CN106520615A - Composite microbial system for quickly degrading maize straw and preparation and pretreatment method thereof - Google Patents

Abstract

The invention discloses a composite bacterial strain for rapidly degrading corn stalks, a preparation method thereof and a pretreatment method for rapidly degrading straw by using the composite bacterial strain. The microbial composite strain for degrading corn stalks provided by the present invention is composed of an optimized combination of fungi, bacteria and actinomycetes, which is more diverse in composition and thus has a better degradation effect on corn stalks. By adopting the pretreatment method of the complex bacterial strain to pretreat corn stalks, the microstructure of the stalks is destroyed, the hydrolysis process is accelerated, the accessibility of lignocellulose is significantly improved, and the efficiency and stability of degrading straws are significantly improved. Further promote the resource utilization of corn stalks.

Description

Composite bacterial strain for rapidly degrading corn stalks, its preparation method and pretreatment method

technical field

The invention relates to the technical field of microbial screening methods, in particular to a composite bacterial strain for rapidly degrading corn stalks, a preparation method thereof, and a pretreatment method for rapidly degrading straw by using the composite bacterial strain.

Background technique

my country is a large agricultural country, and various crop straw resources are very rich, and crop straw is mainly composed of cellulose, hemicellulose and lignin. The cycle of microbial decomposition and transformation is long, so it is difficult to be used as a fertilizer source for seasonal crops. In the past, most of the straw was burned by farmers as fuel when there was a shortage of energy and fuelwood in rural areas. However, with the development of the rural economy and the resolution of rural energy problems, straw is no longer used as fuel, and the phenomenon of concentrated burning of crop straw during the harvest season has become increasingly prominent, which not only pollutes the environment and endangers people's health, but also fills the air with smoke and affects land transportation. and flight safety.

At present, the main utilization methods of straw include straw returning to the field, feed utilization, straw gasification, solidification and carbonization, as a culture material for the production of edible fungi, and industrial applications.

Straw returning includes on-site returning, rapid retting and composting. Among them, returning straw to the field can increase the content of soil organic matter and fertilize the soil, which is of great significance to the sustainable development of agriculture, but there are still many problems that have not been resolved. First, the high carbon-to-nitrogen ratio (C/N) of straw is not conducive to soil microbial degradation, but may also lead to nitrogen deficiency and poor growth of crops; second, straw may also spread some diseases, resulting in crop yield reduction and so on. However, rapid retting and composting have the problem that cellulose is difficult to degrade.

Feed utilization technologies mainly include ammonification of silage, fermentation and production of single-cell protein. There are many kinds of crop straws that can be used as feed in our country, but they have common nutritional characteristics: low content of protein, soluble carbohydrates, minerals and carotene, and high content of crude fiber, so the digestibility is low and the palatability is poor. To improve palatability and increase digestibility, there is a problem of cellulose degradation. Industrial applications include papermaking, biodegradable production of starch, alcohol, acetic acid, etc., which are likely to cause serious environmental pollution, and also have the problem of cellulose degradation.

At this stage, there are many studies on its biomass energy conversion, among which the anaerobic fermentation of straw to produce biogas is one of the current research hotspots. Generally speaking, in the composition of corn stalks, the content of lignin is 17.5%, the content of cellulose is 37.3%, the content of hemicellulose is 20.6%, the content of ash is 6.1%, the content of organic acid salt is 2.0%, other The substance content is 18.5%. Among them, cellulose and hemicellulose are the sources of fermentable sugars, but the presence of lignin makes the hydrolysis process of corn stover slow and the degree of hydrolysis is low, which affects the subsequent acidification and gas production process.

Therefore, it is of great significance to study the appropriate pretreatment method to pretreat corn stalks, thereby destroying the microstructure of the stalks and accelerating the hydrolysis process, which is of great significance for resource utilization such as improving the gas production performance of biogas produced from corn stalks. Become a technical problem to be solved urgently in this field.

Contents of the invention

The technical problem to be solved by the present invention is to overcome the deficiencies of the prior art, and provide a composite bacterial strain capable of rapidly and efficiently degrading corn stalks and a degradation method thereof, so that the efficiency and stability of degrading corn stalks can be significantly improved.

