co-cultivation of microalgae and yeast in yeast wastewater can make full use of the advantages of the two microorganisms, harvest valuable biomass, and purify wastewater at the same time, turning waste into treasure.

At present, although the research on microalgae and yeast co-cultivation to accumulate oil and treat wastewater has received extensive attention, there are relatively few studies on the use of microbial co-culture technology to treat wastewater on a large scale. For example, the Chinese patent application whose publication number is CN102080119 discloses a method of using industrial waste water as a medium to culture yeast and algae to produce microbial oil; Grease (Journal of Beijing University of Chemical Technology, Volume 34 Supplement II, 2007). However, the above-mentioned studies belong to indoor co-cultivation of microorganisms and small-scale treatment of wastewater.

Therefore, it is necessary to develop a method for large-scale treatment of yeast wastewater using outdoor co-cultivation of microorganisms.

Contents of the invention

In order to overcome the deficiencies in the prior art, the present invention provides a method for large-scale treatment of yeast wastewater by utilizing the outdoor co-cultivation mode of chlorella and yeast; Consumption and production costs; it has important practical value in the use of microbial co-cultivation to improve the purification effect of yeast wastewater.

In order to solve the above problems, the technical scheme adopted is as follows:

A kind of chlorella and yeast co-cultivate the method for purifying waste water, comprise the steps:

1) The chlorella and yeast cells are activated and cultured to obtain the chlorella seed liquid I and the yeast seed liquid I;

2) Inoculate the chlorella seed liquid I on the improved basal medium, inoculate the yeast seed liquid I on the medium, place it outdoors, and carry out the seed liquid culture to obtain the chlorella seed liquid II and the yeast seed liquid II;

3) Inoculate the chlorella seed liquid II and the yeast seed liquid II in the yeast wastewater to form a co-cultivation system for outdoor co-cultivation.

Outdoor seed liquid cultivation uses natural light energy to further activate and cultivate the seed liquid on a large scale, and can obtain a large amount of high-density seed liquid in a short time.

Co-cultivation of chlorella and yeast for large-scale wastewater treatment requires a large amount of high-density seed solution. If sufficient seed solution cannot be provided, the inoculation density is too low, which is not conducive to wastewater purification. The inventors of the present application found that direct co-culture of chlorella and yeast cannot obtain high-density seed liquid, so they did not directly co-cultivate chlorella and yeast at the beginning; instead, they used different culture media for chlorella and yeast After activation and seed liquid cultivation, a large amount of high-density seed liquid can be obtained in a short time. Cultivating seed liquid I and seed liquid II are essentially the same, both of which provide high-density seed liquid for outdoor large-scale cultivation. Seed liquid I is cultivated in the laboratory, and it is difficult to meet the needs of large-scale seed liquid in the laboratory. In order to quickly expand the seeds, a large amount of stable high-density seed liquid is provided, first activated and cultivated in the laboratory, and then expanded and cultivated on an outdoor large shaker.

The chlorella includes one of chlorella pyrenoidosa, chlorella vulgaris, chlorella ellipsoides, and chlorella protothecoides; Yeast, Rhodotorula rubrum, Phaffia rhodozyme, Candida tropicalis, Cryptococcus laurentii, Rhodosporidium toruloides.

Preferably, the chlorella is Chlorella pyrenoidosa, and the yeast is Rhodotorula viscosus. After screening and pairing research, the inventors of the present application found that the co-cultivation of Chlorella pyrenoidosa and Rhodotorula viscosus has the best effect on treating yeast wastewater.

The formulation of the improved basal medium is NaNO ₃ 3450-4050mg/L, KH ₂ PO ₄ 1150-1350mg/L, MgSO ₄ ·7H ₂ O 900-1100mg/L, EDTA 450-650mg/L, H ₃ BO ₃ 105-125mg/L, CaCl ₂ 2H ₂ O 101-121mg/L, FeSO ₄ 7H ₂ O 45-55mg/L, ZnSO ₄ 7H ₂ O 80-96mg/L, MnCl ₂ .4H ₂ O 13- 15mg/L, Na ₂ MoO ₄ ·2H ₂ O 11-13mg/L, CuSO ₄ ·5H ₂ O14.5-16.5mg/L, Co(NO ₃ ) ₂ ·6H ₂ O 4.5-5.5mg/L, C ₆ H ₁₂ O ₆ 45-55g/L; the pH of the improved basal medium is 6.10±0.2.

Preferably, the formulation of the improved basal medium is NaNO ₃ 3750mg/L, KH ₂ PO ₄ 1250mg/L, MgSO ₄ ·7H ₂ O 1000mg/L, EDTA 500mg/L, H ₃ BO ₃ 114.2mg/L, CaCl ₂ 2H ₂ O 111mg/L, FeSO ₄ 7H ₂ O 49.8mg/L, ZnSO ₄ 7H ₂ O 88.2mg/L, MnCl ₂ 4H ₂ O 14.2mg/L, Na ₂ M0O ₄ 2H ₂ O 11.92mg/L, CuSO ₄ 5H ₂ O 15.7mg/L, Co(NO ₃ ) ₂ 6H ₂ O 4.9mg/L, C ₆ H ₁₂ O ₆ 50g/L; the pH of the improved basal medium was 6 .10.

