Method for improving yield of methane by delignification through biological method

Abstract

The invention provides a method for increasing the yield of methane by delignification through a biological method, which comprises the following steps: carrying out delignification pretreatment on bamboo powder by using pleurotus ostreatus hypha, and carrying out anaerobic fermentation on the bamboo powder pretreated by the pleurotus ostreatus hypha to generate methane. In the growth process of the pleurotus ostreatus, a large amount of lignocellulolytic enzymes such as laccase, cellulase and chitinase are secreted, the internal structure of lignin in the bamboo powder is destroyed, and part of cellulose and hemicelluloses are decomposed into small molecules; according to the bamboo powder pretreated by the pleurotus ostreatus, substrate degradation flora can be selectively gathered, a microspace ecological niche can be optimized, methanation flora can be more easily and loosely combined with the bamboo powder pretreated by fungi, and the total methane yield of anaerobic fermentation of the bamboo powder pretreated by the pleurotus ostreatus is increased by 16.2% compared with the total yield of direct fermentation of the bamboo powder.

Description

Technical Field

[0001] The invention belongs to the technical field of solid waste treatment and resource utilization, and specifically relates to a method for increasing methane production by biological delignification. Background Art

[0002] Forest resources in my country and even the world are becoming increasingly scarce. Making full use of bamboo materials to replace wood materials is an inevitable trend of development. Bamboo has many varieties and is widely used, but a large amount of waste will be generated during industrial processing. These wastes are mainly composed of cellulose, hemicellulose, and lignin, which have good matrix utilization potential. Lignin, as one of the most important natural complex polymers, is composed of three phenylpropane units: syringyl, guaiacyl, and p-hydroxyphenyl. Because it is a highly renewable carbon source in the biosphere, it has the potential to replace petroleum-derived commodities. However, the carbon-oxygen and carbon-carbon bonds contained in lignin, as well as functional groups such as methoxy, carbonyl, hydroxyl, and carboxyl groups, make its degradation process very complicated.

[0003] The process of decomposing organic matter into methane, carbon dioxide, hydrogen sulfide, water and other substances through the action of facultative bacteria and anaerobic bacteria under anaerobic conditions is called anaerobic fermentation. The anaerobic digestion process is carried out through a biological mode, in which organic matter is degraded by a large number of diverse microbial communities in the absence of oxygen. Anaerobic fermentation is generally divided into the following four stages: the first stage: hydrolysis, fermentation bacteria secrete hydrolases such as amylase and protease to decompose complex macromolecular organic compounds into soluble monomers; the second stage: acidification, the dissolved monomers or oligomers produced by hydrolysis are further degraded into volatile fatty acids, lactic acid, alcohol and other substances; the third stage: acetic acidification, the organic acids and alcohols produced during the acidification process are converted into acetic acid, hydrogen and carbon dioxide by microorganisms; the fourth stage: methanation. Therefore, the combination of resource utilization of bamboo waste and methane production can not only solve the problem of environmental pollution, but also provide a new solution to overcome the energy crisis. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a method for increasing methane production by biological delignification in view of the deficiencies of the above-mentioned prior art. The method firstly uses Pleurotus ostreatus hyphae to pretreat bamboo powder with delignification, and then uses the pretreated bamboo powder for anaerobic fermentation to produce methane. The methane production capacity in the anaerobic fermentation system is improved by optimizing the ecological niche of the methanogenic bacteria, which provides ideas and options for further resource utilization of bamboo waste.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0006] A method for increasing methane production by biological delignification, the method comprising the following steps:

[0007] S1, crushing bamboo waste, air-drying, and sieving to obtain bamboo powder raw materials, drying the bamboo powder raw materials at 50° C.-55° C. for 22-24 hours, and then adding water to a moisture content of 55% to obtain a cultivation material, putting the cultivation material into a fungus bag, and then sterilizing to obtain a fungus bag;

