CN108624506A - The method of microalgae and yeast mixed culture purification biogas slurry coproduction microbial biomass - Google Patents

Abstract

The invention relates to a method for microalgae and yeast mixed culture to purify biogas slurry and co-produce microbial biomass, comprising the following steps: 1) pure culture of microalgae and yeast cells to obtain microalgae seed liquid and yeast seed liquid respectively; 2) Dilute and prepare the supernatant obtained from biogas slurry pretreatment to obtain biogas slurry medium for mixed culture; 3) inoculate the biogas slurry medium with microalgae seed liquid and yeast seed liquid in proportion to establish a mixed culture system; 4) Discharging the purified biogas slurry to harvest microbial biomass. While treating the biogas slurry, the method realizes the recovery of high-protein biomass, minimizes the discharge of waste, achieves the purpose of resource recovery and transformation, energy saving, emission reduction, and efficiency enhancement, and realizes circular economy, turning waste into treasure, and green Production.

Description

Method for the mixed culture of microalgae and yeast to purify biogas slurry and co-produce microbial biomass

technical field

The invention belongs to the field of microbial fermentation, relates to a microalgae and yeast mixed culture technology, and mainly relates to a method for purifying biogas slurry and coproducing microbial biomass through mixed culture of microalgae and yeast.

Background technique

Biogas slurry is a by-product of biogas production, and its treatment and reuse have always been the main bottleneck in the development of biogas industry. In order to avoid the ecological risk and limited treatment capacity of biogas slurry directly used for agricultural irrigation, aerobic biological treatment is often used to reduce nitrogen, phosphorus, organic matter and other components in biogas slurry, but this method produces a large amount of activated sludge and CO _{Grade 2} is difficult to reuse, causing secondary pollution and carbon emissions, and does not meet the requirements of resource recycling.

The biogas slurry has sufficient nitrogen and phosphorus nutrients, which can meet the nitrogen and phosphorus requirements of microalgae growth, and the growth of microalgae can make the biogas slurry achieve the ideal nitrogen and phosphorus removal effect. Adding inorganic carbon sources and simple organic carbon sources (acetate, glycerol, etc.) that microalgae can use to the system can promote the growth of microalgae and the removal of nitrogen and phosphorus. In addition, the further degradation of organic pollutants and the supply of carbon sources can be enhanced through co-cultivation with heterotrophic microorganisms that can utilize organic matter and release CO ₂ , for example, some oily yeasts (such as Yarrowialipolytic) can use the original organic matter in the system to grow In addition, cheap organic carbon sources (such as crude glycerol by-products from biodiesel production) can be used to produce microbial oils and release CO ₂ . Compared with the single culture system, the mixed culture of microalgae and oleaginous yeast has obvious advantages in terms of pollutant removal and microbial biomass accumulation.

At present, the research on the mixed culture of microalgae and yeast has received extensive attention: for example, the Chinese patent application with the publication number CN200910237936.5 discloses a method for producing oil by using the mixed culture of yeast and algae; the Chinese patent application with the publication number CN03105314.9 The patent application discloses a method for producing astaxanthin by mixed culture and fermentation of algae and yeast. The above studies have been used to illustrate the production of oil and astaxanthin, but so far there has been no research on the use of microalgae and yeast mixed culture technology to treat biogas slurry. Therefore, it is necessary to develop a method for biogas slurry treatment and co-production of microbial biomass based on the mixed culture of microalgae and yeast to solve the problem of resource utilization of biogas slurry.

Contents of the invention

In order to solve the above-mentioned problems in the prior art, the present invention provides a method for treating biogas slurry and producing microbial biomass by mixed culture of microalgae and yeast, which can realize the recovery of biomass while processing biogas slurry, and minimize nitrogen and phosphorus emissions.