The amount of NaNO ₃ in the improved basal medium was three times that of the normal medium, and glucose was added as a carbon source. When the concentration of glucose is 50g/L and the concentration of sodium nitrate is 3.75g/L, after 4 days of heterotrophic culture, the glucose can be completely exhausted. At this time, the biomass concentration of Chlorella reaches the highest, and the biomass concentration is 21.31g/L L, significantly higher than the maximum biomass concentration of 13.64g/L that can be achieved when the concentration of sodium nitrate is 1.25g/L. It can be seen that the use of improved basal medium can promote the growth of Chlorella, significantly increase the biomass concentration of Chlorella, achieve rapid plant expansion, and provide stable high-density seed liquid for large-scale treatment of outdoor wastewater.

As a specific embodiment, in step 2), the yeast seed liquid I is inoculated into the medium, and the medium is one of YM medium and wort medium.

Preferably, in step 2), the yeast seed liquid I is inoculated on the medium, and the medium is YM medium.

The formula of the YM medium is casein peptone 5.0g/L, malt extract powder 3.0g/L, glucose 10.0g/L, yeast extract powder 3.0g/L, and the pH value of the YM medium is 6.2 ± 0.2 (25 ℃). Yeast grows faster in YM medium, and can obtain high-density seed liquid in a short time, and its dosage is significantly less than that in wort medium. Considering the demand and cost of large-scale seed liquid, YM medium is more suitable.

The following is the preferred scheme of step 1):

The chlorella was inoculated in the improved basal medium, and the yeast cells were inoculated in the YM medium for cell activation culture. The conditions for cell activation culture are as follows: the culture temperature is a constant temperature of 27-29° C., the rotation speed is 140-160 r/m, the light intensity is 3500-4500 lux, and the culture period is 5-7 days.

Particularly preferably, in step 1), the conditions for the cell activation culture are: the culture temperature is a constant temperature of 28° C., the rotation speed is 150 r/m, the light intensity is 4000 lux, and the culture period is 6 days.

The following is the preferred scheme of step 2):

The chlorella seed liquid I is inoculated in the improved basal medium, the yeast seed liquid I is inoculated in the YM medium, and placed outdoors for seed liquid culture to obtain the chlorella seed liquid II and the yeast seed liquid II; the seed liquid culture The conditions are as follows: the culture temperature is 27-29°C, the chlorella seed liquid culture speed is 150-210r/m, the yeast seed liquid culture speed is 90-150r/m, the light intensity is 1500-3400lux, and the culture cycle is 6- 8 days.

Particularly preferably, in step 2), the conditions for culturing the seed liquid are that the culture temperature is 28°C, the speed of cultivation of the chlorella seed liquid is 180r/m, the speed of cultivation of the yeast seed liquid is 120r/m, and the light intensity is 1500- 3400lux, the culture period is 6-8 days.

The rotation speed of chlorella seed liquid culture is higher than that of yeast seed liquid culture, because the density of chlorella in outdoor shake flasks is high, if the speed is too low, there may be cell sedimentation, which is not conducive to its growth; when yeast is cultivated outdoors, Yeast grows fast and has a high density. Because the medium contains more protein, the yeast grows and produces CO ₂ . If the speed is too high, there will be a lot of foam in the shake flask, which is not conducive to the growth of yeast, so the speed should not be too high. The speed of seed liquid culture can be adjusted according to the actual conditions and cell growth status.

The following is the preferred scheme of step 3):

Option One:

The first stage: take 25%-35% (volume) of the total amount of yeast wastewater, adjust the pH value of yeast wastewater to 5.8-6.3, and inoculate chlorella seed liquid II and yeast seed liquid II therein to form a co-culture system , the culture period is 4-6 days;

The second stage: add 25%-35% (volume) of the total amount of yeast wastewater, and the culture period is 2-4 days;

The third stage: add the remaining yeast wastewater, and the culture period is 2-4 days.

From the first stage to the third stage, the culture temperature is controlled at 26-35°C; the pH value of the culture is controlled at 6.0-7.0; the phosphate and total phosphorus content in the wastewater is sampled every day, when the total phosphorus content of the culture system is less than 40mg /L or when the phosphate root content is less than 10mg/L, according to the remaining phosphate and total phosphorus, add 20-80mg/L PO ₄ ^3- -P phosphate, the phosphate is dipotassium hydrogen phosphate, dihydrogen phosphate At least one of potassium and sodium dihydrogen phosphate.

Phosphorus is an indispensable element for the growth of chlorella and yeast. Phosphorus deficiency or deficiency is not conducive to cell growth, which in turn affects the purification effect of wastewater. The phosphate content in yeast wastewater is not high, in order to ensure the normal growth of cells, phosphate needs to be supplemented.

