CN115677059A - Application of yeast in treatment of dextran industrial wastewater - Google Patents
Application of yeast in treatment of dextran industrial wastewater Download PDFInfo
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Abstract
The invention relates to the technical field of wastewater treatment, and particularly discloses application of yeast in treatment of dextran industrial wastewater, wherein the yeast is candida tropicalis CCIC 32826, and COD of the dextran industrial wastewater is 25000-55000mg/L. In the application of the invention, the candida tropicalis CCIC 32826 is applied to the dextran industrial wastewater, the COD of the dextran industrial wastewater can be effectively removed, the removal rate of the COD of the dextran industrial wastewater can reach more than 70 percent, the dextran industrial wastewater can be treated at low cost, and the wastewater discharge amount is reduced. The dextran industrial wastewater can be used for producing single-cell protein with higher economic value, the maximum single-cell protein yield can reach 8.32g/L and the maximum crude protein content can reach 50.3 percent by optimizing the components and the content of the fermentation culture solution, and the reutilization of waste resources is realized. The method for treating the dextran industrial wastewater has the advantages of simple steps, low cost and good economic and environmental benefits.
Description
Technical Field
The invention belongs to the technical field of wastewater treatment, and particularly relates to application of yeast in treatment of dextran industrial wastewater.
Background
The low molecular dextran is obtained by treating and refining a high molecular glucose polymer generated by fermenting sucrose with leuconostoc mesenteroides; belongs to a blood plasma substitute and is widely applied to the aspects of medicines, foods, industries and the like. However, in the production of dextran, about 52 tons of industrial wastewater are generated at the same time when each ton of dextran is produced, the waste liquid after the dextran is extracted contains high-concentration organic wastewater, and the COD value of the dextran industrial wastewater in a certain factory is determined to be 5.20 x 10 4 mg/L; at present, the main research on the utilization of dextran industrial wastewater is as follows: 1. extracting fructose from the fermentation waste liquid; 2. used for producing a complex enzyme preparation for feed by fermentation; 3. used for producing itaconic acid or succinic acid by fermentation.
Dextran industrial wastewater belongs to high-concentration wastewater (COD (chemical oxygen demand) > 10000mg/L, TDS (total dissolved solids) > 1%); the wastewater has high organic load concentration and high salt content; the traditional treatment process for the wastewater is biochemical treatment after dilution or evaporation desalting, but because the whole organic load of the wastewater is higher, mother liquor is generated by evaporation, the evaporation energy consumption is high, and the cost is high; the dilution method results in an increase in the amount of water to be treated as a whole and a doubling of the treatment cost. Physical and chemical methods such as Fenton oxidation, iron-carbon micro-electrolysis, extraction, resin adsorption and the like have good treatment effect on organic wastewater, but the application range is limited, and the investment cost and the operation cost also greatly limit the engineering of the methods. Therefore, efficient and low-cost treatment methods become the key to the treatment of high-concentration wastewater.
The publication No. CN 114317382A Chinese patent discloses an anaerobic strain applied to removing COD in river water and an application thereof, and provides a strain (Bacillus licheniformis with the preservation number of CCTCC NO: M2022018) for efficiently removing COD, but the COD concentration of the Bacillus licheniformis is over 1000mg/L, the growth is difficult to be good, the high-concentration COD is difficult to be removed, and the removal of COD in dextran industrial wastewater is insufficient; meanwhile, the same strain has different COD removal rates for different waste water, and the strain has close relation with the utilization rate of the waste water, so the method has great value for environmental protection aiming at the research on removing the COD in the dextran industrial waste water.
Disclosure of Invention
The invention provides application of yeast in treatment of dextran industrial wastewater, which can efficiently remove COD in the dextran industrial wastewater, realize efficient low-cost treatment of high-concentration wastewater, efficiently utilize the dextran industrial wastewater to produce single-cell protein, and realize resource recycling.
In order to achieve the purpose, the invention provides application of yeast in treating dextran industrial wastewater, wherein the yeast is candida tropicalis CCIC 32826, and COD of the dextran industrial wastewater is 25000-55000mg/L. Candida tropicalis (Candida tropicalis) was purchased from Guangdong province, microsporus Depositoris, accession number CCIC 32826. By screening strains, the candida tropicalis CCIC 32826 capable of efficiently treating the dextran industrial wastewater is obtained, COD in the dextran industrial wastewater can be removed, the dextran industrial wastewater is efficiently utilized to produce single-cell protein, and the COD in the dextran industrial wastewater is efficiently removed.
Preferably, in the above application, the treating dextran industrial wastewater comprises: adding nutrient components and salt into fermentation culture solution prepared by properly supplementing the dextran industrial wastewater or the dextran industrial wastewater, adding the fermentation culture solution into the candida tropicalis for fermentation culture, removing COD (chemical oxygen demand) of the dextran industrial wastewater, and harvesting the single-cell protein.
Preferably, in the above application, the fermentation broth comprises the following components: 100-975mL/L of dextran industrial wastewater, 2-20g/L of yeast extract powder, 0.2-4g/L of potassium dihydrogen phosphate and 0.02-1g/L of magnesium sulfate heptahydrate, wherein the pH value of the fermentation culture solution is 4.0-6.5.
Preferably, in the above application, the fermentation broth comprises the following components: the fermentation culture solution comprises the following components: 600mL/L of dextran industrial wastewater, 4.18g/L of yeast extract powder, 1g/L of potassium dihydrogen phosphate and 0.1g/L of magnesium sulfate heptahydrate, wherein the pH value of the fermentation culture solution is 5.5.
Preferably, in the above application, sodium hydroxide is used to adjust the pH.