[0008] S2, inoculating the vigorously growing Pleurotus ostreatus liquid spawn into the fungus bag obtained in S1, culturing in the dark at 25° C. to 27° C. for 30 days until the mycelium fills the fungus bag and buds appear, taking out the bamboo powder in the fungus bag, crushing it, and obtaining the Pleurotus ostreatus pretreated bamboo powder;

[0009] S3. Establish an anaerobic fermentation reactor, add bamboo powder pretreated with Pleurotus ostreatus obtained in S2 at 37°C under nitrogen gas condition, and detect methane production; when the daily methane production is 0, it is regarded as the fermentation end point, and the culture is terminated, and microbial community analysis is performed.

[0010] Preferably, the bamboo waste in S1 comes from waste from a bamboo furniture processing plant.

[0011] Preferably, the screening in S1 is through a 50-mesh sieve.

[0012] Preferably, the mycelium biomass in the Pleurotus ostreatus liquid spawn in S2 is 25-30 g / mL, and the ratio of the Pleurotus ostreatus liquid spawn to the culture medium in the spawn bag is 20-25 mL:900 g.

[0013] Preferably, the anaerobic fermentation reactor in S3 is composed of inoculation solution and sterile saline, the volume ratio of the inoculation solution to the sterile saline is 1:0.6; the ratio of the inoculation solution to the bamboo powder pretreated with Pleurotus ostreatus is 1L:10g.

[0014] Preferably, the inoculum is anaerobic sludge.

[0015] The present invention has significant technical effects due to the adoption of the above technical solution:

[0016] 1. The present invention provides a method for increasing methane production by biological delignification, wherein bamboo powder is first pretreated with delignification by using Pleurotus ostreatus hyphae, and then the bamboo powder pretreated with Pleurotus ostreatus is subjected to anaerobic fermentation to produce methane. During the growth process, Pleurotus ostreatus secretes a large amount of cellulose decomposing enzymes such as laccase, cellulase, and chitinase, which destroy the internal structure of lignin in bamboo powder and decompose part of cellulose and hemicellulose into smaller molecules, followed by anaerobic fermentation and cultivation. The bamboo powder pretreated with fungi can selectively aggregate substrate-degrading bacterial communities and optimize the microscopic spatial niche, so that the methanogenic bacterial communities are more likely to loosely combine with the bamboo powder pretreated with fungi, and the methane production is greatly improved.

[0017] 2. After anaerobic fermentation of bamboo powder pretreated with Pleurotus ostreatus in the present invention, the richness of Bacteroidetes was significantly increased by 49%, and its dominant position in the niche distribution of the tightly bound space was also greatly improved. The halophiles, which mainly play a role in methanogenesis, decreased slightly in the tightly bound space, but increased in the loosely bound space. Ultimately, the total methane production of anaerobic fermentation of bamboo powder pretreated with Pleurotus ostreatus was increased by 16.2% compared with the total production of direct fermentation of bamboo powder.

[0018] The present invention is further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is a graph showing changes in the activities of laccase, cellulase and chitinase of Pleurotus ostreatus at different culture periods in Example 1;

[0020] Figure 2 The scanning electron microscope (SEM) images of bamboo powder (BW) and bamboo powder pretreated with Pleurotus ostreatus (FBW);

[0021] Figure 3 FTIR spectra of bamboo powder (BW) and bamboo powder pretreated with Pleurotus ostreatus (FBW);

[0022] Figure 4 X-ray photoelectron spectra of bamboo powder (BW) and bamboo powder pretreated with Pleurotus ostreatus (FBW);

[0023] Figure 5 The change of methane production during anaerobic fermentation of bamboo flour (BW) and bamboo flour (FBW) pretreated with Pleurotus ostreatus, a: total production, b: daily production;

[0024] Figure 6 Microbial community analysis of anaerobic fermentation of bamboo flour (BW) and bamboo flour (FBW) pretreated with Pleurotus ostreatus, a: phylum level, b: genus level;

