Lindnella gedinieri KF-2, microbial agent and application of microbial agent in treatment of coffee primary processing wastewater

Abstract

The invention discloses a strain of lindnella gedinieri KF-2, a microbial agent and application of the microbial agent in treatment of coffee primary processing wastewater, and belongs to the technical field of microorganisms. The strain is preserved in the Guangdong Microbial Culture Collection Center on July 10, 2024, the preservation address is the 5th floor of the building 59, No.100 Courtyard, Xianlie Middle Road, Guangzhou, and the preservation number is GDMCC No: 64858. The invention provides a strain of lindnella gedinieri KF-2, which can directly degrade coffee primary processing wastewater with high chemical oxygen demand without complex pre-treatments such as flocculent precipitation treatment and addition of any medicament, is simple in operation steps, and greatly reduces the production cost. The treated wastewater can be directly put into coffee bean production after being filtered and disinfected. Effective recycling of water resources is achieved, and environmental pollution is reduced.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to a strain of Jardine Seberlindner's yeast KF-2, a microbial agent and application thereof in treating coffee primary processing wastewater. Background Art

[0002] Conventional sewage treatment process steps: pretreatment, primary treatment, secondary treatment, deep treatment and discharge or reuse. Among them, pretreatment usually includes steps such as grid filtration, grit chamber and primary sedimentation tank to remove large particles, floating matter and suspended matter in sewage; the main process of primary treatment is chemical coagulation, sedimentation and oil removal to remove organic matter and suspended matter in sewage; secondary treatment usually removes organic matter and nutrients in sewage through biological reaction tanks such as activated sludge method, A / O method, A2 / O method and secondary sedimentation tank; deep treatment further removes trace organic matter, nutrients and harmful substances in sewage through filtration, disinfection and advanced oxidation. For sewage with strong odor, microbial deodorization process is usually used. Through the biological metabolic capacity of microbial strains, the odorous substances in organic matter (ammonia nitrogen, total nitrogen, hydrogen sulfide, organic sulfide, organic amine and other odor components) are decomposed, thereby converting them into colorless and odorless gases, which float in the air to achieve the effect of removing odor. After the above treatment process, the water quality of sewage is improved and can meet the discharge standards or reuse requirements.

[0003] After picking, fresh coffee berries need to be processed quickly to remove the peel and pulp, and then soak in water, rub and other steps to remove the colloid (mainly composed of sugar and pectin) wrapped around the coffee beans to obtain coffee beans. However, this process consumes a large amount of water and produces a large amount of wastewater with complex composition. The wastewater contains peel, pulp, sugar, pectin, etc., and the concentration of pollutants such as CODcr is high. The CODcr concentration of fermentation wastewater is as high as 50,000 mg / L, and the average comprehensive wastewater is 5,000-16,000 mg / L; the BOD is as high as 3,500 mg / L, and the average comprehensive wastewater is 1,300-3,500 mg / L; the wastewater quality BOD / CODcr>0.21, the wastewater biodegradability is poor; the pH is basically acidic; in particular, the wastewater contains 800-4000 mg / L of pectin. Pipelines will be blocked during wastewater transportation, which is the bottleneck of coffee primary processing wastewater treatment (CN 111410362A). Using conventional sewage treatment methods to treat coffee primary processing wastewater is difficult and costly, which is unacceptable to coffee farmers and processing companies.

[0004] At present, there are mainly the following methods for treating coffee primary processing wastewater. One is coagulation sedimentation and A2 / O (anaerobic-anoxic-aerobic) process. This treatment process is based on the conventional treatment method and adds a pH adjustment step. The second is two-stage filtration, pectin removal by adding drugs, anaerobic fermentation + hydrolysis acidification + AO (anaerobic + aerobic) biochemical process, etc. This type of method is obtained through technical improvements on the basis of conventional treatment methods, but the process steps are still relatively cumbersome, the production cycle is long, and the production cost is high. There is also a relatively simplified membrane treatment process for coffee primary processing wastewater. The pretreatment is activated carbon filtration, and the activated carbon adsorbs impurities and some macromolecular pollutants in the wastewater; special separation membrane separation includes primary reverse osmosis and secondary reverse osmosis, and most of the bacteria and organic solute particles in the wastewater are removed by reverse osmosis filtration; deep ceramic membrane treatment is mesoporous inorganic ceramic membrane filtration and microporous inorganic ceramic membrane filtration (CN108033585A). This type of method uses membrane filtration treatment and has a high cost. Third, electrochemical pretreatment process + biochemical treatment is also used at home and abroad. This process uses MBR membrane filtration (CN111470733A), electrocoagulation-anodic oxidation sequential process and selection of effective microbial treatment process. The main disadvantages of this method are: poor biodegradability in the microbial treatment stage, the strain is a conventional sewage treatment strain, which has not been screened, and the strain has poor adaptability. Wastewater entering microbial treatment needs to go through a large number of process steps to make the chemical oxygen demand of the wastewater less than 500 (after investigating 20 urban sewage treatment plants, when the chemical oxygen demand exceeds 500, the activated sludge wrapped around the strain dies in large quantities and floats on the surface, and the strain dies in large quantities, and can no longer treat sewage, and the treatment effect cannot be achieved). This makes the sewage treatment process complicated, the treatment cost high, and the area occupied large. Summary of the invention

