阅读说明：本技术 一种牛粪和活性污泥共接种强化稻草秸秆产甲烷的方法 (Method for producing methane by using cow dung and activated sludge co-inoculated reinforced straw stalks ) 是由 邓玉营 阮文权 于 2020-04-28 设计创作，主要内容包括：本发明公开了一种牛粪和活性污泥共接种强化稻草秸秆产甲烷的方法,取活性污泥和牛粪,驯化；添加双效缓冲液：NaHCO<Sub>3</Sub>缓冲液,K<Sub>2</Sub>HPO<Sub>4</Sub>·3H<Sub>2</Sub>O和KH<Sub>2</Sub>PO<Sub>4</Sub>缓冲液,使体系初始pH值在6.8～7.1之间变动；添加(NH<Sub>4</Sub>)<Sub>2</Sub>SO<Sub>4</Sub>和瘤胃液；体系在最终产甲烷反应过程中以产乙酸为主,pH维持在6.37～8.12之间。本发明利用牛粪和活性污泥共接种维持秸秆水解和产甲烷之间的平衡；接种物经过驯化,获得高效稳定的接种物；本发明选择合适的发酵条件,维持体系适宜的水解和产甲烷环境,解决秸秆厌氧发酵易受到中间产物VFAs抑制的问题；最终固液相不同功能菌群协同作用实现秸秆发酵产甲烷。(The invention discloses a method for producing methane by inoculating and strengthening straw stalks through cow dung and activated sludge together, which comprises the steps of taking the activated sludge and the cow dung and domesticating; adding a double-effect buffer solution: NaHCO 2 3 Buffer solution, K 2 HPO 4 ·3H 2 O and KH 2 PO 4 Buffering solution to make the initial pH value of the system change between 6.8 and 7.1; addition of (NH) 4 ) 2 SO 4 And rumen fluid; the system mainly produces acetic acid in the final methane production reaction process, and the pH value is maintained between 6.37 and 8.12. The invention maintains the balance between straw hydrolysis and methane production by using cow dung and activated sludge for co-inoculation; domesticating the inoculum to obtain a high-efficiency stable inoculum; the invention has the advantages ofProper fermentation conditions are selected, the proper hydrolysis and methanogenesis environment of the system is maintained, and the problem that the straw anaerobic fermentation is easily inhibited by intermediate products VFAs is solved; finally, the solid-liquid phase realizes the methane production by straw fermentation under the synergistic effect of different functional floras.)

1. A method for producing methane by using cow dung and activated sludge to co-inoculate and strengthen straw stalks is characterized by comprising the following steps:

(1) respectively taking activated sludge and cow dung, wherein the mass fraction ratio of the activated sludge to the cow dung is 6: 1-3: 1;

(2) in the activated sludge and cow dung inoculation system, domestication: standing at room temperature until no gas is generated;

(3) adding the double-effect buffer solution: NaHCO 2₃Buffer solution, K₂HPO₄·3H₂O and KH₂PO₄Buffering solution to make the initial pH value of the system between 6.8 and 7.1;

(4) addition of (NH)₄)₂SO₄Supplementing a nitrogen source; adding nutrient elements required by the rumen fluid to supplement hydrolytic microorganisms, and sterilizing at 121 deg.C for 15min in an autoclave;

(5) the solid-liquid phase flora in the system has a synergistic effect of mainly producing acetic acid in the process of realizing the methane production reaction through straw fermentation, and the pH value is maintained between 6.37 and 8.12.

2. The method for strengthening straw stalk methane production through cow dung and activated sludge co-inoculation as claimed in claim 1, wherein the solid-liquid phase flora in the system comprises:

rumen coccus (Ruminococcus), Fibrobacter (Fibrobacter), Methanosarcina acetoacidophilum (Methanosarcina) and methanobrevibacterium (methanobacter) are present on the solid phase;

the liquid phase contains the genera Aminobacterium (Aminobacterium) and Thermomonas (Syntropimonas).

