A strain of Cyberlindnera jadinii KF-2, a microbial inoculum and its application in treating coffee primary processing wastewater

Abstract

The present invention discloses a strain of Jadinseberlindner's yeast KF‑2, a microbial agent and its application in treating coffee primary processing wastewater, belonging to the field of microbial technology. The strain was deposited in the Guangdong Provincial Microbial Culture Collection Center on July 10, 2024, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No: 64858. The present invention provides a strain of Jadinseberlindner's yeast KF‑2, which can directly degrade coffee primary processing wastewater with high chemical oxygen demand without complex pre-treatment such as flocculation precipitation treatment and any agent addition, and the operation steps are simple, which greatly reduces the production cost. The treated wastewater can be directly put into coffee bean production after filtration and disinfection. The effective recycling of water resources is realized and environmental pollution is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and particularly to a strain of Cyberlindnera jadinii KF-2, a microbial agent, and their application in treating coffee primary processing wastewater. Background Art

[0002] Conventional sewage treatment process steps: pretreatment, primary treatment, secondary treatment, advanced treatment, and discharge or reuse, etc., a total of 5 steps. Among them, pretreatment usually includes steps such as grid filtration, grit chamber, and primary sedimentation tank to remove large particulate matter, floating matter, and suspended matter in the sewage; the main processes of primary treatment are chemical coagulation, sedimentation, and oil removal, etc. to remove organic matter and suspended matter in the sewage; secondary treatment usually removes organic matter and nutrients in the sewage through biological reaction tanks such as activated sludge method, A / O method, A2 / O method, etc. and secondary sedimentation tank; advanced treatment further removes trace organic matter, nutrients, and harmful substances in the sewage through steps such as filtration, disinfection, and advanced oxidation. For sewage with a strong odor, a microbial deodorization process is usually adopted. Through the biological metabolism ability of microbial strains, the odor substances (odor components such as ammonia nitrogen, total nitrogen, hydrogen sulfide, organic sulfides, organic amines, etc.) in organic substances are decomposed, thereby being converted into colorless and odorless gases and floating in the air to achieve the effect of removing odors. After the above treatment process, the water quality of the sewage is improved and can meet the discharge standard or reuse requirements.

[0003] After coffee fresh fruits are picked, they need to be quickly processed to remove the peel and pulp, and then the colloidal substance (mainly composed of sugar and pectin) wrapped outside the coffee beans is removed through steps such as water immersion and rubbing to obtain coffee beans. However, this process uses a large amount of water, generates a large amount of wastewater, and has a complex composition. The wastewater contains peel, pulp, sugars, pectin, etc., and the concentration of pollutants such as CODcr is high. The CODcr concentration of fermentation wastewater is as high as 50000 mg / L, and the average concentration of comprehensive wastewater reaches 5000 - 16000 mg / L; the highest BOD reaches 3500mg / L, and the average concentration of comprehensive wastewater reaches 1300 - 3500mg / L; the BOD / CODcr of the wastewater quality >0.21, and the biodegradability of the wastewater is poor; the pH is basically acidic; especially the wastewater contains 800 - 4000 mg / L of pectin. It will block the pipeline during the wastewater transportation process, which is the bottleneck of coffee primary processing wastewater treatment (CN 111410362A). It is difficult and costly to treat coffee primary processing wastewater using conventional sewage treatment methods, and it is difficult for coffee farmers and processing enterprises to accept.