Preferably, in the above application, the treatment of dextran industrial wastewater comprises the following steps:
(1) Mixing 100-975mL of dextran industrial wastewater, 2-20g of yeast extract powder, 0.2-4g of monopotassium phosphate and 0.02-1g of magnesium sulfate heptahydrate, adding distilled water to dilute to 1000mL, adjusting the pH to 4.0-6.5 by adopting sodium hydroxide, sterilizing at 116 ℃ for 30min, and cooling to room temperature; obtaining a fermentation culture solution;
(2) Inoculating the candida tropicalis into the fermentation culture solution obtained in the step (1) for culture, wherein the culture conditions are as follows: the fermentation temperature is 25-35 ℃, the rotating speed of a shaking table is 200-250 r/min, and the culture time is 24-48 hours;
(3) And (3) centrifuging, washing and drying the fermentation culture solution obtained in the step (2) to obtain the single-cell protein.
Preferably, in the above application, the candida tropicalis is subjected to acclimation culture.
Preferably, in the above application, the candida tropicalis domestication specifically comprises:
(1) inoculating the candida tropicalis strain into a solid culture medium I, and culturing the activated strain for 48 hours in a constant-temperature incubator at 30 ℃; the solid culture medium I is as follows: 40mL of wort, 5g of ammonium sulfate, 10g of peptone, 5g of yeast extract powder, 2g of monopotassium phosphate, 0.1g of magnesium sulfate heptahydrate, 20g of glucose, diluting to 1000mL of distilled water, measuring the pH value to be 5.5, adding 20g of agar powder, sterilizing, heating for dissolving, and cooling to obtain a solid culture medium I;
(2) transferring the strains obtained by the activation in the step (1) into a seed culture medium for amplification culture, and culturing for 48 hours at the temperature of 30 ℃ and at the speed of 220r/min to obtain a bacterial liquid; the seed culture medium is as follows: 40mL of wort, 5g of ammonium sulfate, 10g of peptone, 5g of yeast extract powder, 2g of monopotassium phosphate, 0.1g of magnesium sulfate heptahydrate, 20g of glucose, diluting to 1000mL of distilled water, measuring the pH value to be 5.5, and sterilizing to obtain a seed culture medium;
(3) transferring the bacterial liquid obtained in the step (2) into a fermentation culture medium, and culturing for 72h under the conditions of 30 ℃ and 220 r/min; transferring 100 μ L of the bacterial solution in the fermentation medium, diluting with sterile water to 1.0 x 10 7 Doubling, transferring 25 mu L of the culture medium into a solid culture medium II, uniformly scraping, and culturing for 48 hours in a constant-temperature incubator at 30 ℃; selecting the single colony strain with the optimal growth potential on the solid culture medium II, inoculating the single colony strain to the solid culture medium II in the plane culture dish, carrying out subculture, selecting the single colony strain with the optimal growth potential on the solid culture medium II after 4 times of subculture, inoculating the single colony strain to the solid culture medium I, and culturing the single colony strain in a constant-temperature incubator at 30 ℃ for 48 hours to obtain the strain; after passage is completed on the solid culture medium II, passage is carried out on the solid culture medium I for storage;
the fermentation medium is as follows: 400mL of dextran industrial wastewater, 5g of ammonium sulfate, 10g of peptone, 5g of yeast extract powder, 2g of potassium dihydrogen phosphate and 0.1g of magnesium sulfate heptahydrate, diluting the solution to 1000mL with distilled water, measuring the pH value to be 4.9, and sterilizing the solution;
the solid culture medium II comprises: 400mL of dextran industrial wastewater, 5g of ammonium sulfate, 10g of peptone, 5g of yeast extract powder, 2g of potassium dihydrogen phosphate and 0.1g of magnesium sulfate heptahydrate, diluting the mixture to 1000mL with distilled water, measuring the pH value to be 4.9, adding 20g of agar powder, sterilizing, heating for dissolving, and cooling to obtain a solid culture medium II.
Compared with the prior art, the invention has the following beneficial effects:
in the invention, the candida tropicalis CCIC 32826 capable of efficiently treating the dextran industrial wastewater is obtained by screening strains. The Candida tropicalis CCIC 32826 is applied to the dextran industrial wastewater, the COD of the dextran industrial wastewater can be effectively removed, the removal rate of the COD of the dextran industrial wastewater can reach more than 70%, the dextran industrial wastewater can be treated at low cost, and the wastewater discharge amount is reduced. The method can also produce single-cell protein with higher economic value by utilizing the dextran industrial wastewater, and realizes the reutilization of waste resources by optimizing the components and the content of the fermentation culture solution, wherein the yield of the single-cell protein can reach 8.32g/L at most, and the content of crude protein can reach 50.3 at most. The method for treating the dextran industrial wastewater has the advantages of simple steps, low cost and good economic and environmental benefits.
Drawings
FIG. 1 shows that the response in test example 3 of the present invention is OD 600 Plackett-Burman tests Pareto plots of normalized effects.
FIG. 2 is a graph of Pareto responding to the standardized effect of the Plackett-Burman test on COD removal in test example 3 of the present invention.
FIG. 3 is a graph showing OD at pH 5 maintained in Box-Behnken experiment in test example 3 of the present invention 600 And the contour map of yeast extract powder and waste water.
FIG. 4 is a graph showing OD values at which 0.4g/100mL of yeast extract was maintained in the Box-Behnken experiment in test example 3 of the present invention 600 Contour plot with pH and wastewater.
FIG. 5 is OD at 50mL/100mL of waste water in Box-Behnken experiment in test example 3 of the present invention 600 Contour plots with pH, yeast extract powder.
FIG. 6 is a contour plot of COD removal rate versus yeast extract powder and wastewater when pH was maintained at 5 in the Box-Behnken experiment in test example 3 of the present invention.