3. The method for producing methane by using straw stalks and reinforced straw co-inoculated with cow dung and activated sludge as claimed in claim 2, wherein the flora number ratio of rumenococcus (Ruminococcus), Fibrobacter (Fibrobacter), Methanosarcina acetoacidophilus (Methanosarcina) and methanobrevibacterium (methanobacterbrevibacter) on solid phase is (20-25): (1-2): (20-25): (3-6); the ratio of the number of the bacterial groups between the genus Aminobacter (Aminobacillus) and the genus Zymomonas (Syntropimonas) in the liquid phase is (7-8): 1.

4. the method for producing methane by using the straw stalks and the enhanced straw co-inoculated with the cow dung and the activated sludge as claimed in any one of claims 1 to 3, wherein the step (1): the cow dung is taken from a cow farm, solid residues of the cow dung are filtered by gauze, and the cow dung is stored at the temperature of 4 ℃ for later use; taking the activated sludge out of the anaerobic reactor, and adding glucose at regular time to activate the sludge to improve methanogenic activity; the mass fraction ratio of the activated sludge to the cow dung is 4: 1.

5. The method for producing methane by using the straw stalks and the activated sludge which are strengthened by the co-inoculation of the cow dung and the activated sludge as the raw materials according to any one of the claims 1 to 3, wherein the step (3): said NaHCO₃The final concentration in the system is 5.00-8.00 g.L^-1(ii) a Said K₂HPO₄·3H₂O and KH₂PO₄The final concentration of the buffer solution in the system is 0.59-1.34 g.L^-1And 0.45 to 0.51 g.L^-1(ii) a And storing the domesticated inoculation system in an environment at 4 ℃ for later use.

6. The method for strengthening straw stalk methane production through cow dung and activated sludge co-inoculation as claimed in claim 5, wherein the NaHCO is used₃Final concentration in the system 5.00 g.L^-1(ii) a Said K₂HPO₄·3H₂O and KH₂PO₄The final concentration of the buffer solution in the system is 1.34 g.L^-1And 0.51 g.L^-1。

7. The method for producing methane by using the straw stalks and the activated sludge which are strengthened by the co-inoculation of the cow dung and the activated sludge as the raw materials according to any one of the claims 1 to 3, wherein the step (4): said (NH)₄)₂SO₄The final concentration in the system is 0.26 to 0.3 g.L^-1；

The preparation method of the rumen fluid comprises the following steps: taking from rumen of freshly slaughtered milk cow, placing into a container with a cover, filtering out forage through two layers of gauze within 5h to obtain rumen fluid, and storing at 4 deg.C for use; the percentage by volume of the rumen fluid is 15%.

8. The method for strengthening straw stalk methane production through cow dung and activated sludge co-inoculation as claimed in claim 7, wherein the (NH) is₄)₂SO₄The final concentration in the system was 0.26 g.L^-1。

Technical Field

The invention belongs to the field of anaerobic digestion of agricultural wastes in environmental engineering, relates to a preparation method of an inoculum for producing methane from straw stalks, and particularly relates to a method for strengthening the methane production from the straw stalks by co-inoculation of cow dung and activated sludge.

Background

The rice straw yield is huge every year in China. However, at the present stage, the utilization level is low, and the incineration becomes a main way for the waste water treatment, thereby causing environmental pollution. The anaerobic fermentation for producing methane is an important way for recycling the straws, and the biogas residues and the biogas slurry can be used for returning organic fertilizers to the field, so that the comprehensive benefit is obvious.

The methane production of the straws is closely related to the activity of microorganisms in the system. If the structure of the straw is complex, the lignin and the hemicellulose matrix are wrapped on the outer side of the structure, so that the cellulose is prevented from being adsorbed and degraded by microorganisms, and the change of hydrolytic flora can cause the difference of enzyme activity and influence the degradation of the biomass structure of the straw. Therefore, the efficient hydrolytic bacteria in the inoculum are beneficial to improving the hydrolysis efficiency. The common inoculum for producing methane by anaerobic fermentation of straw stalks is activated sludge of a sewage treatment plant, contains few hydrolytic microorganisms, is easy to cause accumulation of Volatile Fatty Acids (VFAs) in a system, and inhibits the methane production process.

Disclosure of Invention

Aiming at the problem that the efficiency of the current methane-producing inoculum of the rice straws is not high, the invention provides a method for strengthening the methane production of the rice straws by co-inoculating cow dung and activated sludge.