[0005] The purpose of the present invention is to provide a strain of Saccharomyces jadinseberlindnerii KF-2, a microbial agent and the use thereof in treating coffee primary processing wastewater, so as to solve the problems existing in the above-mentioned prior art.

[0006] To achieve the above object, the present invention provides the following solutions: One of the technical solutions of the present invention is a strain of Lindner's yeast ( Cyberlindnera jadinii ) KF-2, the strain was deposited in the Guangdong Provincial Microbiological Culture Collection Center on July 10, 2024, the preservation address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the preservation number is GDMCC No: 64858.

[0007] The second technical solution of the present invention is a microbial agent, comprising the above-mentioned Saccharomyces jedinseberrindae KF-2.

[0008] The third technical solution of the present invention is the use of the Jardine Seiberlindera Saccharomyces cerevisiae KF-2 or the microbial agent in treating coffee primary processing wastewater.

[0009] A fourth technical solution of the present invention is a product for treating coffee primary processing wastewater, comprising the above-mentioned Jardine Seberlindner's yeast KF-2 or the above-mentioned microbial agent.

[0010] A fifth technical solution of the present invention is a method for treating coffee primary processing wastewater, which utilizes the Jardine Seiberrin yeast KF-2 or the microbial agent to treat the coffee primary processing wastewater.

[0011] The sixth technical solution of the present invention is the use of the Jardine Seiberlindera yeast KF-2 or the microbial agent in reducing the acidity of water.

[0012] The seventh technical solution of the present invention is the use of the Jedinseiberlindner yeast KF-2 or the microbial agent in reducing the COD value of water bodies.

[0013] The eighth technical solution of the present invention is the use of the Jardine Seiberrin yeast KF-2 or the microbial agent in reducing the ammonia nitrogen content in water.

[0014] Based on the above technical solution, the present invention has the following technical effects: The present invention provides a strain of Jardine Seberlindner's yeast KF-2, which can directly degrade coffee primary processing wastewater with high chemical oxygen demand without complicated pre-treatment such as flocculation precipitation treatment and any agent addition, has simple operation steps, and greatly reduces production costs. The treated wastewater can be directly put into coffee bean production after filtration and disinfection, thereby achieving effective recycling of water resources and reducing environmental pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0016] Figure 1 This is a picture of KF-2 colonies. DETAILED DESCRIPTION

[0017] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0018] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0019] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0020] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0021] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0022] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0023] The present invention provides a strain of Saccharomyces jadinseberlindnerii ( Cyberlindnera jadinii ) KF-2, the strain was deposited in the Guangdong Provincial Microbiological Culture Collection Center on July 10, 2024, the preservation address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the preservation number is GDMCC No: 64858.

[0024] The embodiment of the present invention also provides a microbial agent, including the above-mentioned Saccharomyces jedinseberlindera KF-2.

[0025] The embodiment of the present invention also provides the use of the Jardine Seiberlindera Saccharomyces KF-2 or the microbial agent in treating coffee primary processing wastewater.

[0026] The embodiment of the present invention also provides a product for treating coffee primary processing wastewater, comprising the Jardine Seiberrin yeast KF-2 or the microbial agent.

[0027] The embodiment of the present invention also provides a method for treating coffee primary processing wastewater, using the Jardine Seiberlindnerella KF-2 or the microbial agent to treat the coffee primary processing wastewater.

[0028] In some specific embodiments, the treated water can be reused for the primary processing of fresh coffee cherries.