Technical purpose of the present invention is achieved by the following technical solutions:

The method for the mixed cultivation of microalgae and yeast of the present invention to purify biogas slurry and co-produce microbial biomass comprises the following steps:

1) Pure culture of microalgae and yeast cells to obtain microalgae seed liquid and yeast seed liquid respectively;

2) diluting and preparing the supernatant obtained from biogas slurry pretreatment to obtain biogas slurry medium for mixed culture;

3) Inoculate the microalgae seed solution and the yeast seed solution in proportion to the biogas slurry medium to establish a mixed culture system;

4) Discharging the purified biogas slurry and harvesting microbial biomass.

Preferably, the microalgae described in step 1) of the present invention are green algae such as Chlorella, Scenedesmus or Coccus; preferably, the microalgae are photoautotrophic or concurrently trophic Chlorella strains, optionally From one of Chlorella vulgaris, Chlorella pyrenoidosa, Chlorella ellipsoides, and Chlorella protothecoides; the yeast cells are selected from aerobic strains using glycerol as a carbon source, and can be selected from Yarrow lipolytica One of yeast, Rhodotorula viscosus, Liposaccharomyces steriensis, Rhodotorula crimson, Rhodotorula Phaffia, and Candida tropicalis. The chlorella described in the present invention is preferably Chlorella vulgaris or Chlorella pyrenoidosa, and the yeast is selected from Yarrowia lipolytica.

More preferably, the pure culture conditions in step 1) are: temperature 25-30°C, light intensity 40-80 μmol photons/m ² /s, rotation speed 150-200 rpm, and culture days 5-8 days.

Specifically, the pretreatment of biogas slurry in step 2) of the present invention includes the following steps: natural sedimentation and centrifugation, and placing in a photobioreactor for disinfection and sterilization.

More specifically, the total nitrogen content in the biogas slurry supernatant in step 2) of the present invention is above 436 mg/L, the ammonia nitrogen content is above 385 mg/L, the total phosphorus content is above 58 mg/L, and the COD content is 1278 mg /L or more, the pH value is 7.3-8.5.

Preferably, the dilution in step 2) of the present invention refers to dilution with tap water, natural water body water or return water from the cultivation process, and the dilution ratio is between 1:1 and 1:5; the deployment refers to the dilution of biogas slurry The pH was adjusted to 7.0 ± 0.5, and the carbon source was supplemented.

Preferably, the carbon source in the present invention refers to pure glycerin or crude glycerin (biodiesel by-product), and the final concentration is 1-5g/L.

Preferably, the mixed culture conditions in step 3) of the present invention are: temperature 25-30°C, light intensity 40-80 μmolphotons/m ² /s, rotation speed 150-200 rpm, and culture days 5-8 days.

Preferably, the inoculation ratio in step 3) of the present invention refers to the ratio of microalgae to yeast cells (3-5):1, and the initial concentration of yeast cells is 0.1-1×10 ⁷ cells/mL.

Preferably, the microbial biomass in step 4) of the present invention is obtained through the following steps: harvesting algae liquid, drying to obtain biomass dry powder; the trigger condition for harvesting culture liquid is: when NH ₃ -N (mg/L) And when the TP (mg/L) content is lower than 5mg/L.

Beneficial effects of the present invention:

Compared with the prior art, the method for the mixed cultivation of microalgae and yeast to purify biogas slurry and co-produce microbial biomass has the following advantages:

1), the method produces no activated sludge, and the introduced glycerin (or crude glycerol) can be converted into microbial biomass without residue;

2) Biomass recovery is achieved while biogas slurry is processed, nitrogen and phosphorus emissions are minimized, resource recovery and transformation, emission reduction, and efficiency enhancement are achieved, and circular economy, waste-to-treasure, and green production are realized.

Description of drawings

Fig. 1 is a graph showing the change and accumulation of biomass in biogas slurry in monoculture and mixed culture of microalgae and yeast in the present invention.