The technical scheme adopted by the invention for solving the technical problems is as follows:

a method for producing methane by using cow dung and activated sludge to co-inoculate and strengthen straw stalks is characterized by comprising the following steps:

(1) respectively taking activated sludge and cow dung, wherein the mass fraction ratio of the activated sludge to the cow dung is 6: 1-3: 1;

(2) domesticating in the activated sludge and cow dung inoculation system, and standing at room temperature until no gas is generated;

(3) adding the double-effect buffer solution: NaHCO 2₃Buffer solution, K₂HPO₄·3H₂O and KH₂PO₄Buffering solution to make the initial pH value of the system between 6.8 and 7.1;

(4) addition of (NH)₄)₂SO₄Supplementing a nitrogen source; adding nutrient elements required by the rumen fluid to supplement hydrolytic microorganisms, and sterilizing at 121 deg.C for 15min in an autoclave;

(5) the solid-liquid phase flora in the system has a synergistic effect of mainly producing acetic acid in the process of realizing the methane production reaction through straw fermentation, and the pH value is maintained between 6.37 and 8.12.

Further, the solid-liquid phase flora in the system comprises: rumen coccus (Ruminococcus), Methanosarcina (Methanosarcina) acetophilus (Fibrobacter) and Methanobrevibacterium (Methanobrevibacterium) are present on the solid phase; the liquid phase contains the genera Aminobacterium (Aminobacterium) and Thermomonas (Syntropimonas).

Further, the number ratio of the colonies among Ruminococcus (Ruminococcus), Cellobacter (Fibrobacter), Methanosarcina acetophila (Methanosarcina), Methanobrevibacterium (Methanobrevibacter) on the solid phase is (20-25): (1-2): (20-25): (3-6); the ratio of the number of the bacterial groups between the genus Aminobacter (Aminobacillus) and the genus Zymomonas (Syntropimonas) in the liquid phase is (7-8): 1.

further, the step (1): the cow dung is taken from a cow farm, solid residues of the cow dung are filtered by gauze, and the cow dung is stored at the temperature of 4 ℃ for later use; taking the activated sludge out of the anaerobic reactor, and adding glucose at regular time to activate the sludge to improve methanogenic activity; the mass fraction ratio of the activated sludge to the cow dung is 4: 1.

Further, the step (3): said NaHCO₃The final concentration in the system is 5.00-8.00 g.L^-1(ii) a Said K₂HPO₄·3H₂O and KH₂PO₄The final concentration of the buffer solution in the system is 0.59-1.34 g.L^-1And 0.45 to 0.51 g.L^-1(ii) a And storing the domesticated inoculation system in an environment at 4 ℃ for later use. Further, said NaHCO₃Final concentration in the system 5.00 g.L^-1(ii) a Said K₂HPO₄·3H₂O and KH₂PO₄The final concentration of the buffer solution in the system is 1.34 g.L^-1And 0.51 g.L^-1。

Further, the step (4): said (NH)₄)₂SO₄The final concentration in the system is 0.26 to 0.3 g.L^-1(ii) a The preparation method of the rumen fluid comprises the following steps: collecting from rumen of freshly slaughtered milk cow, and placing into a coverIn a container, filtering forage by two layers of gauze within 5h to obtain rumen fluid, and storing in an environment at 4 ℃ for later use; the percentage by volume of the rumen fluid is 15%. Further, the (NH)₄)₂SO₄The final concentration in the system was 0.26 g.L^-1。

The present invention can solve the following problems:

(1) cow dung and the methanogenic activated sludge are inoculated together to prepare an inoculum, so that the balance between straw hydrolysis and methanogenesis is maintained, and the real methanogenic potential of the straws is favorably reflected.

(2) The inoculum is domesticated to obtain a high-efficiency stable inoculum, and the problem that no high-efficiency inoculum exists in the measurement of methane production of the straws is solved.

(3) Proper fermentation conditions are selected, the proper hydrolysis and methanogenesis environments of the system are maintained, and the problem that the anaerobic fermentation of the straws is easily inhibited by intermediate products VFAs is solved.

