WO2021062887A1

WO2021062887A1 - Straw biodegradable composite bacterial agent and application thereof

Info

Publication number: WO2021062887A1
Application number: PCT/CN2019/111574
Authority: WO
Inventors: 周辉
Original assignee: 广东普洛宇飞生物科技有限公司
Priority date: 2019-09-30
Filing date: 2019-10-17
Publication date: 2021-04-08
Also published as: CN110577917A

Abstract

Provided is a straw biodegradable composite bacterial agent, said compound bacterial agent being a composition of Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus cereus, Phanerochaete chrysosporium, and Trametes versicolor. Biodegradation of plant straw replaces the chemical pulping method commonly used in current pulping technologies, thus avoiding the environmental pollution caused by the use of a large number of chemical reagents in conventional pulping processes; in the pulping production process, water can be recycled and reused without discharging any sewage containing organic compounds and suspended matter, thus eliminating the sewage treatment phase and reducing production costs; no heating is required during the entire production process, decomposition takes place at ambient temperature, and bleaching takes place at ambient temperature. Also disclosed are a preparation method and application of said straw biodegradable composite bacterial agent.

Description

Straw biodegradable composite microbial agent and its application

Technical field

The invention relates to the technical field of straw degradation, in particular to a straw biodegradable composite fungus agent and its application.

Background technique

Straw cellulose is a very precious biomass energy resource on the earth, and it is widely present in various agricultural production systems. As a large grain-producing country, my country produces nearly 700 million tons of crop straw every year. However, due to the lack of guidance and effective treatment methods, the problem of straw treatment has become increasingly prominent. Most of the straw treatment methods are burning or discarding. This has also become a new source of rural environmental pollution. The current treatment of straw not only wastes valuable renewable resources, but also brings various hazards, causing air pollution, soil mineralization, fires and traffic accidents. How to comprehensively and effectively use crop stalks to avoid the waste of biomass energy and increase the income of farmers has become the focus of solving the stalk problem.

In recent years, with the development of modern industry, the problems of energy shortage and environmental pollution have become increasingly prominent, and the comprehensive utilization of straw resources has gradually become a hot spot that the society pays close attention to. Straw papermaking is one of the important directions. The paper industry is a major source of pollution, and pulping is the most important pollution source. Pulping is the first process in the paper industry. At present, chemical pulping or chemical-mechanical pulping methods are basically used worldwide, mainly alkaline methods, ammonium sulfite methods, etc. Therefore, traditional pulping processes are used in the pulping and cooking process. To a certain extent, Zhongjun will be accompanied by the discharge of a large amount of black liquor waste, which seriously pollutes the environment. With the increasingly stringent environmental protection requirements, traditional pulp and paper companies need to invest a lot of costs to treat sewage containing highly toxic and strong carcinogenic substances generated during the production process, which increases the production cost of the company and reduces the company's products. Market competitiveness. Therefore, it is necessary to improve the traditional process to improve the environmental friendliness of the pulping process.

Since the 1950s, biotechnology has developed by leaps and bounds. The use of biological methods to replace and solve certain processes in industrial production has become a research hotspot. In the field of pulping, biological methods are due to the use of microorganisms themselves. Cellulase is used to degrade lignocellulose, which is highly efficient, economical and safe, and has attracted widespread attention. Therefore, it is necessary to improve the traditional process to improve the environmental friendliness of the pulping process.

Summary of the invention

Based on this, the purpose of the present invention is to overcome the above-mentioned shortcomings of the prior art and provide a straw biodegradable composite microbial agent.

In order to achieve the above objective, the technical solution adopted by the present invention is: a straw biodegradable composite inoculum, the composite inoculum is Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus cereus, Phanerochaete chrysosporium and The compound of Trametes versicolor.

Among the compound bacteria in this application, Phanerochaete chrysosporium and Trametes versicolor mainly remove lignin from straw, and are helpful for bleaching and dye decolorization; Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus cereus Mainly carry out the decomposing of hemicellulose.