In order to achieve the aforementioned object of the invention, the technical solutions adopted in the present invention include:

A complex bacterial strain that rapidly degrades corn stalks, including fungi, bacteria and actinomycetes. Bacillus, Pseudomonas aeruginosa, Actinomycetes including Streptomyces sorrel.

A preparation method of the composite bacterial strain, comprising the steps of:

Prepare liquid medium: wherein, liquid medium comprises nutrient gravy medium, LB medium, PDA medium and yeast powder, starch medium;

Expanded cultivation of a single strain: sterilize the prepared liquid culture medium at high temperature, after cooling to room temperature, use an inoculation loop to take a small amount of each strain for strain cultivation;

Generating complex bacterial strains: Activate the various bacterial strains preserved on the inclined plane through solid-state culture, inoculate the activated different single bacterial strains into different liquid culture media, and place them in a constant temperature shaking incubator for cultivation; then different The bacteria solution of the bacteria was mixed evenly.

Preferably, prepare nutrient gravy medium: take 5 grams of peptone, 30 grams of beef extract, 5 grams of sodium chloride, 1000 ml of distilled water, and sterilize at 121°C for 30 minutes for the cultivation of Bacillus circulans; prepare LB medium : Take 5 grams of yeast extract, 10 grams of peptone, 10 grams of sodium chloride, 1000 ml of distilled water, and sterilize at 121 ° C for 30 minutes for the cultivation of Pseudomonas aeruginosa; prepare yeast powder and starch medium: take yeast Extract 2 g, soluble starch 10 g, distilled water 1000 ml, sterilize at 121 ° C for 30 minutes, used for the cultivation of Streptomyces chestnutus; prepare PDA medium: take 1000 ml of potato extract, 20 g of glucose, at 115 ° C Sterilize for 30 minutes for the cultivation of Phanerochaete chrysosporium, Versicolor versicolor, Trichoderma viride, Aspergillus niger, and Glyophyllum compactus.

Preferably, the expanded cultivation of a single strain refers to sterilizing the prepared culture medium at high temperature for 30 minutes, cooling to room temperature, taking 1 to 2 rings of strains in the Erlenmeyer flask with an inoculation loop, and placing them in In a constant temperature shaking incubator, the culture was continued for 2 days under constant temperature conditions of 30°C and 120r/min.

Preferably, generating a composite strain refers to activating the strains preserved on the inclined plane through solid-state culture respectively, inoculating different single strains after activation into different liquid culture media, and placing them in a constant temperature shaking incubator, Under constant temperature conditions of 30°C and 120r/min, continuous culture was carried out for 2 days, and then the bacterial solutions of different strains were mixed evenly.

A pretreatment method for rapidly degrading corn stalks by using the composite bacteria system. Take 30 grams of straws, put them in a 250 ml Erlenmeyer flask, add the bacteria solution according to the solid-liquid mass ratio of 1:5, mix them evenly and seal them with a sealing film , in order to maintain the moisture content of the substrate in the Erlenmeyer flask; culture at a constant temperature of 30°C for 14 days, and control the humidity above 80%.

Compared with prior art, the advantage of the present invention is:

(1) The microbial complex strain for degrading corn stalks provided by the present invention is an optimized combination of fungi, bacteria and actinomycetes, which can produce destructive effects on the surface coating layer by secreting degrading enzymes, and the straw surface is rough after being pretreated by the complex bacteria There are a lot of protrusions and holes on the surface of rough, bulky and rough structure, and the structure of lignocellulose changes, which increases the exposed area of cellulose, which is conducive to the direct entry of microbial hyphae into the interior of the straw, and degrades the internal lignocellulose by secreting degrading enzymes. degradation. The results showed that the accessibility of lignocellulose was significantly improved during the pretreatment of straw with complex bacteria, and the utilization efficiency of lignocellulose was further significantly improved.

(2) The present invention creatively prepares a composite strain of bacteria. Studies have shown that the pretreatment of corn stalks by the composite strain of the present invention can destroy the microstructure of the straw, accelerate the hydrolysis process, and make its degradation efficiency higher than that of a single strain or Other flora were significantly improved.