Preferably, the ratio of the chlorella seed liquid II and the yeast seed liquid II inoculated in the yeast wastewater is 2-8:1.

Particularly preferably, the ratio of the chlorella seed liquid II and the yeast seed liquid II inoculated in the yeast wastewater is 3-5:1.

Wherein, the ratio of the chlorella seed liquid II and the yeast seed liquid II inoculated in the yeast wastewater is the ratio of the number of cells.

Using the optimal inoculum ratio of Chlorella Seed Solution II and Yeast Seed Solution II can alleviate the impact of pH drop on the growth of Chlorella at the initial stage of cultivation; not only can more useful chlorella and yeast be obtained, but also can be better removed COD, NH ₃ -N, TN, TP and PO ₄ ^3- and other substances in yeast wastewater.

Option II:

The first stage: the yeast seed solution II is inoculated in the yeast wastewater, and the amount of the yeast wastewater is 25%-35% (volume) of the total amount of the yeast wastewater. Cultivate until the pH value rises to 4.7-5.7, insert Chlorella seed liquid II to form a co-culture system, and the culture period is 3-5 days;

The second stage: add 18%-28% (volume) of the total amount of yeast wastewater, and the culture period is 2-3 days;

The third stage: add the remaining yeast wastewater, and the culture period is 5-6 days.

From the first stage to the third stage, the culture temperature is controlled at 26-35°C; the pH value of the culture is controlled at 6.0-7.0; the phosphate and total phosphorus content in the wastewater is sampled every day, when the total phosphorus content of the culture system is less than 20 -40mg/L or when the phosphate content is less than 10mg/L, according to the remaining phosphate and total phosphorus, add 20-80mg/L PO ₄ ^3- -P phosphate, the phosphate is dipotassium hydrogen phosphate, phosphoric acid At least one of potassium dihydrogen and sodium dihydrogen phosphate.

Preferably, the yeast wastewater is molasses yeast wastewater that has been pretreated in three stages of sand filtration, activated carbon filtration, and ultrafiltration. The organic matter content in the original yeast wastewater is high, and the color value of the wastewater is high; if the chlorella is directly cultivated in the original wastewater, due to poor light transmission and low photosynthetic efficiency, the growth of chlorella is slow and the cell density is low. Through pretreatment, the organic matter concentration and chromaticity of yeast wastewater can be reduced, the co-cultivation effect can be improved, and the wastewater purification ability can be improved at the same time. Pretreated yeast wastewater: the pH value is 3.0-5.5; the contents of COD, NH ₃ -N, TN and TP in yeast wastewater are 10000-15000, 300-1000, 450-1800 and 50-200mg/L respectively. However, since the concentration of wastewater may vary from batch to batch, the range fluctuation may be larger.

The preferred solution of step 3) has a higher purification effect of yeast wastewater, and the total removal rate of COD, NH ₃ -N, TN, TP, PO ₄ ^3- and BOD ₅ in yeast wastewater can be as high as 80.98%, 78.54% respectively , 83.21%, 100%, 100% and 87.76%.

Step 3) The steps of the preferred scheme two are simpler, simplifying the step of first adjusting the pH value of the yeast wastewater; and adopting the inoculation of the yeast seed liquid II first, utilizing the growth of the yeast to degrade the macromolecular organic matter (especially the residual protein), only The content of amino nitrogen increased, which increased the pH value of the yeast wastewater; then inoculated with chlorella. Not only the steps are simple, the cost is reduced, and it is more suitable for industrial applications, but also can achieve a good purification effect of yeast wastewater. The total removal rates of COD, NH ₃ -N, TN, TP and PO ₄ ^3- in yeast wastewater can be as high as 81.57%, 67.27%, 76.94%, 100%, and 100%. In addition, preferred schemes 1 and 2 of step 3) all adopt the method of gradually adding yeast wastewater. Gradually adding yeast wastewater is equivalent to using part of the yeast wastewater to cultivate chlorella and yeast seed liquid, and obtain high-density co-cultured cells in a short period of time, and the cells are cells that have been domesticated from yeast wastewater and are more suitable for growth in yeast wastewater . This not only shortens the cultivation period of outdoor co-cultivation, but also improves the purification effect of yeast wastewater. On the other hand, with the prolongation of the culture time, the number of cells of chlorella and yeast in yeast wastewater increases continuously, and more nutrients such as nitrogen and phosphorus are needed. By gradually adding yeast wastewater, more sufficient nutrients can be provided for cell growth. , also increased the wastewater treatment capacity.

After the outdoor co-cultivation, the on-line concentration and wastewater recovery of chlorella and yeast are realized through the ultrafiltration membrane module coupled with the pipeline photobioreactor. Chlorella and yeast concentrate can be used as seed liquid for wastewater treatment, and can also be further concentrated and dried for other purposes; wastewater can be discharged after proper treatment.