Preferably, the ratio of the colony numbers of Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus cereus, Phanerochaete chrysosporium and Trametes versicolor is: (1-3): (1-3): (1) ～3):(1～3):(1～3).

More preferably, the ratio of the number of colonies of Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus cereus, Phanerochaete chrysosporium and Trametes versicolor is 1:1:1:1:1.

At the same time, the present invention also provides a preparation method of the straw biodegradable composite microbial agent, which includes the following steps:

1) Colony activation: Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus cereus, Phanerochaete chrysosporium, and Trametes versicolor are respectively inoculated on solid plates under aseptic conditions to activate the colonies;

2) Seed culture: inoculate the above-mentioned activated strains in liquid culture medium under aseptic conditions, and cultivate in a shaker at 150-180rpm;

3) Fermentation culture: inoculate the above-mentioned seed liquid in a liquid medium, and ferment culture;

4) Preparation of the compound bacterial agent: the above-mentioned fermentation culture broth is mixed according to the ratio to prepare the compound bacterial agent.

Preferably, in step 1), Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus cereus are inoculated on YPD plates and cultured for 1-3 days; Phanerochaete chrysosporium and Trametes versicolor are inoculated on potato culture medium (PDA ) Plate culture for 3-5 days.

Preferably, in the step 2), Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus cereus are inoculated in YPD liquid medium at 25°C-30°C and cultured on a shaker for 2-4 days; Bacteria and Trametes versicolor were inoculated into potato liquid medium and cultured on a shaker for 3-5 days at 25°C-30°C.

Preferably, in the step 3), the inoculation amount of the seed liquid is 0.5-2% of the volume of the liquid culture medium.

More preferably, in the step 3), the inoculation amount of the seed liquid is 1% of the volume of the liquid culture medium.

In addition, the present invention also provides an application of the straw biodegradable composite bacterial agent in a pulping process.

Preferably, the pulping process includes the following steps:

1) Pretreatment of straws: the straws are removed and crushed;

2) Retort and sterilize: the crushed straw is subjected to retort sterilization, the sterilization temperature is 115-121℃, and the sterilization time is 15-20min;

3) Biodegradation: The straw after cooking and sterilization is sent to the degradation tank, and after cooling, the active fermentation broth containing the straw biodegradable compound bacterial agent is added to the degradation tank, mixed evenly, and fermented and degraded until the straw is fluffy to obtain the pulp raw material ；

4) Filtration: Put the pulp raw material filtered by the filter into the pulp tank, add water and stir to form pulp, add the pulping auxiliary agent and then stir and mix, wherein the quality of the pulping auxiliary agent is equal to the quality of the pulp 3%-5%;

5) Refining;

6) Bleaching;

7) Squeeze out the water.

Preferably, in the step 3), the added amount of the active fermentation broth is 20%-40% of the total weight of the plant fiber.

The addition amount of the above-mentioned active fermentation broth in this application is based on comprehensive consideration of the use and degradation effect of the fermentation broth. It is found that the addition amount in the above range can save the usage of the fermentation broth, the processing time is moderate, and the degradation effect is suitable. The choice made, although increasing the amount of use can accelerate the degradation, but the cost is also relatively high.

Preferably, in the step 3), the fermentation temperature is 20-30°C, and the fermentation time is 48-120 hours. The selection of the fermentation temperature and time of the above-mentioned composite bacterial agent in this application can better ensure the effective decomposition of cellulose.

More preferably, when the fermentation temperature is 20-25°C, the fermentation time is 80-120h; when the fermentation temperature is 25-30°C, the fermentation time is 48-80h.

Preferably, in the step 4), the pulping aid includes the following components by weight: 20-30 parts of urea, 10-20 parts of DTPA, 15-25 parts of fatty alcohol polyoxyalkyl ether and fatty alcohol polyoxyethylene 15-25 parts of oxyethylene ether.

More preferably, the pulping auxiliary agent further comprises an excipient in which calcium powder and sodium sulfate are mixed in equal proportions.