[0029] The embodiment of the present invention also provides the use of the Jardine Seiberlindera Saccharomyces KF-2 or the microbial agent in reducing the acidity of water.

[0030] The embodiment of the present invention also provides the use of the Jardine Seiberlindera yeast KF-2 or the microbial agent in reducing the COD value of water bodies.

[0031] The embodiment of the present invention also provides the use of the Jardine Seiberlindera yeast KF-2 or the microbial agent in reducing the ammonia nitrogen content in water.

[0032] In some specific embodiments, the water body is coffee primary processing wastewater. Example 1

[0033] Screening and identification of strains 1. Take the activated sludge from the Chenggong District Water Service Center (Chenggong Sewage Treatment Plant) in Kunming City and let it settle in a measuring cylinder. Continuously pour off the supernatant to reduce the water content and ensure the number of microorganisms. Measure 25mL of sludge and inoculate it into 500mL of coffee primary processing wastewater with a pH of 6.0. Cultivate it in a shaking table at 35℃ and 140r / min. Take samples and measure them every 24 hours until the organic matter is no longer degraded. The experimental results are shown in Table 1.

[0034] Table 1 , 2. After the above residual sewage was settled, the upper liquid was discarded and poured into 500 mL of coffee primary processing wastewater with a pH of 4.5. The mixture was cultured in a shaking table at 35°C and 140 r / min. Samples were taken every 24 hours until the organic matter was no longer degraded. The experimental results are shown in Table 2.

[0035] Table 2 , 3. After the above residual sewage was settled, the upper liquid was discarded and poured into 500 mL of coffee primary processing wastewater with a pH of 3.0. The mixture was cultured in a shaking table at 35°C and 140 r / min. Samples were taken every 24 hours until the organic matter was no longer degraded. The experimental results are shown in Table 3.

[0036] Table 3 , 4. After the microorganisms have been gradually domesticated with coffee primary processing wastewater at pH 6.0, 4.5, and 3.0, they are diluted step by step, and 200 μL of the diluted bacterial solution is taken and spread on the PDA and YPD plate culture media at pH 3.0 with a coating rod. The plate culture medium is inverted in a constant temperature incubator at 35°C, and the growth of microorganisms is observed regularly. When colonies of various forms appear on the plate culture medium, a small amount of bacteria is picked from each colony with an inoculation loop and inoculated on new PDA and YPD (for yeast) plate culture media. Repeat the above process until the morphology of the bacterial colonies on the plate culture medium is consistent in multiple experiments, that is, the strain separation and purification is completed.

[0037] A strain was screened, which was light yellow, round, with moist colonies, raised colonies, and no hyphae. It was identified as belonging to the yeast Jedinseiberlindner ( Cyberlindnera jadinii ), named KF-2.

[0038] Lindnerella jardiniseiber Cyberlindnera jadinii ) KF-2 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on July 10, 2024. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No: 64858. Example 2

[0039] Cyberlindnera jadinii Degradation performance of KF-2 on coffee primary processing wastewater under sterile conditions KF-2 was inoculated on a PDA solid medium with a pH of 3.0, inverted and placed in a constant temperature incubator at 35°C for 48 hours, and two 1 cm bacterial cakes were inoculated in a PDA liquid medium with a pH of 3.0, and cultured in a shaking incubator at 35°C and 140 r / min for 14-16 hours as seed liquid for later use. The seed liquid was added to the primary coffee processing wastewater at 10%, and cultured in a shaking incubator at 35°C and 140 r / min, and samples were taken every 24 hours to test pH and COD. After the degradation was completed, the strain was recovered.

[0040] Under sterile conditions, the degradation effect of the strain on coffee primary processing wastewater is shown in Table 4-5.

[0041] Table 4 Treatment system of coffee primary processing wastewater and KF-2 addition under aseptic system , Table 5 Effect of KF-2 treatment on water pH , In the sewage treatment process, discharging water with an acidic pH value into the environment will affect the growth of plants and animals and also cause their death. Therefore, in the conventional treatment process, the pH value needs to reach 6 to 9 before it can be discharged. From the analysis of the results in Table 5, it can be seen that the water treated with KF-2 can effectively adjust the pH of the water. As the treatment time increases, the pH value of the water increases. When the treatment starts on the fourth day, the pH value of the water is basically maintained at around 7. Moreover, the present invention has found that overly acidic or overly alkaline water has a greater impact on the flavor of coffee when treating coffee pectin. Neutral water is most suitable for coffee pectin treatment. Therefore, when the pH value is neutral, it is conducive to the reuse of water.