FIG. 7 is a contour plot of COD removal rate, pH, and wastewater with the yeast extract powder maintained at 0.4g/100mL in the Box-Behnken experiment in Experimental example 3 of the present invention.
FIG. 8 is a contour plot of COD removal rate, pH and yeast extract powder with the wastewater being maintained at 50mL/100mL in the Box-Behnken experiment in test example 3 of the present invention.
FIG. 9 is an optimization response chart in test example 3 of the present invention.
Detailed Description
The following detailed description of specific embodiments of the invention is provided, but it should be understood that the scope of the invention is not limited to the specific embodiments.
Example 1
The application of the yeast in the treatment of the dextran industrial wastewater in the embodiment is that the yeast is Candida tropicalis, is purchased from China industrial microorganism strain preservation management center, has the strain number of CICC No.32826, and has the preservation address of No. 6 building of Zhonglu 24 Jiuxianqiao of the Inula Britain of the Chaoyang district, beijing City. The COD of the dextran industrial wastewater is 52000mg/L.
The method for treating the dextran industrial wastewater by using the candida tropicalis comprises the following steps:
(1) Mixing 600mL of dextran industrial wastewater, 4.18g of yeast extract powder, 1g of monopotassium phosphate and 0.1g of magnesium sulfate heptahydrate, adding distilled water to dilute to 1000mL, adjusting the pH to 5.5 by adopting sodium hydroxide, sterilizing at 116 ℃ for 30min, and cooling to room temperature to obtain a fermentation culture solution;
(2) Inoculating candida tropicalis to the fermentation culture solution obtained in the step (1), and culturing on a shaking table, wherein the inoculation amount is 20mL, and the culture conditions are as follows: the fermentation temperature is 30 ℃, the rotating speed of a shaking table is 220r/min, and the culture time is 48h;
(3) Centrifuging the fermentation culture solution obtained in the step (2) at the rotation speed of 10000r/min for 10min, detecting the COD removal rate of the supernatant, collecting the precipitate, washing with water for 3 times, and drying at 105 ℃ to constant weight to obtain the single-cell protein.
In the embodiment, the COD removal rate of the dextran industrial wastewater is 73.1 percent, the yield of the single-cell protein is 8.32g/L, and the content of the crude protein is 50.3 percent.
The candida tropicalis in the step (2) is purchased from China industrial microorganism strain preservation management center, the strain number is CICC No.32826, and the strain is activated and domesticated for inoculation.
The Candida tropicalis CICC32826 activation and domestication concretely comprises the following steps:
(1) activating strains: respectively inoculating 1-loop 32826 strain to the solid culture medium I under aseptic operation, and culturing at 30 deg.C in a constant temperature incubator for 48h to recover the strain;
(2) and (3) strain amplification culture: inoculating the strain on the solid culture medium I after 1-ring activation by using an inoculating loop, transferring the strain to a seed culture medium with liquid loading capacity of 50mL/250mL, and culturing for 48h under the conditions of 30 ℃ and 220 r/min;
(3) domestication: measuring 20mL of bacterial liquid in seed culture medium, and transferring the bacterial liquid to a liquid containing quantity of 250mL/1000mLCulturing in fermentation culture medium at 30 deg.C and 220r/min for 72 hr; transferring 100 μ L of the culture solution in the fermentation medium, and diluting with sterile water to 1.0 x 10 7 Doubling, transferring 25 mu L of the culture medium into a solid culture medium II, uniformly scraping, and culturing for 48 hours in a constant-temperature incubator at 30 ℃; and selecting the single colony strain with the optimal growth potential on the solid culture medium II, inoculating the single colony strain with the optimal growth potential on the solid culture medium II onto the solid culture medium II in the ring-1 plane culture dish, carrying out subculture, selecting the single colony strain with the optimal growth potential on the solid culture medium II after 4 times of subculture, inoculating the single colony strain with the optimal growth potential on the solid culture medium II onto the solid culture medium I, and placing the solid culture medium I in a constant-temperature incubator for culturing for 48 hours at 30 ℃.
The solid culture medium I is: 40mL of wort, 5g of ammonium sulfate, 10g of peptone, 5g of yeast extract powder, 2g of monopotassium phosphate, 0.1g of magnesium sulfate heptahydrate, 20g of glucose, diluting to 1000mL of distilled water, measuring the pH value to be 5.5, adding 20g of agar powder, sterilizing, heating for dissolving, and cooling to obtain a solid culture medium I;
the seed culture medium is as follows: 40mL of wort, 5g of ammonium sulfate, 10g of peptone, 5g of yeast extract powder, 2g of monopotassium phosphate, 0.1g of magnesium sulfate heptahydrate, 20g of glucose, diluting to 1000mL of distilled water, measuring the pH value to be 5.5, and sterilizing to obtain a seed culture medium;
the fermentation medium is as follows: 400mL of dextran industrial wastewater, 5g of ammonium sulfate, 10g of peptone, 5g of yeast extract powder, 2g of potassium dihydrogen phosphate and 0.1g of magnesium sulfate heptahydrate, diluting the mixture to 1000mL with distilled water, measuring the pH value to be 4.9, and sterilizing;
the solid culture medium II comprises: 400mL of dextran industrial wastewater, 5g of ammonium sulfate, 10g of peptone, 5g of yeast extract powder, 2g of potassium dihydrogen phosphate and 0.1g of magnesium sulfate heptahydrate, diluting the solution to 1000mL with distilled water, measuring the pH value to be 4.9, adding 20g of agar powder, sterilizing, heating for dissolving, and cooling to obtain a solid culture medium II.