(3) By optimizing specific parameters to prepare a complex bacterial system, a rapid and efficient pretreatment was achieved, which further significantly improved the gas production performance of biogas produced from corn stalks.

Description of drawings

Figure 1 is the microbial growth curve during the pretreatment of corn stalks.

Figure 2 shows the change of pH value during the pretreatment of corn stalks.

Figure 3 shows the change of COD value during the pretreatment of corn stalks.

Figure 4 is a scanning electron microscope image of corn stalks before pretreatment.

Figure 5 is a scanning electron microscope image of corn stalks after natural accumulation pretreatment.

Fig. 6 is a scanning electron microscope image of corn stalks pretreated by complex bacteria.

detailed description

In order to make the above and other objects, features and advantages of the present invention more comprehensible, the preferred embodiments of the present invention are taken as examples below to describe in detail as follows.

According to the main components of corn stalks as shown in Table 1, the present invention creatively prepares a composite bacterial system that rapidly degrades corn stalks by utilizing the characteristics of different bacterial species, the composite bacterial system includes fungi, bacteria and actinomycetes, wherein Fungi include Myxophyllum compacta, Phanerochaete chrysosporium, Versicolor versicolor, Trichoderma viride, and Aspergillus niger, bacteria include Bacillus circulans, Pseudomonas aeruginosa, and actinomycetes include Streptomyces sorrel.

Table 1 Main components of corn stalks

raw material C/% N/% C/N Cellulose/% Hemicellulose/% Lignin/% Total solids TS/% Volatile solid VS/% straw 49.41±1.87 1.48±0.14 33.39 36.53±1.27 27.93±1.62 15.38±0.92 90.92±0.73 87.49±0.31

Corn stover is mainly composed of cellulose, hemicellulose and lignin. The degradation of these substances requires the synergy of a variety of microorganisms. The white rot fungus in this example (Phanerochaete chrysosporium and Versicolor versicolor versicolor in the composite fungus) has good degradation ability to lignin, and can degrade lignin wrapped in cellulose, which is beneficial to cellulose exposed, thereby enhancing the degradation efficiency of other microorganisms to cellulose. At the same time, Aspergillus niger and Trichoderma viride in this example have better degradability to cellulose and hemicellulose in corn stalks. In addition, the actinomycetes and bacteria in this implementation can structurally change the structure of lignocellulose and improve the accessibility of cellulose.

The present invention preferably provides a method for rapidly degrading corn stalks by complex bacterial strains, comprising the following steps:

The preparation method of the liquid medium is as follows: nutrient gravy medium: peptone 5g, beef extract 30g, sodium chloride (NaCl) 5g, distilled water 1000ml, sterilized at 121°C for 30min for the cultivation of Bacillus circulans; LB medium: yeast Extract 5g, peptone 10g, sodium chloride (NaCl) 10g, distilled water 1000ml, sterilize at 121°C for 30 minutes, for the cultivation of Pseudomonas aeruginosa; yeast powder, starch medium: yeast extract 2g, soluble starch 10g , 1000ml of distilled water, sterilized at 121°C for 30min, used for the cultivation of Streptomyces chestnutus; PDA medium: 1000ml of potato extract, 20g of glucose, sterilized at 115°C for 30min, used for Phanerochaete chrysosporium and Versicolor versicolor , Trichoderma viride, Aspergillus niger, and Glycophyllum compact.

Expansion cultivation of a single strain: sterilize each prepared liquid medium at high temperature for 30 minutes, cool to room temperature and place in a conical flask, use an inoculation loop to take 1 to 2 rings of each strain in a conical flask, and put It is placed in a constant temperature shaking incubator, and continuously cultured at 30°C and 120r/min for 2 days, and the various strains after the expanded culture are preserved on a slant.

Composite bacteria pretreatment: activate the strains preserved on the slant after expanded culture through solid-state culture, inoculate the activated different single strains into different liquid media, and place them in a constant temperature shaking incubator Inside, cultured continuously at 120r/min at 30°C for 2 days. Then mix the bacteria liquid of different strains evenly, take 30g of spare straw, put it in a 250mL conical flask, add the bacterial liquid according to the solid-to-liquid mass ratio of 1:5, mix well and seal it with a sealing film to maintain the matrix in the conical flask moisture content. Cultivate at a constant temperature of 30°C for 14 days, and control the humidity above 80%.