(4) Discloses the characteristics of solid-liquid phase flora of a fermentation system and discloses a fermentation methane production strengthening mechanism.

According to the method, the cow dung is added to improve the proportion of the hydrolytic flora, so that the difference of enzyme activity can be caused, and the degradation of the biomass structure of the straw is influenced. VFAs generated after hydrolysis enter a liquid phase, and are subjected to cross-nutrient oxidation to generate acetic acid, so that the acetic acid can be utilized by methanobacteria acetophilus. In addition, the final solid-liquid phase realizes the methane production by straw fermentation under the synergistic effect of different functional floras.

Drawings

FIG. 1 is a comparative schematic of daily methane production for a cow dung addition system and a control system;

FIG. 2 is the individual VFA concentration and pH change during fermentation for cow dung addition system and control system: (a) acetic acid mass concentration; (b) mass concentration of propionic acid; (c) mass concentration of butyric acid; (d) the pH value;

figure 3 is the morphological characteristics of rice straw residue observed with SEM for the cow dung addition system and the control system: (a-1) and (a-2) are control systems; the (b-1) and the (b-2) are cow dung adding systems.

Detailed Description

The invention is described in detail below with reference to the drawings and specific examples.

The content of cellulose, hemicellulose and lignin in the straw is measured by A2000i fiber analyzer (ANKOM, USA), and the residue is dried in a drying oven at 65 deg.C to constant weight. At 12000 r.min^-1The cells were centrifuged for 10 minutes, and the resulting supernatant was used for index analysis, which was repeated three times for each index. VFAs were measured using GC-2010Plus gas chromatography (Shimadzu, Japan) and pH was measured by a DELTA 320pH meter (Mettler-Torledo instruments, USA).

Centrifuging biogas slurry at 800 × g for 15min to obtain solid phase sample, centrifuging supernatant at 27,000 × g for 30min to obtain liquid phase sample, and using PowerDNA kits (Mo Bio, USA) respectively extract DNA of initial inoculum and solid-liquid phase samples, and carry out sequencing and flora analysis.

In the cow dung adding system, the mass fractions of the inoculated sludge, the rice straws and the cow dung in the whole system are respectively 8%, 2% and 2% (the mass fraction of the rice straws is in the range of 1% -4%, preferably 2%), only the activated sludge is in the comparison system, the cow dung is not added, and the rest is consistent with the cow dung adding system. The experiment was performed on a methane potential testing system (AMPTS, Sweden) with an effective volume of 250mL, with addition of NaHCO₃Final concentration 5.00 g.L^-1，K₂HPO₄·3H₂O and KH₂PO₄The final concentration of the buffer solution was 1.34 g.L^-1And 0.51 g.L^-1The initial pH value of the system is changed between 6.8 and 7.1. Addition of (NH)₄)₂SO₄Supplementing nitrogen source to the final concentration of 0.26 g.L^-1Adding 15% rumen fluid to supplement nutrient elements of hydrolytic microorganism, and sterilizing at 121 deg.C for 15min in autoclave.

1 methanogenesis analysis

As shown in FIG. 1, the highest methane yield of the control system occurred at 1d, which was 122.80mL d^-1And then decreases rapidly. The cow dung adding system has two methane producing peaks which are respectively present at the 4 th day and the 38 th day, and the highest methane producing amount reaches 120.35mL·d^-1. The yield of the methane reaches 269.32mL g by calculation^-1VS, 1.35 fold higher than the control system.

2 degradation of rice straw

The rice straw degradation rate of the cow dung adding system is 41.79%, which is higher than 39.32% of that of the control system. The contents of cellulose, hemicellulose and lignin in the rice straw raw material are 30.88%, 24.34% and 11.52% respectively, after fermentation is finished, the reduction rates of the cellulose and the hemicellulose in solid residues respectively reach 53.97% and 56.14%, compared with a control system, the degradation rate of the hemicellulose is not large, the hydrolysis rate of the cellulose is obviously higher than that of the control system, and the lignin is not utilized when the proportion is increased in the whole process, as shown in the following table.