Inoculating the domesticated candida tropicalis into a seed culture medium 1 for amplification culture, wherein the inoculation amount is 20mL, and culturing for 48 hours on a shaking table at the temperature of 30 ℃ and the rotating speed of 220 r/min; the components of the seed culture medium 1 comprise 25g of glucose monohydrate, 2g of peptone, 0.6g of yeast extract powder, 1g of monopotassium phosphate and 0.1g of magnesium sulfate heptahydrate, the components are diluted to 1000mL by distilled water, the measured pH value is =5.5, and the components are packaged: sealing the triangular flask with the volume of 200mL/1000mL by a sealing film, sterilizing at 116 ℃ for 30min, and cooling to room temperature. The candida tropicalis bacterial liquid obtained by culture is used for being inoculated into a fermentation culture solution.
Example 2
The method for producing single-cell protein by using dextran industrial wastewater in the embodiment is the same as the embodiment 1, except that: the fermentation broth of this example had the following composition: 600mL/L of dextran industrial wastewater, 6g/L of yeast extract powder, 1g/L of monopotassium phosphate and 0.1g/L of magnesium sulfate heptahydrate.
Test example 1 bacterial strain screening test for efficiently utilizing dextran industrial wastewater
The concentration of the dextran industrial wastewater is 52000mg/L.
The test method comprises the following steps: respectively adopting monohydrate glucose and dextran industrial wastewater as carbon sources for culture, wherein the monohydrate glucose as the carbon source specifically comprises the following steps: inoculating different strains in the table 1 to a seed culture medium 1 for culture, and then inoculating the strains to a fermentation culture medium 1; the dextran industrial wastewater is used as a carbon source and specifically comprises the following steps: inoculating different strains in the table 1 into a seed culture medium 2, culturing, inoculating into a fermentation culture medium 2, and culturing under the following conditions: the inoculation amount is 20mL, the fermentation temperature is 30 ℃, the rotating speed of a shaking table is 220r/min, and the culture time is 48h.
The fermentation medium 1 (seed medium 1) comprises the following components: 25g of glucose monohydrate, 2g of peptone, 0.6g of yeast extract powder, 1g of potassium dihydrogen phosphate and 0.1g of magnesium sulfate heptahydrate, diluting the mixture to 1000mL with distilled water, and adjusting the pH value to =5.5 with sodium hydroxide. Subpackaging: sealing the triangular flask with the volume of 200mL/1000mL by a sealing film, sterilizing at 116 ℃ for 30min, and cooling to room temperature.
The components of the fermentation medium 2 (seed medium 2) include: 600mL of dextran industrial wastewater, 2g of peptone, 0.6g of yeast extract powder, 1g of potassium dihydrogen phosphate, 0.1g of magnesium sulfate heptahydrate, diluting to 1000mL of distilled water, and adjusting the pH value to =5.5 by sodium hydroxide. Subpackaging: sealing the triangular flask with the volume of 200mL/1000mL by a sealing film, sterilizing at 116 ℃ for 30min, and cooling to room temperature.
TABLE 1 information on the strains
| Strain name | Source |
| 32826 (Candida tropicalis) | Purchased in China center for culture collection and management of industrial microorganisms, activated and domesticated to obtain |
| 1769 (Candida utilis) | Purchased in China center for culture collection and management of industrial microorganisms, activated and domesticated to obtain |
| Alcohol yeast | Separating and purifying alcohol yeast of a certain brewery to obtain |
| Bread yeast | Separating and purifying certain baker's yeast to obtain |
| High-sugar yeast | Separating and purifying Angel high sugar yeast |
| Wine yeast | Separating and purifying Angel wine yeast to obtain |
And (3) performing bacteria density detection on the bacteria liquid after the fermentation medium is cultured, adopting UV detection, taking the culture medium before inoculation as a blank control, and detecting the wavelength of 600nm, wherein the detection results are shown in tables 2 and 3. As can be seen from tables 2 and 3, OD was obtained when glucose monohydrate was used as a carbon source in the formulation 600 The order from large to small is: alcohol yeast>1769>32826>Baker's yeast, however, O when dextran industrial wastewater is used as a carbon source in the formulationD 600 The order from large to small is: 32826>1769>Alcohol yeast>High-sugar yeast>Wine yeast>Baker's yeast; the result shows that the 32826 bacterial strain can be efficiently utilized for the dextran industrial wastewater.
TABLE 2 results of density measurements of different strains cultured with glucose monohydrate
TABLE 3 results of bacterial density detection of different bacterial strains after culturing by dextran industrial wastewater
Test example 2 preliminary screening of fermentation broth components
2.1 optimum content of dextran Industrial waste Water
The test method comprises the following steps: inoculating domesticated candida tropicalis 32826 to a seed culture medium for culture, wherein the culture conditions are as follows: the inoculation amount is 20mL, the fermentation temperature is 30 ℃, the rotating speed of a shaking table is 220r/min, and the culture time is 48h. Then inoculating the bacterial liquid cultured by the seed culture medium into different fermentation culture media (shown in table 4) for culture under the culture conditions that: the inoculation amount is 20mL, the fermentation temperature is 30 ℃, the rotating speed of a shaking table is 220r/min, and the culture time is 48h.
The components of the seed culture medium comprise: 25g of glucose monohydrate, 2g of peptone, 0.6g of yeast extract powder, 1g of potassium dihydrogen phosphate, 0.1g of magnesium sulfate heptahydrate, diluting to 1000mL with distilled water, and adjusting the pH value to =5.5 with sodium hydroxide. Subpackaging: sealing the triangular flask with a sealing film in a 100mL/500mL triangular flask, sterilizing at 116 ℃ for 30min, and cooling to room temperature.