Wherein, the high temperature described in the expanded cultivation of a single strain is above 121°C.

The effect of the above pretreatment methods on the degradation efficiency of corn stalks was studied by using the method of controlled experiments. During the experiment, the natural stacking pretreatment of corn stalks was used as the control group. Samples were taken every 2 days to measure the microbial growth, pH value and COD value in the materials during different pretreatment processes, and the contents of lignin, cellulose and hemicellulose in the straw before and after pretreatment were measured, and the microstructure changes were observed with scanning electron microscope , comparative analysis of the reasons for the change of material characteristics.

Among them, lignocellulose determination method: the content of cellulose, hemicellulose and lignin in the straw is determined by the Paradigm water washing method. Determination of microbial growth: HP8453 (produced by Shanghai Agilent Technology Co., Ltd.) spectrophotometer was used to measure the OD value of the sample at 600nm, which was used as the concentration of microbial cells. Determination of pH value: use PHS-3C (Nanjing Kehuan Analytical Instrument Co., Ltd.) pH meter for determination. Determination of COD value: Take the supernatant of the fermentation broth after standing still, and use the KHCN-200A COD ammonia nitrogen analyzer (manufactured by Nanjing Kehuan Analyzer Co., Ltd.) to measure it.

Scanning electron microscope analysis: Take a small amount of straw samples before and after pretreatment, and observe them with S-3400N II scanning electron microscope (manufactured by HITACHI, Japan), with a magnification of 1500 times.

Test example: The beneficial effect of the complex bacterial strain constructed by the method of the present invention will be specifically described below through the experimental example. In this test example, the complex bacterial strain is inserted into the medium for cultivation at 30°C. Taking the natural accumulation pretreatment as a control, the microbial growth, pH value, COD value, scanning electron microscope image and lignocellulose content were measured respectively.

Test Example 1: Changes in microbial growth curves during pretreatment

The change of OD ₆₀₀ value during straw pretreatment is shown in Figure 1, where the horizontal axis in Figure 1 represents time (d), the vertical axis represents OD ₆₀₀ value, the dots in the curve represent natural accumulation pretreatment, and the square points represent composite Bacterial pretreatment. In the pretreatment process of complex bacteria, 0~2d is the lag period, and the bacteria grow slowly; 2~6d is the logarithmic growth period; 6~10d is the stable period, and the OD ₆₀₀ value of the material is always at a high level, reaching the maximum on the 8th day The value is 1.521; after 10 days, it enters the decay period, and the OD ₆₀₀ value of the material decreases slowly. During the straw pretreatment process, the OD ₆₀₀ values showed a trend of first increasing and then decreasing. The lag period of microorganisms in the process of compound bacteria pretreatment was shorter, indicating that the reproduction of microorganisms was slow in the natural accumulation process, and the compound bacteria could quickly adapt to the growth environment. , so that the straw can begin to degrade in a short period of time; the OD ₆₀₀ value of the composite bacteria during the pretreatment process is always higher than that of the natural accumulation pretreatment, indicating that the concentration of the bacteria during the pretreatment of the straw with the composite bacteria is relatively large, and the growth and metabolism of the bacteria are relatively large. exuberant.

Test Example 2: Changes in pH value during pretreatment

The change of pH value during the straw pretreatment process is shown in Figure 2, where the horizontal axis in Figure 2 represents time (d), the vertical axis represents pH value, the dots in the curve represent natural accumulation pretreatment, and the square points represent complex bacteria pretreatment. deal with. The complex bacteria pretreatment quickly entered the acidification period after the fermentation started, and the pH value reached the minimum value of 5.18 on the 2nd day; the pH value quickly rose to 6.81 within 2-6 days; the pH value of the material showed a slow rising trend after 6 days. The pH value of straw after compound bacteria pretreatment was always higher than that of natural accumulation pretreatment, and the straw experienced a longer acidification period in the process of natural accumulation pretreatment, which was not conducive to the growth of microorganisms; It can basically return to a neutral level. Generally, non-methanogenic microorganisms can adapt to a wide range of pH, while methanogenic bacteria are extremely sensitive to environmental changes, and can only grow and metabolize normally in an environment with a pH of 6.8-7.5. The complex bacteria have a strong pH control ability in the process of straw degradation. From the perspective of pH value, the straw pretreated by the complex bacteria is more suitable for subsequent anaerobic fermentation.