The present invention has following advantage and beneficial effect:

1. The steps of the technical solution of the present invention are simple, adopting indoor seed liquid cultivation and outdoor natural conditions for seed liquid cultivation and co-cultivation, using a small amount of raw materials, utilizing light energy under natural conditions, not only can effectively reduce the co-culture of chlorella and yeast The energy consumption and production cost of cultivation have been reduced, and the large-scale treatment of yeast wastewater by co-cultivation of microorganisms has been realized.

2. Compared with treating yeast wastewater with chlorella or yeast alone, co-cultivation can make full use of the mutualistic symbiotic characteristics of the two microorganisms, and effectively alleviate possible unfavorable factors during the cultivation process, especially in terms of pH and dissolved oxygen adjustment . The growth of chlorella produces oxygen, and too high concentration of dissolved oxygen will cause oxygen damage to chlorella; the growth of yeast produces CO ₂ , and at the same time produces small molecular organic substances such as organic acids. The oxygen released by the photosynthesis of Chlorella in co-culture can be used by yeast cells, and the CO ₂ produced by yeast respiration and the small molecule organic matter produced by growth can be used as carbon sources to promote the growth of Chlorella, so the cultivation process can be well balanced The effects of dissolved oxygen and pH changes in the medium can keep the co-culture system in a relatively stable environment for a certain period of time.

3. The co-cultivation of chlorella and yeast also significantly improves the purification effect of yeast wastewater, not only can remove COD, BOD, nitrogen, phosphorus, heavy metals and other pollutants in yeast wastewater to a certain extent, COD, NH ₃ in yeast wastewater The total removal rates of -N, TN, TP, PO ₄ ^3- and BOD can be as high as 80.98%, 78.54%, 83.21%, 100%, 100% and 87.76% respectively; and it can save fresh water resources, greatly reduce production costs, and at the same time Chlorella and yeast biomass with comprehensive utilization value can also be harvested. It can be seen that the present invention is an effective way to reduce environmental pollution and realize resource utilization of yeast wastewater.

Description of drawings

Figure 1: Changes in biomass dry weight concentration of Chlorella pyrenoidosa and Rhodotorula glutinosa when inoculated with different ratios in yeast wastewater;

Figure 2: Changes in pH value in yeast wastewater when different ratios of Chlorella pyrenoidosa and Rhodotorula viscose were inoculated;

Fig. 3 (A-E): Micrographs of co-culture of Chlorella pyrenoidosa and Rhodotorula viscosus in yeast wastewater (A: Chlorella pyrenoidosa seed liquor; B : Rhodotorula viscosus seed liquor; C, D, E are the co-cultivation of Chlorella pyrenoidosa and Rhodotorula viscosus in yeast wastewater).

detailed description

The technical solution of the present invention will be further described below in conjunction with the accompanying drawings.

The inventor of the present application carried out the following research work when developing a method for co-cultivating and purifying yeast wastewater with chlorella and yeast:

Embodiment 1: investigate the inoculation ratio of Chlorella pyrenoidosa and Rhodotorula viscosus

Experimental steps:

1) Inoculate the cells of Chlorella pyrenoidosa and Rhodotorula viscosus into 250ml Erlenmeyer flasks equipped with improved basal medium and YM medium respectively, and place them in a constant temperature shaker at 28°C, 150r/m, and a light intensity of 4000lux , continuously cultivated for 6 days to obtain Chlorella pyrenoidosa seed liquid I and Rhodotorula viscosus seed liquid I.

Improved basal medium: NaNO ₃ 3750mg/L, KH ₂ PO ₄ 1250mg/L, MgSO ₄ 7H ₂ O 1000mg/L, EDTA 500mg/L, H ₃ BO ₃ 114.2mg/L, CaCl ₂ 2H ₂ O 111mg/L L, FeSO ₄ 7H ₂ O 49.8mg/L, ZnSO ₄ 7H ₂ O 88.2mg/L, MnCl ₂ 4H ₂ O 14.2mg/L, Na ₂ M0O ₄ 2H ₂ O 11.92mg/L, CuSO ₄ · 5H ₂ O 15.7 mg/L, Co(NO ₃ ) ₂ · 6H ₂ O 4.9 mg/L, C ₆ H ₁₂ O ₆ 50 g/L. Adjust the pH to 6.10.

YM medium: casein peptone 5.0g/L, malt extract powder 3.0g/L, glucose 10.0g/L, yeast extract powder 3.0g/L, pH value 6.2±0.2 (25°C).

2) The pH value of the raw yeast wastewater was 5.21, and the contents of COD, NH ₃ -N, TN and TP were 11240±40, 356.50±2.83, 625±7.07 and 48.20±0.69mg/L, respectively. Adjust the pH value of the yeast wastewater to 6.10, and then inoculate the yeast wastewater (pH In a 250ml Erlenmeyer flask with a value of 6.10), the liquid content of yeast wastewater is 50ml, placed in a constant temperature shaker at 28°C, 150r/m, and light intensity of about 8000lux, and continuously cultivated for 6 days.