After the fermentation medium is cultured, bacterial liquid is subjected to bacteria density detection, UV detection is adopted, the medium before inoculation is used as a blank control, the detection wavelength is 600nm, and the detection results are shown in Table 5. As can be seen from Table 5, the higher the density of the 32826 strain is as the content of the dextran industrial wastewater increases, the positive correlation between the density of the 32826 strain and the content of the dextran industrial wastewater is shown. The bacteria density is increased fastest when the content of the dextran industrial wastewater in the formula is less than 60% (V/V), and is increased slowly when the content of the dextran industrial wastewater in the formula is more than 60% (V/V), so that the optimal dextran industrial wastewater content in the formula is about 60% (V/V).
TABLE 4 fermentation culture medium formula table for different dextran industrial wastewater contents
TABLE 5 results of density test of bacteria in bacteria cultured in different fermentation medium formulas
2.2 optimum content of Nitrogen Source
(1) Optimum content of ammonium sulfate as nitrogen source
The test procedure is as in 2.1, and the composition of the fermentation medium is shown in Table 6. The detection method is the same as 2.1, and the detection results are shown in Table 7. As is clear from Table 7, the cell density of the 32826 strain was lower as the ammonium sulfate content increased, and it was found that the cell density of the 32826 strain negatively correlated with the ammonium sulfate content. The bacteria density is highest when the ammonium sulfate content is 0 in the formula, and the ammonium sulfate is not suitable as a nitrogen source.
TABLE 6 fermentation culture medium formula table with different ammonium sulfate contents
TABLE 7 results of density measurement of bacteria in bacteria liquid cultured in fermentation medium with different ammonium sulfate contents
| Fermentation Medium number | Dilution factor | OD 600 |
| 1 | 11 | 1.768 |
| 2 | 11 | 1.730 |
| 3 | 11 | 1.718 |
| 4 | 11 | 1.699 |
| 5 | 11 | 1.672 |
| 6 | 11 | 1.638 |
(2) Optimum content of urea as nitrogen source
The test procedure is as in 2.1, and the composition of the fermentation medium is shown in Table 8. The detection method was the same as 2.1, and the detection results are shown in Table 9. As can be seen from table 9, the density of the strain 32826 was lower as the urea content increased, and it was found that the density of the strain 32826 negatively correlated with the urea content. The density of bacteria is highest when the urea content in the formula is 0, so that urea is not suitable as a nitrogen source.
TABLE 8 fermentation culture medium formula table for different urea contents
TABLE 9 bacterial density test results of cultured bacteria solutions with different urea content fermentation medium formulas
| Fermentation Medium number | Dilution factor | OD 600 |
| 1 | 11 | 1.685 |
| 2 | 11 | 1.624 |
| 3 | 11 | 1.533 |
| 4 | 11 | 1.367 |
(3) Optimum content of yeast extract powder as nitrogen source
The test procedure is as in 2.1, and the composition of the fermentation medium is shown in Table 10. The detection method was the same as 2.1, and the detection results are shown in Table 11.
TABLE 10 fermentation culture medium formula table for different yeast extract powder contents
TABLE 11 Table of density results of bacteria in bacteria culture with different yeast extract content fermentation medium formulas
After the fermentation medium is cultured, the bacterial liquid is centrifuged (10000 r/min and 20 min), and the supernatant is subjected to COD removal rate detection by using a dichromate method, wherein the detection results are shown in Table 12.
The COD removal rate was calculated from the measurement data and the following calculation formula (1):
COD removal rate = [ COD (0) -COD (x) ]/COD (0) × 100% formula (1)
COD (0) is the initial COD of the dextran industrial wastewater culture solution water body of the bacterial strain just inoculated, and COD (x) is the COD of the culture solution sample taken at the x time point.
As can be seen from tables 11 and 12, the density of the 32826 strain increased as the content of the yeast extract increased, and the density of the 32826 strain showed a positive correlation with the content of the yeast extract; the COD removal rate results are increased and then decreased along with the increase of the yeast extract powder content. The COD removal rate is highest when the yeast extract powder content in the formula 3 is high, so that the optimal yeast extract powder content in the formula is about 6g/L.
TABLE 12 COD removal rate after fermentation medium formula culture of different yeast extract powder contents
| Fermentation Medium numbering | COD removal Rate (%) |
| 1 | 66.3 |
| 2 | 68.8 |
| 3 | 69.2 |
| 4 | 68.4 |
| 5 | 67.2 |
The nitrogen source experiments are only partial experiments, and the growth effect of the 32826 strain is not ideal when experiments such as (organic nitrogen) peptone, (nitro nitrogen) potassium nitrate and ammonia (amino nitrogen) are carried out.
In conclusion, the 32826 strain is classified for nitrogen source utilization, and the 32826 strain can promote the growth of thalli and has a remarkable effect on removing COD by adopting nitrogen source-yeast extract powder for culture.
2.3 optimum Potassium dihydrogen phosphate content
The test procedure is as in 2.1, and the composition of the fermentation medium is shown in Table 13. The detection method was the same as 2.1, and the detection results are shown in Table 14. As shown in Table 14, the density of the 32826 strain increased and then decreased with the increase of the concentration of potassium dihydrogen phosphate, and the optimum concentration of potassium dihydrogen phosphate in the formulation was about 1g/L.
TABLE 13 fermentation culture medium formula table for different potassium dihydrogen phosphate contents
TABLE 14 density of bacteria liquid after fermentation culture medium formula culture with different potassium dihydrogen phosphate contents
2.4 optimum content of magnesium sulfate heptahydrate
The test procedure is as in 2.1, and the composition of the fermentation medium is shown in Table 15. The detection method was the same as 2.1, and the detection results are shown in Table 16. As can be seen from Table 16, the density of the 32826 strain increased first and then decreased as the magnesium sulfate heptahydrate content increased. Therefore, the optimum magnesium sulfate heptahydrate content in the formulation is about 0.1g/L.