Test Example 3: Changes in COD during pretreatment

The change of COD value during straw pretreatment is shown in Figure 3, where the horizontal axis in Figure 3 represents time (d), the vertical axis represents COD (mg L ^-1 ), and the dots in the curve represent natural accumulation pretreatment, Box points represent complex bacteria pretreatment. The COD value in the process of straw compound bacteria pretreatment showed an increasing trend from 0 to 8 days, and reached the maximum value of 12865 mg·L ^-1 on the 8th day; after the peak period, the COD value of the material began to decrease slowly, and finally tended to a stable state. Through data comparison, it was found that with the pretreatment process, the organic matter content in the material pretreated by the compound bacteria increased, indicating that the compound bacteria degraded the straw gradually, and accumulated a large amount of organic matter; then decreased, indicating that the microorganisms in the straw Part of the organic matter was consumed during the pretreatment process; the change trend of organic matter content during the pretreatment process of straw natural accumulation indicated that the degradation rate of straw was slow and not complete during the hydrolysis process. In the pretreatment process, if the time is short, the degradation of the straw will not be complete, and if the time is too long, the organic matter will be consumed, and the organic matter produced after straw degradation is the carbon source in the subsequent anaerobic fermentation process. From the analysis of the purpose of utilizing organic matter, the complex bacteria After 8 days of pretreatment, the content of soluble organic matter reached the highest value, which was more suitable for anaerobic fermentation.

Test Example 4: Analysis of Straw Structure Changes

The scanning electron microscope images of the straw before and after pretreatment are shown in Figure 4-6. The surface of the straw before pretreatment is flat and smooth, and the structure is dense and regular. The structure of the straw after natural accumulation pretreatment is lumpy, and exfoliated layers appear on the surface of some structures, indicating that natural accumulation pretreatment can change the structure of some lignocellulose, but the degradation efficiency needs to be improved; There are a few gaps and holes, indicating that the straw with small particle size is degraded earlier in the pretreatment process, which has less damage to the straw structure. After the pretreatment of the compound bacteria, the surface of the straw was rough and bulky, and there were a large number of protrusions and holes on the surface of the rough structure, which indicated that the compound bacteria could have a destructive effect on the surface coating by secreting degradative enzymes; the lignocellulose structure changed, increasing the fiber content. The exposed area of the cellulose is beneficial to the microbial mycelia to directly enter the interior of the straw and degrade the internal lignocellulose by secreting degradative enzymes. The results showed that the accessibility of lignocellulose was improved during the pretreatment of straw with complex bacteria, which was beneficial to improve the utilization efficiency of lignocellulose.

Test Example 5: Changes in lignocellulose content before and after pretreatment

The changes of lignocellulose content before and after pretreatment are shown in Table 2. During straw pretreatment, the contents of neutral detergent fiber, cellulose and hemicellulose in straw were significantly reduced (P<0.05), indicating that neutral detergent fiber, cellulose and hemicellulose in corn straw were significantly reduced (P<0.05). Detergent fiber, cellulose, and hemicellulose are relatively easy to degrade; compared with natural accumulation pretreatment, the acidic detergent fiber and lignin content of corn stalks in the process of pretreatment with complex bacteria were significantly reduced (P<0.05), indicating that the complex bacteria have a strong effect on acidic detergent. Fiber and lignin have strong degradation ability.

Table 2 Chemical composition of corn stalks before and after pretreatment

Corn stalks Neutral detergent fiber/% Acid detergent fiber/% Cellulose/% Hemicellulose/% Lignin/% unprocessed 73.76±0.39a 45.83±0.14a 36.53±1.27a 27.93±1.62a 15.38±0.92a Natural accumulation pretreatment 65.38±1.12b 43.02±0.54a 31.17±1.52b 22.36±0.58b 13.46±1.35a Complex bacteria pretreatment 54.13±2.08c 38.61±1.05b 23.78±0.63c 15.52±0.76c 9.35±1.21b

Note: Different lowercase letters in the same row in the table indicate significant differences (P<0.05).