[0004] At present, the relevant reports on the treatment methods of coffee primary processing wastewater mainly include the following methods. One is coagulation sedimentation and A2 / O (anaerobic - anoxic - aerobic) process, and this treatment process is based on the conventional treatment method with the addition of a pH adjustment step. The second is two - stage filtration, adding medicine to remove pectin, anaerobic fermentation + hydrolysis acidification + AO (anaerobic + aerobic) biochemical process, etc. This type of method is obtained through technical improvement on the basis of the conventional treatment method, 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 coffee primary processing wastewater treatment process, the membrane treatment method. The pretreatment is activated carbon filtration, and impurities and some macromolecular pollutants in the wastewater are adsorbed by activated carbon; special separation membrane separation includes first - stage reverse osmosis and second - stage reverse osmosis, and most 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 the cost is high. Thirdly, electrochemical pretreatment process + biochemical treatment is also adopted at home and abroad. This process uses MBR membrane filtration (CN111470733A), electro - flocculation - anodic oxidation sequential process, and selective effective microorganism treatment process, etc. The main disadvantages of this type of method are: the biodegradability is poor in the microorganism treatment stage, the strains are conventional sewage treatment strains without screening, and the adaptability of the strains is poor. The wastewater entering the microorganism treatment needs to go through a large number of process steps to make the chemical oxygen demand less than 500 (after investigating 20 urban sewage treatment plants, when the chemical oxygen demand exceeds 500, a large amount of activated sludge wrapped with strains dies and floats on the surface, a large number of strains die, and the sewage cannot be treated anymore, failing to achieve the treatment effect). As a result, the sewage treatment process is complex, the treatment cost is high, and the floor area is large. Summary of the Invention

[0005] The object of the present invention is to provide a strain of Cyberlindnera jadinii KF - 2, a microbial inoculum and their application in treating coffee primary processing wastewater to solve the problems existing in the above - mentioned prior art.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] One of the technical solutions of the present invention is a strain of Cyberlindnera jadinii ( Cyberlindnera jadinii ) KF - 2, which was deposited at the Guangdong Provincial Microbial Culture Collection Center on July 10, 2024. The deposit address is the 5th floor of Building 59, No. 100 Yard, Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No: 64858.

[0008] Another technical solution of the present invention is a microbial inoculum, including the strain of Cyberlindnera jadinii KF - 2.

[0009] Embodiment 3 of the technical solution of the present invention: Application of the Saccharomyces cerevisiae KF-2 or the microbial agent in treating coffee primary processing wastewater.

[0010] Embodiment 4 of the technical solution of the present invention: A product for treating coffee primary processing wastewater, comprising the Saccharomyces cerevisiae KF-2 or the microbial agent.

[0011] Embodiment 5 of the technical solution of the present invention: A method for treating coffee primary processing wastewater, using the Saccharomyces cerevisiae KF-2 or the microbial agent to treat the coffee primary processing wastewater.

[0012] Embodiment 6 of the technical solution of the present invention: Application of the Saccharomyces cerevisiae KF-2 or the microbial agent in reducing the acidity of water bodies.

[0013] Embodiment 7 of the technical solution of the present invention: Application of the Saccharomyces cerevisiae KF-2 or the microbial agent in reducing the COD value of water bodies.

[0014] Embodiment 8 of the technical solution of the present invention: Application of the Saccharomyces cerevisiae KF-2 or the microbial agent in reducing the ammonia nitrogen content of water bodies.

[0015] Based on the above technical solutions, the present invention has the following technical effects:

[0016] The present invention provides a strain of Saccharomyces cerevisiae KF-2, which can directly degrade coffee primary processing wastewater with high chemical oxygen demand without complicated pretreatment such as flocculation precipitation treatment and addition of any chemicals. The operation steps are simple, and the production cost is greatly reduced. The treated wastewater can be directly put into coffee bean production after filtration and disinfection. It realizes the effective recycling of water resources and reduces environmental pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0018] Figure 1 It is a colony map of KF-2. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0019] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0020] It should be understood that the terms described in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can 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 related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0022] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of this application are merely exemplary.

[0023] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0024] The technical solutions described in the present invention are all conventional solutions in the art unless otherwise specified, and the reagents or raw materials used are all purchased from commercial channels or have been made public unless otherwise specified.

[0025] An embodiment of the present invention provides a strain of Cyberlindnera jadinii ( Cyberlindnera jadinii ) KF-2, which was deposited at the Guangdong Provincial Culture Collection of Microorganisms on July 10, 2024. The deposit address is the 5th floor of Building 59, No. 100 Yard, Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No: 64858.

[0026] An embodiment of the present invention also provides a microbial inoculant, including the Cyberlindnera jadinii KF-2.

[0027] An embodiment of the present invention also provides the application of the Cyberlindnera jadinii KF-2 or the microbial inoculant in treating coffee primary processing wastewater.

[0028] An embodiment of the present invention also provides a product for treating coffee primary processing wastewater, including the Cyberlindnera jadinii KF-2 or the microbial inoculant.