Among them, DTPA can eliminate the influence of heavy metal ions on subsequent hydrogen peroxide; urea can promote the stable and rapid decomposition of hydrogen peroxide; fatty alcohol polyoxyalkyl ether and fatty alcohol polyoxyethylene ether play a role in osmotic emulsification.

Preferably, the refining is that the pulp is brought into a high-consistency refiner through a conveyor belt, and 30-45° C. water is added, and the fibers in the slurry are decomposed through a high-speed refining sheet.

Preferably, the bleaching is: adding hydrogen peroxide to the pulp after refining for bleaching treatment for 2-3 hours, and raising the temperature to 90° C. to decompose the hydrogen peroxide after the bleaching treatment.

Preferably, the process of squeezing the moisture is: transporting the bleached pulp to the filter screen to filter out part of the moisture in the pulp, wherein the water is discharged to the reservoir at the lower part of the filter screen, and the filtered pulp passes through The squeezing machine squeezes out 40-50% of the moisture, and then passes through the baler to squeeze out 10% of the moisture, and the dryness of the finished pulp is 50-60%.

Compared with the prior art, the beneficial effects of the present invention are:

The invention belongs to an environmentally-friendly technology, which uses Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus cereus, Phanerochaete chrysosporium, and Trametes versicolor compound inoculants to process crop straws, thereby avoiding the traditional pulping process The use of a large number of chemical reagents pollutes the environment. During the pulping production process, water can be recycled without discharging any sewage containing organic compounds and suspended solids, avoiding the process of sewage treatment, saving production costs, and reducing energy. Consumption and consumption, no heating required during all production processes, decomposition at room temperature, and bleaching at room temperature.

The technology has novel and unique design, clear thinking, strong adaptability and universality, and a high degree of industrialization; the use of composite microbial agents for pulping avoids the use of a large number of chemical agents and solves the problem of wastewater treatment in the traditional pulping process , Which reduces the production cost of the enterprise, has advanced technology, and is environmentally friendly, in line with the national energy-saving emission reduction strategy.

Description of the drawings

Figure 1 is a flow chart of the pulping process of the invention.

Detailed ways

In order to better illustrate the objectives, technical solutions and advantages of the present invention, the present invention will be further described below with reference to the accompanying drawings and specific embodiments.

The preparation of the culture medium in the embodiment:

The components of potato culture medium (PDA) are as follows: potato extract 1.0L, glucose 20.0g, KH ₂ PO ₄ 3.0 g, MgSO ₄ .7H ₂ O 1.5 g, agar 15.0 g (no need for liquid medium), pH 6.0. The preparation method of the potato extract: take 200g of peeled potatoes, cut into small pieces, add 1.0L of water and boil for 30min, filter out the potato pieces, and make up the filtrate to 1.0L;

The composition of YPD medium is as follows: yeast extract 1%, tryptone 2%, glucose 2%, agar 1% (liquid medium is not required).

Example 1

An embodiment of the straw biodegradable composite inoculum of the present invention, the preparation method of the straw biodegradable composite inoculum in this embodiment is as follows:

1) Slant culture: Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus cereus were inoculated on YPD plates and activated for 3 days; Phanerochaete chrysosporium and Trametes versicolor were inoculated on potato medium (PDA) plates and activated for 4 days.

2) The above-mentioned activated strains were respectively inoculated into the corresponding liquid medium under aseptic conditions, and cultured on a shaker at 30°C and 180 rpm. Among them, Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus cereus were cultured for 2 days; Phanerochaete chrysosporium and Trametes versicolor were cultured for 5 days;

3) Inoculating the above seed liquid in a liquid medium, and fermenting it. The inoculation amount was 1% of the volume of the liquid medium, and the fermentation time was 7 days.

4) According to the number of colony forming units of Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus cereus, Phanerochaete chrysosporium, and Trametes versicolor, the number of colony forming units 3:1:3:1:3 is mixed to obtain a straw-degrading bacterial system.