During the cultivation process, samples were taken every day, and the cell morphology and growth state were observed with a microscope; the cell number was measured by flow cytometry; the dry weight of biomass was measured; the water quality of COD, NH ₃ -N, TN, PO ₄ ^3- , TP, etc. in the wastewater was measured Indicators for evaluating cell growth status and yeast wastewater purification effect.

See Figure 1, Figure 2 and Table 1 for the experimental results.

Figure 1 shows the change of biomass dry weight concentration in yeast wastewater when inoculated with different ratios of Chlorella pyrenoidosa and Rhodotorula viscose. It can be seen from Figure 1 that the growth rate of Rhodotorula viscosus cultured alone in yeast wastewater was significantly greater than that of Chlorella pyrenoidosa alone; the inoculation ratios of Rhodotorula glutinosa and Chlorella pyrenoidosa were 1:2 and 1:3, and after 6 days of culture, The dry weight of biomass can reach 5.38g/L and 5.23g/L respectively, which is significantly higher than that of Rhodotorula viscosus and Chlorella pyrenoidosa cultured alone, or when the inoculation ratio of Rhodotorula glutinosa and Chlorella pyrenoidosa is 1:1. The resulting maximum biomass dry weight.

Figure 2 shows the change of pH value in yeast wastewater when inoculated with different proportions of Chlorella pyrenoidosa and Rhodotorula viscose. It can be seen from Figure 2 that under the inoculation conditions of Rhodotorula viscosus alone and Rhodotorula viscosus: Chlorella pyrenoidosa = 1:1, the pH dropped significantly at the initial stage of culture, and then gradually increased; The ratios were 1:2 and 1:3, and the pH change was basically the same as that of Chlorella cultured alone, indicating that increasing the inoculation ratio of yeast and Chlorella for co-cultivation can effectively alleviate the pH drop at the initial stage of culture and may affect the growth of Chlorella. Impact.

Table 1: Purification effect of yeast wastewater under inoculation of different proportions of Rhodotorula viscosus and Chlorella pyrenoidosa co-culture

It can be seen from Table 1 that when Rhodotorula viscosus is cultured alone, the removal rate of COD in yeast wastewater is significantly higher than that of Chlorella pyrenoidosa alone, but the removal rate of TN, TP and NH ₃ -N in yeast wastewater is far lower than that of protein Chlorella nucleatum was cultured alone. The removal rates of NH ₃ -N and TN in yeast wastewater were significantly higher when Rhodotorula viscosus and Chlorella pyrenoidosa were cultured together than when Rhodotorula glutinosa or Chlorella pyrenoidosa were cultured alone.

Embodiment 2: Step 3) Adopt preferred scheme one, Chlorella pyrenoidosa and Rhodotorula viscosus co-cultivate and process yeast wastewater in an outdoor 700L pipeline photobioreactor

Experimental steps:

1) Inoculate the cells of Chlorella pyrenoidosa and Rhodotorula viscosus into 250ml Erlenmeyer flasks equipped with improved basal medium and YM medium respectively, and place them in a constant temperature shaker at 28°C, 150r/m, and a light intensity of 4000lux , continuously cultivated for 6 days to obtain Chlorella pyrenoidosa seed liquid I and Rhodotorula viscosus seed liquid I.

Improved basal medium: NaNO ₃ 3750mg/L, KH ₂ PO ₄ 1250mg/L, MgSO ₄ 7H ₂ O 1000mg/L, EDTA 500mg/L, H ₃ BO ₃ 114.2mg/L, CaCl ₂ 2H ₂ O 111mg/L L, FeSO ₄ 7H ₂ O 49.8mg/L, ZnSO ₄ 7H ₂ O 88.2mg/L, MnCl ₂ 4H ₂ O 14.2mg/L, Na ₂ MoO ₄ 2H ₂ O 11.92mg/L, CuSO ₄ · 5H ₂ O 15.7 mg/L, Co(NO ₃ ) ₂ · 6H ₂ O 4.9 mg/L, C ₆ H ₁₂ O ₆ 50 g/L. Adjust the pH to 6.10.

YM medium: casein peptone 5.0g/L, malt extract powder 3.0g/L, glucose 10.0g/L, yeast extract powder 3.0g/L, pH value 6.2±0.2 (25°C).

2) Inoculate Chlorella pyrenoidosa seed solution I and Rhodotorula viscosus seed solution I into 2L large triangular flasks equipped with improved basal medium and YM medium respectively, place them in an outdoor large shaker, and control the temperature at At about 28°C, the light intensity is 1500-3400lux, and the rotation speed of Chlorella pyrenoidosa seed liquid is 180r/m. The rotation speed of Rhodotorula viscosus seed solution was 120r/m, and cultured for 7 days to obtain Chlorella pyrenoidosa seed solution II and Rhodotorula viscosus seed solution II.