TABLE 1 variation of fermentation parameters during degradation of Rice straws

Variation of 3VFAs concentration and pH

The accumulation and consumption of individual VFAs reflects the balance between fermentative acidogenesis and methanogenesis. As shown in FIG. 2, the acetic acid concentration in the control system reached a peak of 3.69 g.L at 4d^-1And then gradually decreases. The concentration of acetic acid in the cow dung adding system has two peak values at the 4 th d and the 32 th d respectively, and the mass concentration reaches 4.95 g.L respectively^-1And 3.89 g.L^-1And 36d are then fully utilized. The data show that the cow dung adding system is mainly used for producing acetic acid and fermenting, is consumed through an acetophilic methanogenesis way after being accumulated for a short time, the concentration of the methane production way is closely related to the methanogenesis activity, and the phenomenon is consistent with the metabolic type of methane production through fermenting other straws.

The cow dung adding system generates propionic acid accumulation at the beginning of fermentation, and the highest mass concentration reaches 3.65 g.L in 12 days^-1The concentration dropped to 0 after 20d, whereas the butyric acid dropped to 0 after 16 d. The highest concentration of the two acids in the control system respectively reaches 4.42 g.L^-1And 1.98 g.L^-1. It is believed that propionic and butyric acids are products that are not utilized in an anaerobic system to reduce hydrogen and only overcome heat by means of cross-nutrient oxidationMechanical obstacle, if the oxidizing bacteria grow slowly, the acidification of the system is easy to cause. In the research, the pH value is reduced to 6.73 at the 20 th day due to the transient accumulation of two acids in the cow dung adding system, but the pH value is changed within the range in the whole fermentation process due to the fact that the alternate oxidation flora contained in the inoculum of the system utilizes propionic acid and butyric acid.

4 functional microbial analysis

4.1 solid-phase functional flora

Changes of specific functional microorganisms derived from inoculated sludge and cow dung in the solid phase are shown in table 2, Clostridium is a main hydrolytic bacterium in all systems, and the relative abundance of efficient cellulose hydrolytic bacteria Ruminococcus and Fibrobacter in the system is higher than that of a control, which explains the phenomenon that the activity and the degradation rate of the rice straw cellulose hydrolytic enzyme are obviously improved after cow dung is added. Lutispora, sedimentary bacillus (Sementibacter) and the like belong to polysaccharide degrading acetogenic bacteria, and the improvement of relative abundance in a cow dung adding system shows that the acetogenic metabolic pathway is strengthened, which is beneficial to methane production. The acetophilic Methanosarcina (Methanosarcina) in the cow dung adding system is the main type, and the methanobrevibacterium (Methanobrevibacter) derived from cow dung is the second type; the relative abundance of Methanosaeta was highest in the control system.

TABLE 2 variation of the relative abundance of specific functional bacteria and Methanobacterium species on the solid phase

As shown in fig. 3, observation of rice straw residue with SEM also reflects a synergistic effect, functional microorganisms form a compact structure with the broken substrate in the cow dung addition system, which can accelerate the degradation of rice straw, while similar morphological characteristics are not observed in the control system.

4.2 liquid-phase functional flora

As shown in table 3, Butyrivibrio, Treponema, Prevotella and the like in the liquid phase belong to acid-producing microorganisms, and the acid-producing microorganisms in the control system have high relative abundance and can accumulate the accumulation of butyric acid, lactic acid and succinic acid, so that the acidification of the system inhibits the activity of methane production. The oxidizing bacteria from the inoculum can form a mutual-nutrition oxidizing bacteria group with the companion bacteria, and is beneficial to reducing the concentration of VFAs. In the cow dung adding system, the relative abundance of bacillus (Aminobacterium) and butyric acid type alteromonas (Syntrophomonas) is high, and the concentrations of propionic acid and butyric acid can be reduced through the alterogenic action. The propionic acid oxidizing bacteria such as Syntrophotobacter and Pelotomaculum mainly exist in a control system.

TABLE 3 variation of relative abundance of functional bacteria on liquid phase

Table 3 Changes in relative abundances of selected function bacteriain the liquid fraction

The above description is only a preferred embodiment of the present invention, and should not be construed as limiting the scope of the present invention. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the scope of the claims of the present invention.