[0042] Table 6 Effect of KF-2 treatment on COD value of water body , COD value is an important indicator to measure the degree of organic pollution in water, and its value directly reflects the pollution status of water body. The higher the COD value means the higher the content of reducing substances in the water body. Excessive reducing substances will consume dissolved oxygen in the water, causing aquatic organisms to die due to lack of oxygen, thus affecting the ecological balance. At the same time, when the COD value is high, it will cause the water body to turn black and stink, affecting the reuse of water. As can be seen from Table 6, with the increase of KF-2 treatment time, the COD value of the water body gradually decreases. After 9 days of degradation, the COD value of the water body decreased from 13048 to 1469, and the degradation rate reached 89%. Example 3

[0043] Degradation performance of domesticated strains on coffee primary processing wastewater under natural conditions KF-2 was inoculated on a PDA solid medium with a pH of 3.0, inverted and incubated in a constant temperature incubator at 35°C for 48 hours, and two 1 cm bacterial cakes were inoculated in a PDA liquid medium with a pH of 3.0, and cultured in a shaking incubator at 35°C and 140 r / min for 14 to 16 hours as seed liquid for later use. The seed liquid was added to coffee primary processing wastewater [non-sterilized and sterilized (121°C 15min)] at 10%, and cultured in a shaking incubator at 35°C and 140 r / min, and samples were taken every 24 hours to test pH, COD, and ammonia nitrogen. After the degradation was completed, the strain was recovered.

[0044] Table 7 , Table 8 Effect of KF-2 treatment on water pH under different conditions , From the results in Table 8, it can be seen that under natural conditions, the water treated with KF-2 can still effectively adjust the pH of the water. As the treatment time is extended to the 9th day, the pH value of the water reaches 6.85, which is close to neutral. Compared with the aseptic environment, the adjustment speed is slower, which may be due to the presence of a large number of other microorganisms in the unsterilized water, which has an impact on the adjustment speed of KF-2, but does not affect the final result.

[0045] Table 9 Effect of KF-2 treatment on COD value of water under different conditions , From the results in Table 9, it can be seen that under natural conditions, the COD value of water treated with KF-2 can still be reduced. As the treatment time increases, the COD value in the water decreases significantly, from the initial 11501 to 2085, and the degradation rate can reach 81%. The degradation speed and degradation rate are lower than those in the sterile environment. It may be that other microorganisms in the unsterilized water affect the degradation effect of KF-2, but it does not affect the reuse of the water. Example 4

[0046] 1 Effect of reuse of treated wastewater on coffee flavor Coffee sensory evaluation: The sensory evaluation panel consists of 10 people (5 men and 5 women), all of whom are specialty coffee quality graders (Q-Graders) who can accurately identify the quality of coffee. The sensory evaluation is carried out in accordance with the sensory evaluation standards established by the Specialty Coffee Association of America (SCA), and the aroma, flavor, acidity, body, uniformity, clean cup, balance, sweetness and other indicators are selected for evaluation to determine the comprehensive score of the sample. All samples are provided at room temperature of 20°C and evaluated in random order.

[0047] Each treatment was tested five times independently and samples were taken for evaluation by human tasters, who rated the intensity of the attribute using a 0-10 scale with 0.25 point increments (0 = none; 10 = extremely strong).

[0048] Fresh coffee fruit processing: Fresh coffee fruit needs to be processed quickly after picking, the peel and pulp are removed, and then soaked in clean water, wastewater that has been treated for 1 cycle, wastewater that has been treated for 2 cycles, wastewater that has been treated for 3 cycles, wastewater that has been treated for 4 cycles, and wastewater that has been treated for 5 cycles for 12 hours overnight, and rubbed to remove the colloid wrapped around the coffee beans to obtain coffee beans. After roasting, the beans are provided for sensory evaluation, and the results are shown in Table 10.