[0029] An embodiment of the present invention also provides a method for treating coffee primary processing wastewater, using the Saccharomyces cerevisiae KF-2 or the microbial agent to treat the coffee primary processing wastewater.

[0030] In some specific embodiments, the treated water body can be reused for the primary processing of coffee fresh fruits.

[0031] An embodiment of the present invention also provides the application of the Saccharomyces cerevisiae KF-2 or the microbial agent in reducing the acidity of the water body.

[0032] An embodiment of the present invention also provides the application of the Saccharomyces cerevisiae KF-2 or the microbial agent in reducing the COD value of the water body.

[0033] An embodiment of the present invention also provides the application of the Saccharomyces cerevisiae KF-2 or the microbial agent in reducing the ammonia nitrogen content of the water body.

[0034] In some specific embodiments, the water body is coffee primary processing wastewater. Example 1

[0035] Screening and identification of strains

[0036] 1. Take the activated sludge from the Chenggong Water Service Center (Chenggong Sewage Treatment Plant) in Kunming City and settle it in a graduated cylinder. Continuously pour off the supernatant to reduce the moisture content and ensure the number of individual microorganisms. Measure 25 mL of sludge and inoculate it into 500 mL of coffee primary processing wastewater with a pH of 6.0. Incubate it on a shaker at 35 °C and 140 r / min. Take samples every 24 hours for measurement until the organic matter is no longer degraded. The experimental results are shown in Table 1.

[0037] Table 1

[0038] ,

[0039] 2. After settling the remaining sewage above, discard the upper liquid and pour it into 500 mL of coffee primary processing wastewater with a pH of 4.5. Incubate it on a shaker at 35 °C and 140 r / min. Take samples every 24 hours for measurement until the organic matter is no longer degraded. The experimental results are shown in Table 2.

[0040] Table 2

[0041] ,

[0042] 3. Decant the supernatant after sedimentation of the above remaining sewage, pour it into 500 mL of coffee primary processing wastewater with a pH of 3.0, and incubate it on a shaker at 35 °C and 140 r / min. Take samples every 24 hours for measurement until the organic matter is no longer degraded. The experimental results are shown in Table 3.

[0043] Table 3

[0044] ,

[0045] 4. The microorganisms acclimated step by step with coffee primary processing wastewater with pH values of 6.0, 4.5, and 3.0 are diluted step by step. Then, 200 μL of the diluted bacterial solution is taken and spread on PDA and YPD plate media with a pH of 3.0 using a spreading rod. The plate media are inverted and placed in an incubator at 35 °C, and the growth of microorganisms is observed regularly. When colonies of various morphologies appear on the plate media, a small amount of bacterial cells are picked from each colony with an inoculation loop and inoculated onto new PDA and YPD (yeast-specific) plate media. Repeat the above process until the colony morphologies on the plate media are consistent in multiple experiments, indicating that the strain isolation and purification are completed.

[0046] A strain was screened out, light yellow, round, with relatively moist colonies, convex colonies, and no hyphae growing. After identification, this strain belongs to Cyberlindnera jadinii ), and it is named KF-2.

[0047] Cyberlindnera jadinii ), KF-2 was deposited at the Guangdong Provincial Microbial Culture Collection Center on July 10, 2024. The deposit address is the 5th floor of Building 59, No. 100 compound, Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No: 64858. Example 2

[0048] Cyberlindnera jadinii Degradation performance of KF-2 on coffee primary processing wastewater under aseptic conditions

[0049] Inoculate KF-2 onto PDA solid medium with a pH of 3.0, invert it and incubate it in an incubator at 35 °C for 48 h. Take two 1-cm agar discs and inoculate them into PDA liquid medium with a pH of 3.0, and incubate them on a shaker at 35 °C and 140 r / min for 14 - 16 hours as the seed solution for standby. Add the seed solution to the coffee primary processing wastewater at 10%, and incubate it on a shaker at 35 °C and 140 r / min. Take samples every 24 hours to detect the pH and COD. After degradation is completed, the strain is recovered.

[0050] Under aseptic conditions, the degradation effect of the strain on coffee primary processing wastewater is shown in Tables 4 - 5.