The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any form and in essence. Those who are familiar with this profession can use the above The disclosed technical content, and the equivalent changes of some changes, modifications and evolutions are all equivalent embodiments of the present invention; at the same time, any equivalent changes made to the above embodiments according to the substantive technology of the present invention Alteration, modification and evolution all still belong to the scope of the technical solution of the present invention.

Claims (6)

1. a kind of composite microbial system of fast degradation corn straw, it is characterised in that：

Composite microbial system includes funguses, antibacterial and actinomycetes, and wherein funguses include gloeophyllum trabeum, Phanerochaete chrysosporium, variegated cloud Sesame, Trichoderma viride and aspergillus niger, antibacterial include Bacillus circulans, Pseudomonas aeruginosa, and actinomycetes include Streptomyces badius.

2. a kind of preparation method of composite microbial system as claimed in claim 1, it is characterised in that comprise the steps：

S1）Prepare fluid medium：Wherein, fluid medium include nutrient culture medium, LB culture medium, PDA culture medium with And yeast powder, starch culture-medium；

S2）Single culture amplification culture：Each fluid medium to preparing carries out high temperature sterilize, after being cooled to room temperature, with connecing Kind ring takes a small amount of each strain and carries out spawn culture；

S3）Generate composite microbial system：Each strain of slant preservation is activated by solid state rheology respectively, the difference after activation Single culture is inoculated into different liquids culture medium respectively, and places it in culture in isothermal vibration incubator；Then by different bacterium The bacterium solution mix homogeneously planted.

3. the preparation method of composite microbial system according to claim 2, it is characterised in that：

Prepare nutrient culture medium：Take 5 grams of peptone, 30 grams of Carnis Bovis seu Bubali cream, 5 grams of Sodium Chloride, 1000 milliliters of distilled water, 121 DEG C sterilizing 30 minutes, for the culture of Bacillus circulans；

Prepare LB culture medium：Take 5 grams of yeast extract, 10 grams of peptone, 10 grams of Sodium Chloride, 1000 milliliters of distilled water, at 121 DEG C Sterilizing 30 minutes, for the culture of Pseudomonas aeruginosa；

Prepare yeast powder, starch culture-medium：Take 2 grams of yeast extract, 10 grams of soluble starch, 1000 milliliters of distilled water, 121 DEG C sterilizing 30 minutes, for the culture of Streptomyces badius；

Prepare PDA culture medium：1000 milliliters of Rhizoma Solani tuber osi extracting solution is taken, 20 grams of glucose sterilizes 30 minutes at 115 DEG C, for Huang The flat lead fungi of archespore hair, Coriolus Versicolor, Trichoderma viride, aspergillus niger, the culture of gloeophyllum trabeum.

4. the preparation method of the composite microbial system according to Claims 2 or 3, it is characterised in that：

Single culture amplification culture refers to the culture medium that will be prepared, and sterilizes 30 minutes at high temperature, after being cooled to room temperature, with connecing Plant ring and 1～2 ring of strain is taken in triangular flask, and place it in isothermal vibration incubator, 120r/min under 30 DEG C of constant temperatures Continuous culture 2 days.

5. the preparation method of composite microbial system according to claim 4, it is characterised in that：

Generation composite microbial system is referred to and is activated each strain of slant preservation respectively by solid state rheology, the difference after activation Single culture is inoculated into different liquids culture medium respectively, and places it in isothermal vibration incubator, under 30 DEG C of constant temperatures 120r/min is continuously cultivated 2 days, then by the bacterium solution mix homogeneously of different strain.

6. the preprocess method of the composite microbial system fast degradation corn straw described in a kind of utilization claim 1-5, its feature exist In：

30 grams of straw is taken, is placed in 250 milliliters of triangular flasks, according to solid-liquid mass ratio 1：5 add bacterium solution, with sealing after mix homogeneously Film is sealed, to maintain the moisture content of triangular flask mesostroma；Cultivate 14 days under 30 DEG C of constant temperatures, humid control is more than 80%.