Detailed ways

The present invention takes the biogas slurry of a dairy farm as an example, and is also applicable to all types of biogas slurry. The present invention implements concrete steps as follows:

(1) Pretreatment of dairy farm biogas slurry: natural sedimentation and centrifugation to remove solids in the biogas slurry, thereby reducing light permeability and facilitating the polytrophic growth of microalgae. Then place it in a photobioreactor (including shake flask, columnar photobioreactor, flat plate photobioreactor, pipeline photobioreactor and open pool) for disinfection and sterilization.

The supernatant obtained after pretreatment has a total nitrogen content of more than 436mg/L, an ammonia nitrogen content of more than 385mg/L, a total phosphorus content of more than 58mg/L, a COD content of more than 1278mg/L, and a pH value of 7.3-8.5 .

Dilute and adjust the treated biogas slurry to meet the growth needs of microalgae and yeast. Dilution is to use tap water or natural water body water or return water from the cultivation process to dilute the pretreated supernatant, and the dilution ratio is between 1:1 and 1:5; the preparation refers to adding 1N HCl The pH of the solution was adjusted to 7.0±0.5, and glycerol or crude glycerol was added at a final concentration of 1-5 g/L as a carbon source.

(2) Inoculate logarithmic growth microalgae and yeast cells in a photobioreactor with pretreated dairy farm biogas slurry for mixed culture. Microalgae and yeast need to meet a specific inoculation ratio: the concentration ratio of chlorella and yeast cells (3-5): 1, and the initial concentration of yeast cells is 0.1-1×10 ⁷ cells/mL.

The yeast strain used is an aerobic strain that can use glycerol as a carbon source (one of Yarrow lipolytica, Rhodotorula viscosus, Liposaccharomyces steerii, Rhodotorula rubrum, Phaffia rhodotorula, and Candida tropicalis). The coccus is a photosynthetic autotroph or a combination of bacteria strains (one of Chlorella vulgaris, Chlorella pyrenoidosa, Chlorella ellipsoides, and Chlorella protothecoides). Preferably, the mixed culture of Chlorella vulgaris/Chlorella pyrenoidosa and Yarrow lipolytica has an ideal effect on biogas slurry treatment.

The mixed culture is as follows: the temperature is 25-30° C., the light intensity is 40-80 μmol photons/m ² /s, the rotation speed is 150-200 rpm, and the culture is 4-8 days. Harvesting can be carried out when the content of NH ₃ (mg/L) and TP (mg/L) is lower than 5mg/L.

(3) After the cultivation, the algae cells are harvested by an ultrafiltration device to prepare a concentrated solution, centrifuged to obtain wet mud, and dried to obtain dry biomass powder. This process can convert nutrients such as nitrogen and phosphorus in biogas slurry together with low-value glycerol added to the system into microbial biomass, and the obtained microbial biomass contains protein, oil, and carbohydrates, which can be used as single-cell protein, single-cell oil and chemical products. Raw materials have achieved nitrogen and phosphorus emission reduction, biogas slurry purification and resource utilization of wastewater.

The present invention will be described in further detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

Example 1

Microalgae-yeast mixed culture using cattle farm biogas slurry (glycerol concentration 2g/L)

1.1 Activation of algal species and preparation of seed solution

Pick a single algae colony of Chlorella vulgaris from the solid plate of activated BG11 medium, and inoculate it into a 250mL Erlenmeyer flask containing BG11 medium, with a liquid volume of 100mL. They were placed in a constant temperature shaker at a temperature of 28°C, a light intensity of 40 μmol photons/m ² /s, and 150 rpm for 6 days, and 2% sterile CO ₂ was introduced during the cultivation process. Pick a single colony of Yarrowia lipolytica (Yarrowia lipolytica) from the activated YPD medium solid plate, and inoculate it into a 250mL Erlenmeyer flask containing YPD medium, with a liquid volume of 100mL. Place them in a constant temperature shaker at 28°C and 150 rpm for 24 hours.