[0051] ​Table 4 Coffee primary processing wastewater treatment system and KF-2 addition amount under sterile system

[0052] ,

[0053] Table 5 Influence of KF-2 treatment on water body pH value

[0054] ,

[0055] During the sewage treatment process, discharging water bodies with acidic pH values into the environment will affect the growth of animals and plants and can also cause the death of animals and plants. Therefore, during the conventional treatment process, the pH value needs to reach 6 - 9 before discharge. From the analysis of the results in Table 5, it can be seen that the water body treated with KF-2 can effectively adjust the acidity and alkalinity of water. As the treatment time increases, the pH value of the water body rises. When the treatment reaches the fourth day, the pH value of the water body basically remains around 7. Moreover, the present invention finds that water that is too acidic or too alkaline has a greater impact on the coffee flavor when treating coffee pectin. Neutral water is most suitable for treating coffee pectin. Therefore, when the pH value is neutral, it is beneficial for the reuse of the water body.

[0056] Table 6 Influence of KF-2 treatment on water body COD value

[0057] ,

[0058] The COD value is an important indicator to measure the degree of organic matter pollution in water, and the level of its value directly reflects the pollution status of the water body. The higher the COD value, the higher the content of reducing substances in the water body. Excessive reducing substances will consume the dissolved oxygen in the water, leading to the death of aquatic organisms due to lack of oxygen, and further affecting the ecological balance. At the same time, when the COD value is high, the water body will turn black and emit a stench, affecting the reuse of water. It can be seen from Table 6 that as the KF-2 treatment time increases, the COD value of the water body gradually decreases. After 9 days of degradation, the COD value of the water body decreases from 13048 to 1469, and the degradation rate reaches 89%. Example 3

[0059] Degradation performance of domesticated strains on coffee primary processing wastewater under natural conditions

[0060] Inoculate KF-2 onto a PDA solid medium with a pH of 3.0, invert it and culture it in a constant temperature incubator at 35°C for 48 h. Take two 1-cm agar discs and inoculate them into a PDA liquid medium with a pH of 3.0, and culture them in a shaker at 35°C and 140 r / min for 14 - 16 hours as the seed liquid for standby. Add the seed liquid to coffee primary processing wastewater [non-sterilized and sterilized (121°C, 15 min)] at 10%, and culture it in a shaker at 35°C and 140 r / min. Take samples every 24 hours to detect pH, COD, and ammonia nitrogen. After the degradation is completed, the strains are recovered.

[0061] Table 7

[0062] ,

[0063] Table 8. Influence of KF-2 treatment on pH value of water under different conditions

[0064] ,

[0065] It can be seen from the results analysis of Table 8 that under natural conditions, the water body treated with KF-2 can still effectively adjust the acidity and alkalinity of water. As the treatment time extends to the 9th day, the pH value of the water body reaches 6.85, approaching neutrality. Compared with the adjustment speed in the sterile environment, it slows down, probably because there are a large number of other microorganisms in the non-sterilized water body, which affects the adjustment speed of KF-2, but does not affect the final result.

[0066] Table 9. Influence of KF-2 treatment on COD value of water under different conditions

[0067] ,

[0068] It can be seen from the results analysis of Table 9 that under natural conditions, the water body treated with KF-2 can still reduce the COD value of the water body. As the treatment time extends, the COD value in the water body 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. Probably because the other microorganisms existing in the non-sterilized water body affect the degradation effect of KF-2, but it does not affect the repeated reuse of the water body. Example 4

[0069] 1 Influence of repeated utilization of treated wastewater on coffee flavor

[0070] Coffee sensory evaluation: The sensory evaluation panel consists of 10 people (5 males and 5 females), all of whom are Q-Graders (qualified coffee quality graders) and can accurately identify coffee quality. The sensory evaluation is carried out according to the sensory evaluation standards formulated by the Specialty Coffee Association of America (SCA). Select indicators such as aroma, flavor, acidity, body, uniformity, clean cup, balance, and sweetness 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.

[0071] Each treatment was independently repeated 5 times, and samples were taken for evaluation by tasters. The tasters evaluated the intensity of the attributes on a scale of 0 - 10, with an increment of 0.25 points (0 = none; 10 = extremely strong).