3) The molasses yeast wastewater pretreated by three stages of sand filtration, activated carbon filtration and ultrafiltration is yeast wastewater, the pH value of yeast wastewater is 5.21, and the contents of COD, NH ₃ -N, TN, PO ₄ ^3- and TP in yeast wastewater They were 10910±70, 307.0±0.5, 490±10, 35±0 and 82.0±0.4 mg/L, respectively. The first stage: adjust the pH value of the yeast wastewater to 6.08; in the outdoor 700L pipeline photobioreactor, first add 200L yeast wastewater (pH value is 6.08), and the Chlorella pyrenoidosa seed liquid II and Rhodotorula viscosum seeds Solution II was inoculated in yeast wastewater at a ratio of 4.6:1 to form a co-culture system and cultured for 5 days; the initial cell densities of Chlorella pyrenoidosa and Rhodotorula viscosus were 4.2×10 ⁶ cells/ml and 9.0×10 ⁵ cells/ml. The second stage: add 200L of yeast wastewater (pH value is 6.08) and cultivate for 3 days. The third stage: add yeast wastewater (pH value is 6.08) to the capacity of pipeline photobioreactor 700L, and cultivate for 3 days. The whole culture period is 11 days. The spray system cools down the entire system, and the temperature is controlled between 27.1-32.7°C; the in-situ carbon replenishment-pH feedback control device adjusts the pH for the entire system, and the pH in the later stage of cultivation is around 6.0-7.0. ₂ to adjust; when the co-culture system lacks phosphorus sources (total phosphorus content is less than 40mg/L or phosphate content is less than 10mg/L), according to the remaining phosphate and total phosphorus, add 20-80mg/L PO ₄ ^3- -P dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium dihydrogen phosphate.

During the cultivation process, samples were taken every day, and the cell morphology and growth state were observed with a microscope; the cell number was measured by flow cytometry; the dry weight of biomass was measured; COD, BOD, NH ₃ -N, TN, PO ₄ ^3- and TP in wastewater were measured It is used to evaluate the growth status of cells and the purification effect of yeast wastewater.

See Table 2 for the experimental results.

Table 2 step 3) using preferred scheme one, Chlorella pyrenoidosa and Rhodotorula viscosus co-cultivate wastewater purification effect in outdoor 700L pipeline photobioreactor

Removal rate (%) COD NH ₃ -N TN BOD preprocessing 22.29±0.08 30.66±0.67 21.59±0.40 53.06 The first stage (200L) 69.84±0.12 58.39±0.48 74.50±0.71 52.17 The second stage (400L) 48.82±0.08 48.59±0.16 54.01±0.23 - The third stage (700L) 22.16±0.41 52.56±0.24 55.34±1.67 - The whole training process 75.53±0.09 69.06±0.04 78.58±0.82 73.91 The whole process 80.98±0.05 78.54±0.18 83.21±0.56 87.76

It can be seen from Table 2 that after the pretreatment of the original yeast wastewater, the step-by-step co-cultivation of Chlorella pyrenoidosa and Rhodotorula viscosus in an outdoor 700L pipeline photobioreactor, the COD, NH ₃ -N, The total removal rates of TN and BOD ₅ can reach 80.98%, 78.54%, 83.21% and 87.76%, respectively. In addition, during the cultivation process, phosphorus deficiency occurs in the co-cultivation system, and phosphate needs to be supplemented, so the total removal rate of TP and PO ₄ ^3- in the whole process of yeast wastewater is 100%.

Embodiment 3: Step 3) Adopt preferred scheme two, Chlorella pyrenoidosa and Rhodotorula viscosus co-cultivate and process yeast wastewater in an outdoor 1300L pipeline photobioreactor

Experimental steps:

1) Inoculate the cells of Chlorella pyrenoidosa and Rhodotorula viscosus into 250ml Erlenmeyer flasks equipped with improved basal medium and YM medium respectively, and place them in a constant temperature shaker at 28°C, 150r/m, and a light intensity of 4000lux , continuously cultivated for 6 days to obtain Chlorella pyrenoidosa seed liquid I and Rhodotorula viscosus seed liquid I.

Improved basal medium: NaNO ₃ 3750mg/L, KH ₂ PO ₄ 1250mg/L, MgSO ₄ 7H ₂ O 1000mg/L, EDTA 500mg/L, H ₃ BO ₃ 114.2mg/L, CaCl ₂ 2H ₂ O 111mg/L L, FeSO ₄ 7H ₂ O 49.8mg/L, ZnSO ₄ 7H ₂ O 88.2mg/L, MnCl ₂ 4H ₂ O 14.2mg/L, Na ₂ MoO ₄ 2H ₂ O 11.92mg/L, CuSO ₄ · 5H ₂ O 15.7 mg/L, Co(NO ₃ ) ₂ · 6H ₂ O 4.9 mg/L, C ₆ H ₁₂ O ₆ 50 g/L. Adjust the pH to 6.10.

YM medium: casein peptone 5.0g/L, malt extract powder 3.0g/L, glucose 10.0g/L, yeast extract powder 3.0g/L, pH value 6.2±0.2 (25°C).

