WO2023284891A1

WO2023284891A1 - Low-temperature acid-resistant saccharomyces cerevisiae and screening method and use therefor

Info

Publication number: WO2023284891A1
Application number: PCT/CN2022/113806
Authority: WO
Inventors: 朱廷恒; 丁国春; 易蒲红; 李芩萍; 林路成; 徐志伟; 丁晓艳; 李季
Original assignee: 中农新科(苏州)有机循环研究院有限公司
Priority date: 2021-07-15
Filing date: 2022-08-22
Publication date: 2023-01-19
Also published as: CN114231428B; CN114231428A

Abstract

A low-temperature acid-resistant Saccharomyces cerevisiae. The Saccharomyces cerevisiae is classified and named as Saccharomyces cerevisiae DW, is preserved in the China General Microbiological Culture Collection Center, and the preservation number is: CGMCC No.22786. Also disclosed are a screening method and use for the Saccharomyces cerevisiae. The obtained Saccharomyces cerevisiae DW can grow at a low temperature condition (4° C.) and has good acid resistance; when the saccharomyces cerevisiae DW and a Bacillus subtilis are added in proportion under different aerobic composting conditions, compared with the use of only the Bacillus subtilis, the reactor can rapidly increase the pH value, is started earlier, enters the high-temperature period earlier, and prolongs the high-temperature period duration.

Description

A low-temperature acid-resistant Saccharomyces cerevisiae and its screening method and application

technical field

The invention relates to the technical field of kitchen waste treatment, in particular to a low-temperature acid-resistant Saccharomyces cerevisiae and its screening method and application.

Background technique

my country produces about 1.3 billion tons of kitchen waste organic waste every year, and the output is gradually increasing. Food waste is the main part of municipal solid waste. Its generation and accumulation seriously pollute the environment and cause food safety problems.

At present, the treatment methods of food waste mainly include landfill, incineration, anaerobic fermentation, aerobic composting, etc. Landfill not only occupies a large amount of land, but also produces a large amount of leachate that pollutes soil and groundwater. The moisture content of food waste is as high as 70%-90%, and its calorific value is low, so it is not suitable for incineration; and incineration will produce a lot of toxic and harmful gases, polluting the air. The operation cost of anaerobic fermentation is high, there are many graded treatments, the generated biogas residues need to be treated twice, and the purity of biogas products is low. Aerobic composting is not only low in cost, easy to operate, and less polluting, but also can recycle nutrients in kitchen waste. The finished product can be used as organic fertilizer, soil improver, flower substrate, etc.

During the aerobic composting process, the composting efficiency can be improved by adding exogenous microbial agents. According to reports, the main microorganisms in compost are generally bacteria. However, generally bacteria need to grow normally when the pH is greater than 6.0 and the temperature is above 20°C, which is unfavorable for aerobic composting in northern regions and severe cold seasons. The microbial flora in the composting process almost stops growing at low temperatures, it is difficult to decompose the organic matter in the food waste, and cannot generate enough heat to start the composting process, resulting in composting failure. In addition, food waste is easily acidified and corrupted during the stacking process, and the pH can be reduced to below 5.0, which affects the growth of bacteria; in addition, some food waste has a high oil content, with a wet basis oil content as high as 20%-30%, and oil is easy to wrap in The surface of the material makes it difficult for microorganisms to attach, and forms an anaerobic zone, leading to further acidification, etc. The research by Nakasaki et al. (2013) showed that by inoculating Pichia pastoris RB1 into compost materials added with different types of organic acids, organic acids can be degraded, and the pH can be raised above neutral, thereby stimulating the growth and reproduction of bacteria, and realizing the violent degradation of organic matter. degrades and accelerates the composting process. Choi and Park (1998) showed that yeast inoculation can act as an activator of compost failure. Therefore, it is particularly important to develop a strain with strong acid resistance, which can overcome the anaerobic environment and enable the food waste to start the aerobic composting heating process under low temperature conditions; The application effect in the low-temperature aerobic composting process of food waste under different conditions is more realistic and instructive.

Contents of the invention

Purpose of the invention: In order to overcome the deficiencies in the background technology, the first purpose of the present invention is to disclose a low-temperature acid-resistant Saccharomyces cerevisiae; the second purpose is to disclose the screening method of the Saccharomyces cerevisiae; Application of kitchen waste in low-temperature aerobic composting.