[0072] Fresh coffee fruit treatment: After the fresh coffee fruit is picked, it needs to be processed quickly. The peel and pulp are removed, and then it is soaked in clean water, wastewater recycled once, wastewater recycled twice, wastewater recycled three times, wastewater recycled four times, and wastewater recycled five times for 12 hours overnight. Rub and knead to remove the colloid wrapped outside the coffee beans to obtain coffee beans. After roasting, it is used for sensory evaluation, and the results are shown in Table 10.

[0073] Table 10 Effects of the number of recycling times of coffee primary processing wastewater on coffee quality

[0074] ,

[0075] From the analysis of the results in Table 10, it can be seen that the average scores of coffee beans treated with clean water, wastewater recycled once, wastewater recycled twice, wastewater recycled three times, wastewater recycled four times, and wastewater recycled five times are 7.5 ± 0.20, 7.475 ± 0.18, 7.4 ± 0.17, 7.35 ± 0.13, 7.35 ± 0.12, 7.35 ± 0.12. Compared with the coffee beans treated with clean water, there is no significant difference in the coffee beans treated with wastewater recycled once, wastewater recycled twice, wastewater recycled three times, wastewater recycled four times, and wastewater recycled five times (p = 0.77, 0.25, 0.07, 0.07, 0.07 respectively). Therefore, the coffee primary processing wastewater treated with KF-2 can be recycled.

[0076] 2 Effect of the pH value of the treated wastewater on coffee flavor

[0077] After the fresh coffee fruit is picked, it needs to be processed quickly. The peel and pulp are removed, and then it is soaked in clean water for 12 hours. Rub and knead to remove the colloid wrapped outside the coffee beans, and filter to obtain coffee primary processing wastewater.

[0078] KF-2 was inoculated onto a PDA solid medium with a pH of 3.0 and incubated in an incubator at 35°C for 48 h. Two 1-cm agar discs were taken and inoculated into a PDA liquid medium with a pH of 3.0 and shaken at 35°C and 140 r / min for 14 hours to be used as the seed solution. The seed solution was added to the coffee primary processing wastewater at 10% and shaken at 35°C and 140 r / min for 9 days to obtain the treated coffee primary processing wastewater.

[0079] After treatment with KF-2, the pH value was adjusted to 2.0 with hydrochloric acid to obtain acidic wastewater; the pH value was adjusted to 13 with sodium hydroxide to obtain alkaline wastewater.

[0080] Fresh coffee fruit processing: After the fresh coffee fruit is picked, it needs to be processed quickly to remove the peel and pulp. Then, it is 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 kneaded to remove the colloidal substance wrapped around the coffee beans to obtain coffee beans. After roasting, it is used for sensory evaluation, and the results are shown in Table 11.

[0081] Table 11 Effects of coffee primary processing wastewater with different pH values on coffee quality

[0082] ,

[0083] From the analysis of the results in Table 11, it can be seen that the average scores of coffee beans processed by clean water, coffee primary processing wastewater without pH adjustment, acidic wastewater, and alkaline wastewater are 7.5±0.20, 7.475±0.18, 6.43±0.12, and 6.4±0.13 respectively. Compared with the coffee beans processed by clean water, coffee primary processing wastewater with different pH values has a significant impact on coffee quality (coffee primary processing wastewater without pH adjustment: 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 for recycling.

[0084] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, rather than limiting the implementation manners of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation manners here. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. Application of Cyberlindnera jadinii KF-2 in treating wastewater from coffee primary processing, characterized in that, Cyberlindnera jadinii 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 product for treating the wastewater from the primary processing of coffee, characterized in that, The invention comprises the Jedinseiberlindera yeast KF-2 as claimed in claim 1.

3. A method for treating coffee primary processing wastewater, characterized in that The coffee primary processing wastewater is treated by using the yeast Jedinseiberlindnerella KF-2 described in claim 1.

4. Use of the yeast Saccharomyces jedinseberlindera KF-2 described in claim 1 in reducing the acidity of coffee primary processing wastewater.

5. Use of the yeast Saccharomyces jadinseberlindera KF-2 described in claim 1 in reducing the COD value of coffee primary processing wastewater.

6. Use of the yeast Saccharomyces jadinseberlindera KF-2 described in claim 1 in reducing the ammonia nitrogen content of coffee primary processing wastewater.

Images