1.2 Biogas slurry pretreatment and inoculation

After sedimentation and centrifugation pretreatment, the cattle farm biogas slurry was diluted with an equal volume of tap water, adjusted to pH 7.04 with 1N hydrochloric acid, filtered through a 0.22 μm sterile filter membrane, and then put into a 250 mL Erlenmeyer flask with a volume of 100 mL, and then add sterile glycerol so that its final concentration is 2 g/L. The seed solution of Chlorella vulgaris and Y. lipolytica was seeded into the culture system, so that the concentration of algal cells at the beginning of the culture was 24.5×10 ⁶ cells/mL, and that of yeast was 8.05×10 ⁶ cells/mL. The initial index of the culture medium is: TN 218mg/L, NH ₃ -N 192.5mg/L, TP 29mg/L.

1.3 Culture method

The mixed culture is as follows: temperature 27°C, light intensity 40 μmol photons/m ² /s, rotation speed 170 rpm, and culture for 6 days.

1.4 Test method

1.3.1 Determination of TN, NH ₃ -N, TP content in wastewater

Using a special kit from HACH Company in the United States, the measurement ranges of TN, NH ₃ -N, and TP are 0-150 mg/L, 0-50 mg/L, and 0-3.5 mg/L, respectively. Follow the standard operating procedures of the kit. The average value of each sample was repeated three times, and the measured reading was multiplied by the dilution factor of the sample, which was the content of TN, NH ₃ -N, and TP in the water sample to be tested.

1.5 Biomass determination

When the content of NH ₃ -N (mg/L) and TP (mg/L) is lower than 5 mg/L, the culture solution is centrifuged, the obtained wet mud is resuspended with deionized water, and the biomass is obtained by repeated centrifugation. Transfer the biomass to a pre-weighed 1.5mL centrifuge tube, centrifuge at a high speed (12000 rpm/centrifuge for 5 minutes), remove the supernatant, dry it in a 60°C oven and weigh it.

1.6 Result Analysis

The removal rate of TN, NH ₃ -N, and TP is used as an index to reflect the purification of wastewater. As shown in Table 1, the removal rate of TP in mixed culture before and after cultivation can reach 100%, and the removal rates of TN and NH ₃ -N are 88.26% and 99.74%, respectively.

Table 1: Comparison of purification effects of biogas slurry under single culture and mixed culture of microalgae and yeast

Table 2: Comparison of biomass obtained by monoculture and mixed culture of microalgae and yeast

Biomass productivity (g/L/d) Maximum biomass concentration (g/L) Single algal culture 0.13±0.010 0.85±0.098 single yeast culture 0.09±0.003 0.92±0.005 mixed culture 0.21±0.026 1.62±0.196

Table 3: Biomass nutrient comparison obtained by monoculture and mixed culture of microalgae and yeast

Figure 1 and Table 2 show the variation of biomass and the final yield in the monoculture and mixed culture systems. Analysis shows that through 144h cultivation, the maximum biomass concentration (1.62g/L) obtained by mixed culture is greater than that of single algae culture system (0.85g/L) and single yeast culture system (0.92g/L), and the biomass of mixed culture system The yield (0.21g/L/d) was higher than that of single algae culture (0.13g/L/d) and single yeast (0.09g/L/d) culture system.

The nutrient analysis and the output situation of the biomass obtained by single culture and mixed culture system are as in Table 3, and the analysis shows that mixed culture obtains the output (0.31g/L and 0.51g/L) of oil and protein, which is greater than the single algae culture system ( 0.28g/L and 0.22g/L) and single yeast culture system (0.04g/L and 0.18g/L). Considering the removal of nitrogen and phosphorus, biomass output, oil and protein output, the mixed culture system has obvious advantages compared with the single culture system.

Apparently, the above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, rather than limiting the implementation of the present invention. For those of ordinary skill in the art, other changes or changes in different forms can be made on the basis of the above description. All the implementation manners cannot be exhaustively listed here. All obvious changes or variations derived from the technical solutions of the present invention are still within the 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.