TABLE 15 fermentation culture medium formula table for different magnesium sulfate heptahydrate contents
TABLE 16 bacterial liquid density detection after fermentation culture of different magnesium sulfate heptahydrate content fermentation medium formula
2.5pH optimum
The test procedure is as in 2.1, and the composition of the fermentation medium is shown in Table 17. The detection method was the same as 2.1, and the detection results are shown in Table 18. As shown in Table 18, the density of 32826 strain increased and then decreased with increasing pH, and the optimum pH of the formulation was about 5.5.
TABLE 17 fermentation Medium formulation Table with different pH values
TABLE 18 bacterial liquid density measurements after fermentation medium formula cultivation at different pH values
From the above, the basic formula obtained according to the optimum content of each component in the formula is as follows: 600mL/L of dextran industrial wastewater, 6g/L of yeast extract powder, 1g/L of monopotassium phosphate, 0.1g/L of magnesium sulfate heptahydrate and pH of 5.5.
Test example 3 optimization of fermentation broth composition content
The components of the seed culture medium comprise 25g of glucose monohydrate, 2g of peptone, 0.6g of yeast extract powder, 1g of potassium dihydrogen phosphate and 0.1g of magnesium sulfate heptahydrate, distilled water is diluted to 1000mL, and the pH value of sodium hydroxide is adjusted to be =5.5. Subpackaging: sealing with a sealing film in a 100mL/500mL triangular flask, sterilizing at 116 deg.C for 30min, and cooling to room temperature. The culture conditions are as follows: the inoculation amount is 20mL, the fermentation temperature is 30 ℃, the rotating speed of a shaking table is 220r/min, and the culture time is 48 hours.
The primary formulation (the base formulation obtained in experimental example 1) was optimized using the Plackett-Burman design and the Box-Behnken design using Minitab software.
3.1Plackett-Burman design test
Screening for primary obvious factors on strain growth or COD removal, plackett-Burman designed the fermentation medium formulation as shown in Table 19.
TABLE 19 Plackett-Burman design fermentation Medium formulation Table with Minitab software
The preparation, inoculation and culture tests were carried out according to the above designed test formula, and the test method was the same as that in 2.1 of test example 2. After the fermentation medium is completely cultured, the bacterial liquid is subjected to bacteria density detection, and the detection results are shown in table 20, table 21 and figure 1.
Table 20 shows the density of bacteria in the bacterial liquid after the fermentation medium formulation is cultured
TABLE 21 response as OD 600 Plackett-Burman experiment factor level and main effect analysis table thereof
Centrifuging the bacterial liquid (10000 r/min, 20 min) after the fermentation medium is cultured, detecting the COD removal rate of the supernatant, and detecting by a dichromate method, wherein the detection results are shown in table 22, table 23 and figure 2.
Table 22 shows COD removal rate of supernatant after centrifugation of bacterial liquid cultured in fermentation medium formula
TABLE 23 response is COD Plackett-Burman experimental factor level and its main effect analysis table
As can be seen from tables 20 to 23, when the running serial number of the formula of the fermentation medium is 1 to 12 when the 32826 strain is subjected to Plackett-Burman design by adopting Minitab software, the credibility of the 2 factors of the dextran industrial wastewater and the yeast extract powder in the bacteria density result is more than 90 percent, and the credibility of the 1 factor of the dextran industrial wastewater in the COD removal rate result is more than 90 percent; because the bacterium density and the COD removal rate meet less than 3 with the reliability of more than 90%, in order to carry out Box-Behnken design, 3 factors with the highest reliability are selected for experimental design, and the experimental design respectively comprises the following steps: dextran industrial wastewater (A), yeast extract powder (B) and pH value (E).
3.2Box-Behnken design test
3 factors with obvious strain growth or COD removal in the formula are optimized
The formula of the Box-Behnken designed fermentation medium is shown in Table 22, and the preparation, inoculation and culture tests are carried out according to the designed test formula. After the fermentation medium was cultured, the bacterial liquid was subjected to density measurement, and the results are shown in tables 25 to 27 and FIGS. 3 to 5.
TABLE 24 formula of Box-Behnken designed fermentation medium using Minitab software
Table 25 shows density of bacteria in bacterial liquid test after fermentation with fermentation medium formula
TABLE 26 Box-Behnken Experimental response as OD 600 Table of estimated regression coefficients
TABLE 27 Box-Behnken Experimental response as OD 600 Analysis of variance table
After the fermentation medium is cultured, the bacterial liquid is centrifuged (10 000r/min and 20 min), and the supernatant is subjected to COD removal rate detection by a dichromate method, wherein the detection results are shown in tables 28-30 and FIGS. 6-8.
TABLE 28 COD removal Rate after cultivation by design of fermentation Medium formulation
| Fermentation medium operating sequence | COD removal Rate (%) |
| 1 | 69.9 |
| 2 | 69.2 |
| 3 | 78.1 |
| 4 | 75.7 |
| 5 | 71.9 |
| 6 | 84.5 |
| 7 | 82.5 |
| 8 | 84.0 |
| 9 | 82.9 |
| 10 | 78.1 |
| 11 | 78.4 |
| 12 | 61.8 |
| 13 | 80.7 |
| 14 | 76.4 |
| 15 | 70.9 |
TABLE 29 estimated regression coefficient Table for Box-Behnken Experimental response as COD
TABLE 30 Box-Behnken ANOVA TABLE with Experimental response as COD
In the result, A is dextran industrial wastewater, B is yeast extract powder, and E is pH value. As can be seen from tables 25 to 27 and FIGS. 3 to 5, when the running number of the formula of the Box-Behnken fermentation medium design with the Minitab software for the 32826 strain is 1 to 15, the RSM analysis screens the optimal level of the important factors, and the level of the important factors used for the RSM analysis is shown in Table 24; the results are shown in Table 25. Performing secondary regression analysis by using the OD600 level as a response value according to an experimental result designed by Box-Behnken in a table 25, wherein a regression equation is as follows:
OD600= 0.74-0.0020A + 0.366B-0.137C + 0.000046A + 0.071B + 0.0129C + 0.00256A + B + 0.00105C + C-0.0500B. The estimated regression coefficients for OD600 and the analysis of variance results of the regression equations are shown in tables 26 and 27. Determining coefficient R of the model based on regression analysis result 2 =98.10%, indicating that the model can account for the change in density of the bacteria from the 98.10% experiment, indicating a better fit of the equation. In table 27, the mismatch term F =5.69, indicating that the mismatch is not significant and the regression is significant. Contour plots are further drawn, see FIGS. 3-5.