Technical solution: The invention discloses a low-temperature acid-resistant Saccharomyces cerevisiae, which is classified as Saccharomyces cerevisiae DW, and is preserved in the China General Microorganism Culture Collection Management Center, and the preservation number is: CGMCC No.22786.

The screening method for the above-mentioned low-temperature acid-resistant Saccharomyces cerevisiae comprises the following steps:

S1. Take refrigerated fermented rice wine grains, add deionized water, shake and mix well, then centrifuge to take the upper liquid in the acidic bean sprouts juice medium, enrich and cultivate, transfer to new acidic bean sprouts juice medium every few days, and continue Transfer multiple times to obtain enriched bacterial liquid;

S2. Aspirate the enriched bacterial solution for 10-fold concentration gradient dilution, take the diluted solution and spread it on the YPD solid plate, and continuously transfer the isolated single colonies to the YPD solid plate for isolation, purification, and culture, and finally obtain the strain DW.

Wherein, the components of the acidic bean sprout juice medium include: soybean sprouts 200g/L, sucrose 30g/L, lactic acid 0.5%, deionized water to volume, pH natural, sterilized at 115°C for 30 minutes;

YPD solid medium: peptone 20g/L, glucose 20g/L, yeast extract 10g/L, agar 20g/L, deionized water to volume, pH natural, sterilized at 115°C for 30min.

The application of the above-mentioned low-temperature acid-resistant Saccharomyces cerevisiae, using Saccharomyces cerevisiae as a starting agent for low-temperature aerobic composting of kitchen waste, is particularly effective when the temperature is 6-13°C.

At 6-8°C, mix Saccharomyces cerevisiae and Bacillus subtilis at a volume ratio of 1:1, and apply it to start low-temperature aerobic composting of acidified food waste.

At 10-13°C, mix Saccharomyces cerevisiae and Bacillus subtilis at a volume ratio of 1:2, and apply it to start acidification-low-temperature aerobic composting of high-oil kitchen waste.

The food waste is the acidified material formed by natural storage of fresh food waste after filtering for 1-3 days, and the acidified-high-oil material formed by adding 2% soybean oil from an external source.

Beneficial effect: compared with prior art, the advantages of the present invention are:

1. The obtained Saccharomyces cerevisiae DW can grow under low temperature conditions (4°C) and has good acid resistance;

2. When Saccharomyces cerevisiae DW and Bacillus subtilis are added in proportion under different aerobic composting conditions, compared with the use of single Bacillus subtilis, the compost can rapidly increase the pH value, start the temperature rise earlier, and enter the high temperature period earlier , and prolong the high temperature period;

3. Saccharomyces cerevisiae DW and its compound bacterial agent can make food waste aerobic composting spontaneously generate heat and heat up under low temperature conditions to drive the activities of other indigenous microorganisms without external heating, thereby saving costs;

4. The dosage of the bacterial agent is small, only need to add 2% bacterial solution with a concentration of 1×10 ⁷ cfu/mL to achieve obvious effect;

5. The microbial agent has a simple composition, Saccharomyces cerevisiae and Bacillus subtilis are easy to obtain from the environment, and the screening cost is low.

Description of drawings

Fig. 1 is the morphological figure of Saccharomyces cerevisiae DW colony of the present invention;

Fig. 2 is the morphological figure of Saccharomyces cerevisiae DW cell of the present invention;

Fig. 3 is the Saccharomyces cerevisiae DW phylogenetic tree of the present invention;

Fig. 4 is the Saccharomyces cerevisiae DW low temperature test of the present invention;

Figure 5 shows the changes in temperature, pH, and C/N ratio of acidified kitchen waste in low-temperature aerobic composting;

Figure 6 shows the temperature, pH, and C/N ratio changes of acidification-high-oil food waste low-temperature aerobic composting.

detailed description

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

Culture medium preparation

Acidic bean sprouts juice medium: soybean sprouts 200g/L, sucrose 30g/L, lactic acid 0.5%, deionized water to volume, pH natural. Sterilize at 115°C for 30 minutes.

YPD solid medium: peptone 20g/L, glucose 20g/L, yeast extract 10g/L, agar 20g/L, deionized water to volume, pH natural. Sterilize at 115°C for 30 minutes.

YPD liquid medium: peptone 20g/L, glucose 20g/L, yeast extract 10g/L, deionized water to volume, pH natural. Sterilize at 115°C for 30 minutes.