2) Inoculate Chlorella pyrenoidosa seed solution I and Rhodotorula viscosus seed solution I into 2L large triangular flasks equipped with improved basal medium and YM medium respectively, place them in an outdoor large shaker, and control the temperature at At about 28°C, the light intensity is 1500-3400lux, and the rotation speed of Chlorella pyrenoidosa seed liquid is 180r/m. The rotation speed of Rhodotorula viscosus seed solution was 120r/m, and cultured for 7 days to obtain Chlorella pyrenoidosa seed solution II and Rhodotorula viscosus seed solution II.

3) The molasses yeast wastewater pretreated by three stages of sand filtration, activated carbon filtration and ultrafiltration is yeast wastewater, the pH value of yeast wastewater is 3.64, and the contents of COD, NH ₃ -N, TN, PO ₄ ^3- and TP in yeast wastewater They were 13460±40, 847±13, 1480±20, 0 and 54.6±1.4 mg/L, respectively. The first stage: In the outdoor 700L pipeline photobioreactor (the outer diameter of the plexiglass tube is 5cm), add 400L yeast wastewater (pH value is 3.64) first, inoculate the yeast wastewater II with red yeast seed liquid II, and wait until the pH value When the temperature rises to 5.2, add Chlorella pyrenoidosa seed solution II to form a co-cultivation system, and cultivate for 4 days; among them, Rhodotorula viscosus seed solution is inserted first, and the initial cell density is 1.7×10 ⁵ cells/ml. When the pH value rose to 5.2, Chlorella pyrenoidosa was added, and the initial cell density was 7.5×10 ⁵ cells/ml. The second stage: add 300L of yeast wastewater to the capacity of the pipeline photobioreactor to 700L, and cultivate for 2 days. The third stage: Transfer all the cultures from the 700L pipeline photobioreactor to the 1300L pipeline photobioreactor (the outer diameter of the plexiglass tube is 10cm), and then add 600L yeast wastewater to the capacity of the pipeline photobioreactor 1300L. Co-cultivate outdoors for 6 days. The whole culture period is 12 days in total. The spray system cools down the entire system, and the temperature is controlled between 29.0-33.6°C; the in-situ carbon replenishment-pH feedback control device adjusts the pH for the entire system, and the pH in the later stage of cultivation is around 6.0-7.0. ₂ to adjust; when the co-culture system lacks phosphorus sources (total phosphorus content is less than 40mg/L or phosphate content is less than 10mg/L), according to the remaining phosphate and total phosphorus, add 20-80mg/L PO ₄ ^3- -p dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium dihydrogen phosphate.

During the cultivation process, samples were taken every day, and the cell morphology and growth state were observed with a microscope; the cell number was measured by flow cytometry; the dry weight of biomass was measured; the water quality of COD, NH ₃ -N, TN, PO ₄ ^3- , TP, etc. in the wastewater was measured Indicators for evaluating cell growth status and yeast wastewater purification effect.

See Table 3 for the experimental results.

Table 3 step 3) using preferred scheme two, Chlorella pyrenoidosa and Rhodotorula viscosus co-cultivate wastewater purification effect in outdoor 1300L pipeline photobioreactor

Removal rate (%) COD NH ₃ -N TN preprocessing 24.13±0.04 19.87±1.42 17.78±0.28 The first stage (400L) 47.55±0.01 18.70±1.35 42.57±0.14 The second stage (expansion 300L) 25.58±0.09 16.56±3.43 25.00±2.72 The third stage (expanded to 1300L) 52.26±0.50 34.53±0.46 51.73±0.77 The whole training process 75.71±0.01 59.15±0.14 71.96±0.06 The whole process 81.57±0.01 67.27±0.47 76.94±0.03

It can be seen from Table 3 that the original yeast wastewater was pretreated, and the step-by-step co-cultivation of Chlorella pyrenoidosa and Rhodotorula viscosus was carried out in an outdoor 1300L pipeline photobioreactor. The COD, NH ₃ -N and The total removal rate of TN can reach 81.57%, 67.27% and 76.94%, respectively. In addition, during the cultivation process, phosphorus deficiency occurs in the co-cultivation system, and phosphate needs to be supplemented, so the total removal rate of TP and PO ₄ ^3- in the whole process of yeast wastewater is 100%.

The detection method adopted in the embodiment of the present invention can be carried out with reference to the following description:

(1) The determination of the dry weight concentration of microbial biomass is as follows:

Take an appropriate amount of microbial culture solution, centrifuge at 8000r/m for 10 minutes, wash repeatedly with sterile water for 3 times, dry at 60°C to constant weight, weigh with an electronic balance and calculate the difference. Each sample has 3 parallel samples. Expressed as mean ± standard deviation.

(2) Determination of yeast wastewater BOD ₅ , COD, total nitrogen, and total phosphorus concentration adopts the following methods:

(1) Determination of BOD ₅ in yeast wastewater:

According to the amount of organic matter in the water sample and the source of the wastewater, choose the appropriate dilution factor. The difference of dissolved oxygen in the solution before and after culturing at 20±1°C for 5 days was measured. Expressed as BOD ₅ .