The straw biodegradable composite microbial agent prepared above is applied to plant straw biodegradable pulping, and the specific method is shown in Figure 1:

1. Use a shredder to cut the straw into sections with a length of 5-10cm. After cutting, clean the straw to remove impurities on the straw, and then dry it after cleaning;

2. Use a vertical crusher to crush the straw;

3. The crushed plant stalks are subjected to steaming sterilization treatment, the steaming sterilization time is 20 minutes, and the temperature is 120°C;

4. The boiled and sterilized plant stalks are sent to the degradation tank. After cooling, add 30% of the dry weight of wheat straw stalks to the degradation tank, mix them evenly, and carry out fermentation and degradation at 30°C for 72 hours until the stalks are fluffy. The shape is the pulp raw material;

5. Use a filter screen to discharge the filtrate in the degradation tank to the storage tank, and the degradation liquid in the storage tank can continue to be put into the compound bacteria fermentation tank for recycling;

6. Put the pulp raw material filtered by the filter into the pulping tank, add water and stir to form pulp, add 3% pulping auxiliary agent to it, stir and mix, based on 100 parts by weight, the pulping auxiliary agent The ingredients are: 20 parts of urea, 10 parts of DTPA, 15 parts of fatty alcohol polyoxyalkyl ether and 25 parts of fatty alcohol polyoxyethylene ether. The balance is an excipient mixed with calcium powder and sodium sulfate in equal proportions;

7. The above-mentioned pulp is sent to the high-consistency refiner for refining. The refining is the pulp that is brought into the high-consistency refiner through a conveyor belt, and water at 30°C is added to make the fibers in the slurry through high-speed refining. break down;

8. After refining, add 20% hydrogen peroxide of dry weight of straw to the pulp for bleaching treatment. The treatment time is 2h. After bleaching, the temperature is increased to 90°C to decompose the hydrogen peroxide;

9. Transport the bleached pulp in step 8 to the filter screen to filter out part of the water in the pulp, where the water is drained to the reservoir at the bottom of the filter screen, and the filtered pulp is then passed through a squeezer and extruded 40-50% of the moisture is passed through the baler to squeeze out 10% of the moisture, and the dryness of the finished pulp will be 50-60%.

Example 2

An embodiment of the straw biodegradable composite inocula of the present invention, the preparation method of the straw biodegradable composite inocula in this embodiment is as follows:

1) Slant culture: Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus cereus were inoculated on YPD plates and activated for 1 day; Phanerochaete chrysosporium and Trametes versicolor were inoculated on potato medium (PDA) plates and activated for 3 days.

2) The above-mentioned activated strains were respectively inoculated into the corresponding liquid medium under aseptic conditions, and cultured on a shaker at 30°C and 180 rpm. Among them, Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus cereus were cultured for 3 days; Phanerochaete chrysosporium and Trametes versicolor were cultured for 3 days;

3) Inoculating the above seed liquid in a liquid medium, and fermenting it. The inoculation amount is 0.5% of the volume of the liquid medium, and the fermentation time is 5 days.

4) According to the 1:3:1:3:1 mixture of Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus cereus, Phanerochaete chrysosporium, and Trametes versicolor, the number of colony forming units is 1:3:1:3:1 to obtain a straw-degrading bacterial system.

The straw biodegradable composite inoculum prepared above is applied to plant straw biodegradable pulping, and the specific method is as follows:

2. Use a vertical crusher to crush the straw;

4. The boiled and sterilized plant stalks are sent to the degradation tank. After cooling, add 20% of the dry weight of the wheat straw stalks to the degradation tank, mix them evenly, and carry out fermentation and degradation at 25°C for 48 hours until the stalks are fluffy. The shape is the pulp raw material;

6. Put the pulp raw material filtered out by the filter into the pulping tank, add water and stir to form pulp, add 4% pulping auxiliary agent to it, stir and mix, based on 100 parts by weight, the pulping auxiliary agent The ingredients are: 30 parts of urea, 20 parts of DTPA, 25 parts of fatty alcohol polyoxyalkyl ether and 15 parts of fatty alcohol polyoxyethylene ether. The balance is an excipient mixed with calcium powder and sodium sulfate in equal proportions;