LB liquid medium: peptone 10g/L, yeast powder 5g/L, sodium chloride 10g/L, deionized water to volume, pH natural. Sterilize at 121°C for 20 minutes.

LB solid medium: peptone 10g/L, yeast powder 5g/L, sodium chloride 10g/L, agar 20g/L, deionized water to volume, pH natural. Sterilize at 121°C for 20 minutes.

Preparation of Saccharomyces cerevisiae

The Saccharomyces cerevisiae is classified as Saccharomyces cerevisiae DW, and is preserved in the China General Microorganism Culture Collection Management Center, and the preservation number is: CGMCC No.22786.

Sampling and enrichment: Samples were collected from rice wine distillers grains, and refrigerated immediately after retrieval for future use. Take 2g of refrigerated fermented distiller's grains, add 18ml of deionized water, shake with a vortex shaker at the highest speed for 10min; centrifuge at a speed of 6000rpm for 10min in a centrifuge.

Take 1mL of the supernatant in the centrifuge tube and put it in 50mL acidic bean sprouts juice medium, place it in a shaker at 28°C for culture, transfer it to another new acidic bean sprouts juice medium every three days, and separate after three consecutive transfers.

Separation of strains: Separate the culture medium from the final transfer using the dilution coating plate method. Serially dilute to 1×10 ^-1 , 1×10 ^-2 , 1×10 ^-3 , 1×10 ^-4 , 1×10 ^-5 , 1×10 ^-6 , 1×10 ^-7 concentrations according to 10-fold concentration gradient liquid. 100uL of 1×10 ^-3 , 1×10 ^-5 , and 1×10 ^-7 concentration liquids were respectively applied on YPD plates, and cultured in a biochemical incubator at 28°C for 2-3 days.

Purification of strains: Select smooth, round and milky white single colonies from the cultivated YPD plate, transfer them to new YPD plates several times in a row for streak separation, and culture them in a biochemical incubator at 28°C for 2-3 days. In the meantime, closely observe the colony shape and observe whether the cell shape is consistent with a microscope to ensure that the last isolation is a pure bacterial colony.

Strain preservation: pick and purify a single colony on YPD slant medium, culture at 28°C for 2-3 days, and store in refrigerator at 4°C for later use.

Identification of Saccharomyces cerevisiae

Colony morphology: Use an inoculation loop to pick a small amount of the above-mentioned slant-preserved strains, streak them on a YPD plate, and culture them in a biochemical incubator at 28°C for 2-3 days.

As shown in Figure 1, the colonies are round, with smooth surfaces and edges, milky white, opaque, sticky, and easy to stir up.

As shown in Figure 2, the shape of the bacteria: spherical, oval cells, etc. under the microscope.

Molecular identification:

Template preparation: Dip a single colony with a sterile pipette tip into a 1.5mL centrifuge tube with 10uL sterile ddH ₂ O, blow and mix well and use it as a DNA template. Amplification was performed using a fungal identification kit.

The amplification system is (50uL): DNA template 50-100ng, Forward Primer 0.5uL, Reverse Primer 0.5uL, PCR Premix 25uL, ddH ₂ O to make up to 50uL.

The reaction program was: pre-denaturation at 94°C for 5min, denaturation at 94°C for 1min, annealing at 54°C for 1min, extension at 72°C for 1min, 30 cycles, 72°C for 5min, and storage at 4°C.

After the obtained sequence was uploaded to NCBI, BLAST was used to search and compare, and the rDNA sequence with high similarity to the sequence was selected, and a phylogenetic tree was constructed with MEGA7.0, as shown in Figure 3. The results showed that the strain DW was similar to S. Saccharomyces cerevisiae) have the highest homology and belong to the same branch.

Low-temperature growth test: Inoculate a loop of the preserved strain DW into a YPD liquid medium test tube, measure the OD value after 10 hours of cultivation, adjust the initial concentration of the bacterial solution to 10 ⁷ cfu/mL, and dilute to 1×10 ⁷ , 1 ×10 ⁶ , 1×10 ⁵ , and 1×10 ⁴ cfu/mL were used for drop plate experiments, cultured at 4°C for 6 days, and the growth of DW at 2 days, 4 days and 6 days was recorded. The results showed that the strain could grow under the low temperature condition of 4° C. in a short period of time ( FIG. 4 ).