In the result, A is dextran industrial wastewater, B is yeast extract powder, and E is pH value. As can be seen from tables 28 to 30 and FIGS. 6 to 8, when the running number of the formula of the Box-Behnken designed fermentation medium of the 32826 strain is 1 to 15 by adopting Minitab software, the RSM analysis screens the optimal level of the important factors, and the level of the important factors used for the RSM analysis is shown in Table 24; the results are shown in Table 28. Performing secondary regression analysis by using the COD removal rate level as a response value according to an experimental result designed by Box-Behnken in a table 28, wherein a regression equation is as follows:
OD600=490-4.70 × A +75 × B-134 × C +0.0419 × A +4.1 × B +14.4 × C-0.07 × A + B +0.115 × A-18.5 × B + C. The estimated regression coefficients of the COD removal rates and the analysis of variance results of the regression equations are shown in tables 29 and 30. Determining coefficient R of the model based on regression analysis result 2 =61.63%, indicating that the model can account for the change in density of the bacteria from the 61.63% experiment, indicating a better fit of the equation. In table 30, the mismatch term F =0.27, indicating that the mismatch is not significant and the regression is significant. Contour plots are further plotted, see FIGS. 6-8.
The uncoded values (60.0, 0.4182 and 5.50) of the main factors (A, B and E) and the optimal levels of dextran industrial wastewater, yeast extract powder and pH value (60.0 g/100mL, 0.4182g/100mL and 5.50 respectively) are obtained by optimizing response analysis, the OD600 of the fermentation liquor (with the dilution multiple of 21 times) is 0.8859, and the COD removal rate is 84.44 percent, which is shown in figure 9.
Test example 4
The strain is as follows: 32826
The components of the seed culture medium comprise 25g of glucose monohydrate, 2g of peptone, 0.6g of yeast extract powder, 1g of potassium dihydrogen phosphate and 0.1g of magnesium sulfate heptahydrate, the components are diluted to 1000mL by distilled water, and the pH value of sodium hydroxide is adjusted to be =5.5. Subpackaging: sealing the triangular flask with a sealing film in a 100mL/500mL triangular flask, sterilizing at 116 ℃ for 30min, and cooling to room temperature.
The formula of the initial fermentation culture solution is as follows: 400mL of dextran industrial wastewater, 5g of ammonium sulfate, 10g of peptone, 5g of yeast extract powder, 2g of potassium dihydrogen phosphate, 0.1g of magnesium sulfate heptahydrate and distilled water are diluted to 1000ml, and the pH value is 4.9.
The optimized fermentation culture solution comprises 600mL of dextran industrial wastewater, 4.18g of dextran industrial wastewater, 1.0g of potassium dihydrogen phosphate and 0.1g of magnesium sulfate heptahydrate, wherein the dextran industrial wastewater is diluted to 1000mL by distilled water, and the pH value of the fermentation culture solution is adjusted to be =5.5 by sodium hydroxide. Subpackaging: sealing with a sealing film in a 100mL/500mL triangular flask, sterilizing at 116 deg.C for 30min, and cooling to room temperature.
The test method comprises the following steps: inoculating domesticated candida tropicalis 32826 to a seed culture medium for culture, wherein the culture conditions are as follows: the inoculation amount is 20mL, the fermentation temperature is 30 ℃, the rotating speed of a shaking table is 220r/min, and the culture time is 48h. Then inoculating the bacterial liquid decibel cultured by the seed culture medium into an initial fermentation culture medium and an optimized fermentation culture medium for culture, wherein the culture conditions are as follows: the inoculation amount is 20mL, the fermentation temperature is 30 ℃, the rotating speed of a shaking table is 220r/min, and the culture time is 48h; detecting the COD removal rate, centrifugally collecting microbial biomass, washing with water, and drying to obtain the single-cell protein. The results are shown in Table 31.
As can be seen from Table 31, the biomass, crude protein content and COD removal rate of the optimized fermentation broth were as follows: 8.18g/L, 50.3g/100g, 73.1%, has reached the optimization target level; the level of biomass obtained in the optimized medium was increased by about 9.7% compared to the initial fermentation broth; the level of crude protein content increased by about 13.5%; the level of COD removal rate is improved by about 38.7%; the improvement amplitude is obvious.
TABLE 31 removal rates of biomass, crude protein, COD
| Culture medium | Biomass (g/L) | Crude protein content (g/100 g) | COD removal Rate (%) |
| Optimized fermentation broth | 8.32 | 50.3 | 73.1 |
| Initial fermentation broth | 7.58 | 44.3 | 52.7 |
The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.
Claims (8)
1. The application of the yeast in treatment of the dextran industrial wastewater is characterized in that the yeast is Candida tropicalis (Candida tropicalis) CCIC 32826, and COD of the dextran industrial wastewater is 25000-55000mg/L.
2. The use of claim 1, wherein the treating dextran industrial wastewater comprises: adding nutrient components and salt into fermentation culture solution prepared by properly supplementing nutrient components and salt into dextran industrial wastewater or dextran industrial wastewater, and adding the fermentation culture solution into the candida tropicalis for fermentation culture to remove COD (chemical oxygen demand) in the dextran industrial wastewater and obtain single-cell protein.