7. The above-mentioned pulp is sent to a high-consistency refiner for refining. The refining is the pulp that is brought into the high-consistency refiner through a conveyor belt, and water at 45°C is added to make the fibers in the slurry through high-speed grinding. break down;

Example 3

1) Slant culture: Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus cereus were inoculated on YPD plates and activated for 2 days; Phanerochaete chrysosporium and Trametes versicolor were inoculated on potato medium (PDA) plates and activated for 5 days.

2) The above-mentioned activated strains were respectively inoculated into the corresponding liquid medium under aseptic conditions, and cultured on a shaker at 30°C and 180 rpm. Among them, Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus cereus were cultured for 4 days; Phanerochaete chrysosporium and Trametes versicolor were cultured for 4 days;

3) Inoculating the above seed liquid in a liquid medium, and fermenting it. The inoculation amount was 2% of the volume of the liquid medium, and the fermentation time was 6 days.

4) According to the number of colony forming units of Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus cereus, Phanerochaete chrysosporium, and Trametes versicolor, the number of colony forming units is 1:1:1:1:1 to obtain a straw-degrading bacteria system.

2. Use a vertical crusher to crush the straw;

3. The crushed plant straws are subjected to steaming and sterilization treatment, the steaming and sterilization time is 15 minutes, and the temperature is 118°C;

4. The boiled and sterilized plant stalks are sent to the degradation tank. After cooling, add the composite bacteria yeast solution of 40% of the dry weight of wheat straw stalks to the degradation tank, mix well, and carry out fermentation and degradation at 20°C for 120 hours until the straws are fluffy. The shape is the pulp raw material;

5. Use a filter screen to discharge the filtrate in the degradation tank to the storage tank, and the degradation liquid in the storage tank can continue to be put into the compound fermentation tank for recycling;

6. Put the pulp raw material filtered by the filter into the pulping tank, add water and stir to form pulp, add 5% pulping auxiliary agent to it, stir and mix well, based on 100 parts by weight, the pulping auxiliary agent The ingredients are: 25 parts of urea, 15 parts of DTPA, 22 parts of fatty alcohol polyoxyalkyl ether and 18 parts of fatty alcohol polyoxyethylene ether. The balance is an excipient mixed with calcium powder and sodium sulfate in equal proportions;

7. The above-mentioned pulp is sent to a high-consistency refiner for refining. The refining is the pulp that is brought into the high-consistency refiner through a conveyor belt, and water at 40°C is added to make the fibers in the slurry through high-speed grinding. break down;

8. After refining, add hydrogen peroxide of 20% dry weight of straw to the pulp for bleaching. The treatment time is 2h. After bleaching, the temperature is raised to 90°C to decompose hydrogen peroxide;

9. Transport the bleached pulp in step 8 to the filter screen to filter out part of the water in the pulp. The water is drained to the reservoir at the bottom of the filter screen. The filtered pulp is then passed through a squeezer and extruded. 40-50% of the moisture is passed through the baler to squeeze out 10% of the moisture, and the dryness of the finished pulp will be 50-60%.

The pulp yield, whiteness, breaking length and ring pressure index of the paper pulp prepared under the conditions of the straw bioenzymatic degradation pulping process of the present invention are respectively tested, and the formula for calculating the pulp yield: (m ₁ /m)*100% (m ₁ refers to the dry weight of coarse pulp, m refers to the dry weight of raw materials), the whiteness and the breaking length of the paper and the ring pressure index were measured with a whiteness measuring instrument and a paper tensile strength measuring instrument. The test results As shown in Table 1 below.