Application of Saccharomyces cerevisiae DW in low-temperature aerobic composting of acidified kitchen waste

Bacteria preparation:

Use an inoculation loop to pick a small amount of strain DW preserved on a slant and place it in a test tube containing 5mL of YPD liquid medium, culture at 28°C and 180rpm for 10-12h to obtain seed liquid; inoculate 500uL of seed liquid into 500mL of YPD liquid medium, place Expand culture in a shaker at 28°C and 180rpm for 10-12h to obtain a fermentation broth. The strain used in the control group was Bacillus subtilis, provided by the School of Bioengineering, Zhejiang University of Technology. Similarly, use an inoculation loop to pick a small amount of Bacillus subtilis preserved on a slant and place it in a test tube containing 5 mL of LB liquid medium, and culture it at 37°C and 180 rpm for 14-16 hours to obtain seed liquid; inoculate 500uL of seed liquid into 500mL of LB liquid culture medium, placed in a shaker at 37°C, 180rpm and expanded for 14-16h to obtain a fermentation broth. After culturing separately, the fermentation broth was centrifuged at 8000×g for 10 min to collect the bacterial cells, and the obtained bacterial cells were washed with sterile water and resuspended. After the bacteria were resuspended, 1 mL was drawn for 10-fold gradient dilution, counted with a hemocytometer, and the bacteria solution was diluted to 1×10 ⁷ cfu/mL according to the counting results.

Food waste pretreatment:

The food waste comes from the staff canteen of the Organic Recycling Research Institute (Suzhou) of China Agricultural University. Filter the collected food waste to remove water, pick out eggshells, bones, paper towels, etc., store the food waste for 1-3 days, and naturally acidify it to a pH of about 5.0. Finally, crush it with a pulverizer so that the length of the material is about 1cm.

The room temperature for aerobic composting of acidified food waste is 6-8°C. Five treatments were designed in the experiment (T1V _{Saccharomyces cerevisiae} : V _{Bacillus subtilis} =1:0, T2V _{Saccharomyces cerevisiae} : V _{Bacillus subtilis} =0:1, T3V _{Saccharomyces cerevisiae} : V _{Bacillus subtilis=1:1, T4V Saccharomyces cerevisiae: V Bacillus subtilis} =1:1, T4V _{Saccharomyces cerevisiae} : V _{Bacillus _subtilis} =2:1, T5V _{Saccharomyces cerevisiae} : V _{Bacillus subtilis} =1:2), each process is repeated three times, each repetition is 2kg, wherein the ratio of kitchen waste and sawdust is 4:1, The initial moisture is 60%, the C/N ratio is about 30, and the inoculation amount of bacteria is 2%.

During the composting process, the temperature was regularly recorded every day; the turning frequency was set to 2 times a day, 2 minutes each time; the sampling time was once every 2 days. The pH value of the fresh sample was measured with a pH meter, and the C/N ratio of the air-dried sample was measured with an elemental analyzer.

The measurement results were compared with the control group respectively.

As shown in Figure 5, the results show that:

(1) In the first 96 hours, the treatment with Saccharomyces cerevisiae DW (T1, T3, T4, T5) was started by heating up first, and the temperature was increased by 5-10°C compared with the treatment with only Bacillus subtilis (T2).

(2) Within 120-216 hours, T3 and T4 entered the 50°C high temperature period first, 72 hours earlier than T2; followed by T5 and T1, 42 hours earlier than T2.

(3) The duration of the high temperature period above 50℃ in the treatment of adding Saccharomyces cerevisiae was longer than 1.0d, while T2 was only maintained for 0.95d.

(4) Within 120 hours, the pH value of Saccharomyces cerevisiae treatment increased rapidly to 5.3-6.25, while the pH value of T2 changed the slowest, which was 4.7.

(5) Within 120-192h, the pH value of Saccharomyces cerevisiae treatment increased rapidly to about 8.5, while the pH value of T2 was about 7.1.

(6) The change of temperature and pH value indicated that adding Saccharomyces cerevisiae DW was better than adding only a single Bacillus subtilis inoculum in the process of acidifying food waste low-temperature aerobic composting, and Saccharomyces cerevisiae DW had a better effect on acidification The low-temperature aerobic composting of food waste has obvious promoting effect.

(7) In the process of low-temperature aerobic composting of acidified kitchen waste, the composition of the bacterial agent is the best when V _{Saccharomyces cerevisiae} : V _{Bacillus subtilis} = 1:1.