[0049] Table 10 Effect of coffee primary processing wastewater recycling times on coffee quality , From the results analysis in Table 10, it can be seen that the average scores of coffee beans treated with clean water, wastewater after 1 cycle, wastewater after 2 cycles, wastewater after 3 cycles, wastewater after 4 cycles and wastewater after 5 cycles are 7.5±0.20, 7.475±0.18, 7.4±0.17, 7.35±0.13, 7.35±0.12 and 7.35±0.12. Compared with coffee beans treated with clean water, there is no significant difference in coffee beans treated with wastewater after 1 cycle, 2 cycles, 3 cycles, 4 cycles and 5 cycles (p=0.77, 0.25, 0.07, 0.07 and 0.07 respectively). Therefore, the coffee primary processing wastewater treated with KF-2 can be recycled.

[0050] 2 Effect of pH value of treated wastewater on coffee flavor After picking, fresh coffee berries need to be processed quickly to remove the peel and pulp, then soak them in clean water for 12 hours, rub them to remove the colloid wrapped around the outside of the coffee beans, and filter them to obtain coffee primary processing wastewater.

[0051] KF-2 was inoculated on a PDA solid medium with a pH of 3.0, inverted and incubated in a constant temperature incubator at 35°C for 48 hours, and two 1 cm bacterial cakes were inoculated in a PDA liquid medium with a pH of 3.0, and cultured in a shaker at 35°C and 140 r / min for 14 hours as seed liquid. The seed liquid was added to the coffee primary processing wastewater at 10%, and cultured in a shaker at 35°C and 140 r / min for 9 days to obtain the treated coffee primary processing wastewater.

[0052] After being treated with KF-2, the pH value was adjusted to 2.0 with hydrochloric acid to obtain acidic wastewater; and the pH value was adjusted to 13 with sodium hydroxide to obtain alkaline wastewater.

[0053] Fresh coffee fruit processing: Fresh coffee fruit needs to be processed quickly after picking, the peel and pulp are removed, and then soaked in clean water and coffee primary processing wastewater with different pH values ​​(wastewater without pH adjustment, acidic wastewater and alkaline wastewater) for 12 hours overnight, and rubbed to remove the colloid wrapped around the coffee beans to obtain coffee beans. After roasting, it is provided for sensory evaluation. The results are shown in Table 11.

[0054] Table 11 Effects of coffee primary processing wastewater with different pH values ​​on coffee quality , From the results analysis in Table 11, it can be seen that the average scores of coffee bean quality treated with clean water, unadjusted pH coffee primary processing wastewater, acidic wastewater, and alkaline wastewater are 7.5±0.20, 7.475±0.18, 6.43±0.12, and 6.4±0.13. Compared with coffee beans treated with clean water, coffee primary processing wastewater with different pH values ​​has a significant effect on coffee quality (unadjusted pH coffee primary processing wastewater: p>0.05; acidic wastewater: p <0.05; alkaline wastewater: p <0.05). Therefore, the treated coffee primary processing wastewater needs to be kept neutral before it can be recycled.

[0055] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A strain of Lindnerella jardiniseiber Cyberlindnera jadinii ) KF-2, characterized in that, The strain was deposited in the Guangdong Provincial Microbiological Culture Collection Center on July 10, 2024. The collection address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the collection number is GDMCC No: 64858.

2. A microbial agent, characterized in that: The invention comprises the Jardine Seiberrin yeast KF-2 according to claim 1.

3. Use of the yeast Saccharomyces jedinseberlindera KF-2 as described in claim 1 or the microbial agent as described in claim 2 in treating coffee primary processing wastewater.

4. A product for treating coffee primary processing wastewater, characterized in that: It comprises the Jedinseiberlindera yeast KF-2 described in claim 1 or the microbial agent described in claim 2.

5. A method for treating coffee primary processing wastewater, characterized in that: The coffee primary processing wastewater is treated using the Jedinseiberlindnerella KF-2 described in claim 1 or the microbial agent described in claim 2.

6. The processing method according to claim 5, characterized in that: The treated water can be reused for the primary processing of fresh coffee cherries.

7. Use of the Saccharomyces jedinseberlindera KF-2 according to claim 1 or the microbial agent according to claim 2 in reducing the acidity of water.

8. Use of the yeast Saccharomyces jedinseberlindera KF-2 according to claim 1 or the microbial agent according to claim 2 in reducing the COD value of water bodies.

9. Use of the Saccharomyces jedinseberlindera KF-2 according to claim 1 or the microbial agent according to claim 2 in reducing the ammonia nitrogen content in water.

10. The use according to any one of claims 7 to 9, characterized in that: The water body is coffee primary processing wastewater.

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