Table 1 The properties of pulp prepared under the conditions of the straw bioenzymatic degradation pulping process of the present invention

To	实施例1Example 1	实施例2Example 2	实施例3Example 3
浆得率(％)Pulp yield (%)	75.275.2	74.374.3	77.677.6
白度(％ISO)Whiteness (%ISO)	8686	8585	8989
断裂长(Km)Break length (Km)	4.714.71	4.854.85	4.684.68
环压指数(N.m/g)Ring pressure index (N.m/g)	11.211.2	11.311.3	11.511.5

It can be seen from Table 1 that the method for biological pulping of straw using composite biological inoculants provided in the embodiments of the present invention has a pulp yield of more than 70%, whiteness of more than 85%, and a break length of more than 70%. 4.6Km, the ring pressure index is higher than 11.2Nm/g, and when the ratio of the bacteria is 1:1:1:1:1, the pulp yield and whiteness are 77.6% and 89%, respectively, and the effect is good. It shows that the method for biological pulping with a composite biological inoculum provided by the present invention meets the requirements in terms of pulp yield and whiteness, and the mechanical properties of the prepared paper are also excellent.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that, The technical solution of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solution of the present invention.

Claims

A straw biodegradable composite inoculum is characterized in that the composite inoculum is a combination of Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus cereus, Phanerochaete chrysosporium, and Trametes versicolor.
The straw biodegradable composite inoculum according to claim 1, wherein the ratio of the number of colonies of Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus cereus, Phanerochaete chrysosporium and Trametes versicolor is : (1～3):(1～3):(1～3):(1～3):(1～3).
The straw biodegradable compound inoculum according to claim 2, wherein the ratio of the colony numbers of Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus cereus, Phanerochaete chrysosporium and Trametes versicolor is :1:1:1:1:1.
A method for preparing a straw biodegradable composite microbial agent according to any one of claims 1 to 3, characterized in that it comprises the following steps:

1) Colony activation: Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus cereus, Phanerochaete chrysosporium, and Trametes versicolor are respectively inoculated on solid plates under aseptic conditions to activate the colonies;

2) Seed culture: inoculate the above-mentioned activated strains into liquid culture medium under aseptic conditions, and cultivate in a shaker at 150-180rpm;

3) Fermentation culture: inoculate the above-mentioned seed liquid in a liquid medium, and ferment culture;

4) Preparation of the compound bacterial agent: the above-mentioned fermentation culture broth is mixed according to the ratio to prepare the compound bacterial agent.
The method for preparing a straw biodegradable composite inoculum according to claim 4, wherein in the step 3), the inoculum of the seed liquid is 0.5-2% of the volume of the liquid culture medium.
An application of the straw biodegradable composite bacterial agent according to any one of claims 1 to 3 in a pulping process.
The application according to claim 6, wherein the pulping process includes the following steps:

1) Pretreatment of straws: the straws are removed and crushed;

2) Retort and sterilize: the crushed straw is subjected to retort sterilization, the sterilization temperature is 115-121℃, and the sterilization time is 15-20min;

3) Biodegradation: The straw after cooking and sterilization is sent to the degradation tank, and after cooling, the active fermentation broth containing the straw biodegradable compound bacterial agent is added to the degradation tank, mixed evenly, and fermented and degraded until the straw is fluffy to obtain the pulp raw material ；

4) Filtration: Put the pulp raw material filtered by the filter into the pulp tank, add water and stir to form pulp, add the pulping auxiliary agent and then stir and mix, wherein the quality of the pulping auxiliary agent is equal to the quality of the pulp 3%-5%;

5) Refining;

6) Bleaching;

7) Squeeze out the water.
The application according to claim 7, characterized in that, in the step 3), the added amount of the active fermentation broth is 20%-40% of the total weight of the plant fiber.
The application according to claim 7 or 8, characterized in that, in the step 3), the temperature of fermentation is 20-30°C, and the time of fermentation is 48-120 hours.
The application according to claim 7, characterized in that, in the step 5), the pulping aid contains the following components by weight: 20-30 parts of urea, 10-20 parts of DTPA, fatty alcohol polyoxane 15-25 parts of base ether and 15-25 parts of fatty alcohol polyoxyethylene ether.