Application of Saccharomyces cerevisiae DW in low-temperature aerobic composting of acidification-high-oil kitchen waste

Bacteria preparation: same as above.

Food waste pretreatment: the same as above, and each repetition added 2% edible soybean oil from an external source and mixed evenly.

The room temperature of acidification-high-oil kitchen waste for aerobic composting is 10-13°C. There are 5 treatments designed in the experiment (each treatment is the same as above), and each treatment is repeated three times, and each repetition is 2kg. The ratio is 4:1, the initial moisture is 63%, the C/N ratio is about 28, and the inoculum inoculum is 2%.

The measurement results were compared with the control group respectively.

As shown in Figure 6, the results show that:

(1) In the first 72 hours, the treatment with Saccharomyces cerevisiae DW (T1, T3, T4, T5) was first started with temperature rise compared with the treatment with only Bacillus subtilis (T2), and the temperature was increased by 4-14°C.

(2) In the first 72 hours, the temperature of the treatment with Saccharomyces cerevisiae was raised first, reaching 30-41°C; while the temperature of T2 changed the slowest, about 27°C.

(3) Within 72-192h, the treatment of adding Saccharomyces cerevisiae entered the high temperature period of 45℃ at the earliest, which was 38-56h earlier than T2.

(4) Within 48 hours, the pH value of Saccharomyces cerevisiae treatment increased rapidly to 5.5; while the pH value of T2 was 4.7.

(5) Within 48-192 hours, the pH value of the treatment with Saccharomyces cerevisiae rose to about 7.2 48 hours earlier than T2.

(6) The change of temperature and pH value showed that the treatment of adding Saccharomyces cerevisiae DW was better than adding only a single Bacillus subtilis inoculum in the acidification-high-oil food waste low-temperature aerobic composting process, and Saccharomyces cerevisiae DW It has a significant promoting effect in the process of acidification-low-temperature aerobic composting of high-oil food waste.

(7) In the process of acidification-high-oil kitchen waste low-temperature aerobic composting, the composition of the bacterial agent is as follows: V _{Saccharomyces cerevisiae} : V _{Bacillus subtilis} = 1:2, the effect is the best.

Claims

A low-temperature acid-resistant Saccharomyces cerevisiae, characterized in that: the Saccharomyces cerevisiae is classified as Saccharomyces cerevisiae DW, and is preserved in the China General Microorganism Culture Collection Management Center, and the preservation number is: CGMCC No.22786.
The screening method of low-temperature acid-resistant Saccharomyces cerevisiae as claimed in claim 1, is characterized in that, comprises the following steps:

S1. Take refrigerated fermented rice wine grains, add deionized water, shake and mix well, then centrifuge to take the upper liquid in the acidic bean sprouts juice medium, enrich and cultivate, transfer to new acidic bean sprouts juice medium every few days, and continue Transfer multiple times to obtain enriched bacterial liquid;

S2. Aspirate the enriched bacterial solution for 10-fold concentration gradient dilution, take the diluted solution and spread it on the YPD solid plate, and continuously transfer the isolated single colonies to the YPD solid plate for isolation, purification, and culture, and finally obtain the strain DW.
The screening method of low-temperature acid-resistant Saccharomyces cerevisiae according to claim 2, is characterized in that:

The components of the acidic bean sprout juice medium include: soybean sprouts 200g/L, sucrose 30g/L, lactic acid 0.5%, deionized water to volume, pH natural, sterilized at 115°C for 30 minutes;
YPD solid medium: peptone 20g/L, glucose 20g/L, yeast extract 10g/L, agar 20g/L, deionized water to volume, pH natural, sterilized at 115°C for 30min.
The application of the low-temperature acid-resistant Saccharomyces cerevisiae according to claim 1 is characterized in that: Saccharomyces cerevisiae is used as a starting agent for low-temperature aerobic composting of kitchen waste.
The application of the low-temperature acid-resistant Saccharomyces cerevisiae according to claim 5, characterized in that: Saccharomyces cerevisiae and Bacillus subtilis are mixed according to the volume ratio of 1:1, and then applied to start low-temperature aerobic composting of acidified kitchen waste.
The application of the low-temperature acid-resistant Saccharomyces cerevisiae according to claim 5, characterized in that: Saccharomyces cerevisiae and Bacillus subtilis are mixed according to the volume ratio of 1:2, and then applied to start the low-temperature aerobic composting of acidification-high-oil kitchen waste.