(2) Determination of COD, total nitrogen and total phosphorus concentration of yeast wastewater:

Use the special kit of HACH Company, according to the operation steps of the kit, dilute the sample to the measurement range, add reagents and then digest it on the digester DRB200 at different temperatures. After the digestion is complete and cooled, read in the DR2700 spectrophotometer.

(3) The determination of the concentration of NH ₃ -N and PO ₄ ^3- in yeast wastewater is as follows:

The multi-parameter water quality analyzer of Italy Hana Company was used for determination. According to the operating instructions of the instrument, dilute the sample to the measurement range, add the sample and corresponding reagents to the cuvette, place it in the instrument for measurement and read.

The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form, so any simple modification made to the above embodiments according to the technical essence of the present invention does not depart from the content of the technical solution of the present invention. , equivalent changes and modifications all still belong to the scope of the technical solution of the present invention.

Claims (10)

1. a method for the co-cultivation of chlorella and yeast to purify yeast waste water, is characterized in that, comprises the steps: 1) The chlorella and yeast cells are activated and cultured to obtain the chlorella seed liquid I and the yeast seed liquid I; 2) Inoculate the chlorella seed liquid I on the improved basal medium, inoculate the yeast seed liquid I on the medium, place it outdoors, and carry out the seed liquid culture to obtain the chlorella seed liquid II and the yeast seed liquid II; 3) Inoculate the chlorella seed solution II and the yeast seed solution II into the yeast wastewater to be treated to form a co-cultivation system for outdoor co-cultivation. 2. The method according to claim 1, characterized in that the formulation of the improved basal medium comprises the following components: NaNO ₃ 3450-4050 mg/L, KH ₂ PO ₄ 1150-1350 mg/L, MgSO ₄ ·7H ₂ O 900-1100mg/L, EDTA 450-650mg/L, H ₃ BO ₃ 105-125mg/L, CaCl ₂ 2H ₂ O 101-121mg/L, FeSO ₄ 7H ₂ O 45-55mg/L, ZnSO ₄ 7H ₂ O 80-96mg/L, MnCl ₂ 4H ₂ O 13-15mg/L, Na ₂ MoO ₄ 2H ₂ O 11-13mg/L, CuSO ₄ 5H ₂ O 14.5-16.5mg/L, Co(NO ₃ ) ₂ ·6H ₂ O 4.5-5.5 mg/L, C ₆ H ₁₂ O ₆ 45-55 g/L; the pH of the improved basal medium is 6.10±0.2. 3. The method according to claim 1, characterized in that, the conditions for the cultivation of the chlorella seed liquid I and the yeast seed liquid I in step 2) are: the cultivation temperature is 27-29° C., the light intensity is 1500-3400 lux, The culture period is 6-8 days; the culture speed of the chlorella seed liquid I is 150-210r/m, and the culture speed of the yeast seed liquid I is 90-150r/m. 4. method according to claim 1, is characterized in that, step 3) carries out according to following method: The first stage: adjust the pH value of yeast wastewater to 5.8-6.3, take 25%-35% of the total amount of yeast wastewater, and inoculate Chlorella seed liquid II and yeast seed liquid II into it to form a co-culture system, and the culture cycle 4-6 days; The second stage: add 25%-35% of the total amount of yeast wastewater, and the culture period is 2-4 days; The third stage: add the remaining yeast wastewater, and the culture period is 2-4 days. 5. The method according to claim 4, characterized in that the inoculation ratio of the chlorella seed liquid II and the yeast seed liquid II in step 3) is 2-8:1. 6. The method according to claim 1, characterized in that step 3) is carried out as follows: The first stage: Inoculate yeast seed liquid II in yeast wastewater, and the amount of yeast wastewater is 25%-35% of the total amount of yeast wastewater, and the pH value of the yeast wastewater to be inoculated with yeast seed liquid II rises to 4.7-5.7 , insert the chlorella seed solution II to form a co-cultivation system, and the culture period is 3-5 days; The second stage: add 18%-28% of the total amount of yeast wastewater, and the culture period is 2-3 days; The third stage: add the remaining yeast wastewater, and the culture period is 5-6 days. 7. The method according to any one of claims 1, 4, 6, characterized in that, in step 3), the culture temperature is 26-35°C. 8. The method according to any one of claims 1, 4, and 6, characterized in that, in step 3), when the total phosphorus content of the co-cultivation system is less than 40 mg/L or the phosphate content is less than 10 mg/L When L, add phosphate. 9. The method according to claim 1, wherein the chlorella comprises one of Chlorella pyrenoidosa, Chlorella vulgaris, Chlorella ellipsoides, and Chlorella protothecoides; Including one of Rhodotorula viscoscens, Liposporidium steerii, Saccharomyces lipolytica, Rhodotorula rubrum, Phaffia rhodozyma, Candida tropicalis, Cryptococcus laurentii, Rhodosporidium toruloides. 10. The method according to claim 1, characterized in that, in step 2), the yeast seed liquid I is inoculated in the substratum, and the substratum is one of YM substratum and wort substratum.