3. Use according to claim 2, wherein the fermentation broth comprises the following components: 100-975mL/L of dextran industrial wastewater, 2-20g/L of yeast extract powder, 0.2-4g/L of monopotassium phosphate and 0.02-1g/L of magnesium sulfate heptahydrate, wherein the pH value of the fermentation culture solution is 4.0-6.5.
4. Use according to claim 3, wherein the fermentation broth comprises the following components: the fermentation culture solution comprises the following components: 600mL/L of dextran industrial wastewater, 4.18g/L of yeast extract powder, 1g/L of potassium dihydrogen phosphate and 0.1g/L of magnesium sulfate heptahydrate, wherein the pH value of the fermentation culture solution is 5.5.
5. Use according to claim 3 or 4, characterized in that the pH is adjusted with sodium hydroxide.
6. The use of claim 2, wherein the treatment of dextran industrial wastewater comprises the steps of:
(1) Mixing 100-975mL of dextran industrial wastewater, 2-20g of yeast extract powder, 0.2-4g of monopotassium phosphate and 0.02-1g of magnesium sulfate heptahydrate, adding distilled water to dilute to 1000mL, adjusting the pH to 4.0-6.5 by adopting sodium hydroxide, sterilizing at 116 ℃ for 30min, and cooling to room temperature; obtaining a fermentation culture solution;
(2) Inoculating the candida tropicalis into the fermentation culture solution obtained in the step (1) for culture, wherein the culture conditions are as follows: the fermentation temperature is 25-35 ℃, the rotating speed of a shaking table is 200-250 r/min, and the culture time is 24-48 h;
(3) And (3) centrifuging the fermentation culture solution obtained in the step (2), removing COD in the supernatant, collecting the precipitate, washing and drying to obtain the single-cell protein.
7. The use according to claim 6, wherein said Candida tropicalis is acclimatized.
8. The use according to claim 7, wherein said Candida tropicalis acclimation comprises in particular:
(1) inoculating the candida tropicalis strains into a solid culture medium I, and culturing activated strains for 48 hours in a constant-temperature incubator at 30 ℃; the solid culture medium I is as follows: 40mL of wort, 5g of ammonium sulfate, 10g of peptone, 5g of yeast extract powder, 2g of monopotassium phosphate, 0.1g of magnesium sulfate heptahydrate, 20g of glucose, diluting to 1000mL of distilled water, measuring the pH value to be 5.5, adding 20g of agar powder, sterilizing, heating for dissolving, and cooling to obtain a solid culture medium I;
(2) transferring the strains obtained by activation in the step (1) into a seed culture medium for amplification culture, and culturing for 48 hours at the temperature of 30 ℃ and at the speed of 220r/min to obtain a bacterial liquid; the seed culture medium is as follows: 40mL of wort, 5g of ammonium sulfate, 10g of peptone, 5g of yeast extract powder, 2g of monopotassium phosphate, 0.1g of magnesium sulfate heptahydrate, 20g of glucose, diluting to 1000mL with distilled water, measuring the pH value to be 5.5, and sterilizing to obtain a seed culture medium;
(3) transferring the bacterial liquid obtained in the step (2) into a fermentation medium II, and culturing for 72h at the temperature of 30 ℃ and at the speed of 220 r/min; transferring 100 μ L of the bacterial solution in the fermentation medium, diluting with sterile water to 1.0 x 10 7 Doubling, transferring 25 mu L of the culture medium into a solid culture medium II, uniformly scraping, and culturing for 48 hours in a constant-temperature incubator at 30 ℃; selecting the single colony strain with the optimal growth potential on the solid culture medium II, inoculating the single colony strain to the solid culture medium II in the plane culture dish, carrying out subculture, selecting the single colony strain with the optimal growth potential on the solid culture medium II after 4 times of subculture, and inoculating the single colony strain to the solid culture medium I in the step (1)Culturing in a constant temperature incubator at 30 deg.C for 48 hr to obtain;
the fermentation medium comprises: 400mL of dextran industrial wastewater, 5g of ammonium sulfate, 10g of peptone, 5g of yeast extract powder, 2g of potassium dihydrogen phosphate and 0.1g of magnesium sulfate heptahydrate, diluting the mixture to 1000mL with distilled water, measuring the pH value to be 4.9, and sterilizing;
the solid culture medium II comprises: 400mL of dextran industrial wastewater, 5g of ammonium sulfate, 10g of peptone, 5g of yeast extract powder, 2g of potassium dihydrogen phosphate and 0.1g of magnesium sulfate heptahydrate, diluting the solution to 1000mL with distilled water, measuring the pH value to be 4.9, adding 20g of agar powder, sterilizing, heating for dissolving, and cooling to obtain a solid culture medium II.
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| CN117363499A (en) * | 2023-12-08 | 2024-01-09 | 万华化学集团股份有限公司 | Method for producing single-cell protein by utilizing industrial wastewater and application thereof |
| CN117568195A (en) * | 2024-01-16 | 2024-02-20 | 安琪酵母（滨州）有限公司 | Method for improving activity of low-sugar yeast |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117363499A (en) * | 2023-12-08 | 2024-01-09 | 万华化学集团股份有限公司 | Method for producing single-cell protein by utilizing industrial wastewater and application thereof |
| CN117363499B (en) * | 2023-12-08 | 2024-03-01 | 万华化学集团股份有限公司 | Method for producing single-cell protein by utilizing industrial wastewater and application thereof |
| CN117568195A (en) * | 2024-01-16 | 2024-02-20 | 安琪酵母（滨州）有限公司 | Method for improving activity of low-sugar yeast |
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