[0025] Figure 7Figure 3 Differences in spatial distribution of anaerobic fermentation microbial communities of bamboo powder (BW) and bamboo powder pretreated with Pleurotus ostreatus (FBW) at the phylum level, where T represents tightly bound space, L represents loosely bound space, and S represents suspended bound space. DETAILED DESCRIPTION

[0026] The bamboo waste of the invention comes from a bamboo furniture processing factory in Fuzhou, Jiangxi Province; the Pleurotus ostreatus is provided by the strain resource library of the Engineering Research Center for Edible and Medicinal Fungi of the Ministry of Education of Jilin Agricultural University, and the mycelium biomass in the liquid strain of Pleurotus ostreatus is 25-30 g / mL; the inoculum liquid is anaerobic sludge collected from the Ulu Panlan Sewage Treatment Plant in Singapore.

[0027] Example 1

[0028] This embodiment is a method for increasing methane production by biological delignification, comprising the following steps:

[0029] S1. Bamboo waste treatment

[0030] The bamboo waste is crushed, naturally air-dried, and then passed through a 50-mesh sieve to obtain a bamboo powder raw material. The bamboo powder raw material is dried at 50° C. for 24 hours, and then water is added to a moisture content of 55% to obtain a cultivation material. 900 g of the cultivation material is put into a mushroom bag, which is then sterilized to obtain a mushroom bag.

[0031] S2. Inoculation of Pleurotus ostreatus

[0032] 20 mL of the liquid culture of Pleurotus ostreatus with vigorous growth was inoculated into the fungus bag obtained in S1, and cultured in the dark at 25°C for 30 days. On the 5th day of culture, the fungus bag was cut open to take out 10 g of culture material, and the bag was sealed after sampling and continued to be cultured. The sampling was repeated at the same position of the fungus bag every 5 days until the mycelium covered the fungus bag and buds appeared. The fungus bag was opened and crushed to obtain bamboo powder (FBW) pretreated with Pleurotus ostreatus.

[0033] The results showed that the mycelium of Pleurotus ostreatus grew well during the culture period. No obvious mycelium was observed on the 5th day, small mycelium was observed in many places in the culture medium on the 10th day, the mycelium had basically grown all over the mushroom bag but no buds appeared on the 15th day, and buds appeared in several places on the 20th day.

[0034] The chitinase, laccase and cellulase activities of the sampled culture medium were determined as follows: 0.1 g of the culture medium sample was added with 1 mL of the extract, homogenized in an ice bath at 4°C, and centrifuged at 10000 g and 4°C for 10 min. The supernatant was collected as a crude enzyme sample and tested with the Solebold laccase activity test kit, Solebold chitinase activity test kit and Solebold cellulase activity test kit. The results are as follows Figure 1As shown. The chitinase activity gradually increased with the increase of culture time, with the largest increase from the 10th to the 15th day, and reached a maximum value of 2.97U / g on the 20th day, indicating that Pleurotus ostreatus grew well. The laccase activity reached a maximum of 3.29U / g on the 15th day, and maintained an upward trend throughout the 15 days, but dropped sharply on the 20th day, which may be due to the budding of mycelium, which greatly reduced the dependence of Pleurotus ostreatus on lignin. The cellulase activity maintained an upward trend throughout the 20 days, with the largest increase from the 15th to the 20th day, reaching a peak of 1.54U / g on the 20th day. Combined with the changes in laccase activity, this phenomenon may be due to the transformation of mycelium's dependence on lignin to dependence on cellulose after budding. Therefore, the budding period can be regarded as an important node for the transformation of Pleurotus ostreatus's strategy for decomposing lignocellulose.

[0035] S3. Anaerobic fermentation

[0036] An anaerobic fermentation reactor (effective volume of 1.6L, inoculum 1L, sterile physiological saline 0.6L) was established. Under nitrogen insufflation at 37°C, bamboo powder (FBW) pretreated with Pleurotus ostreatus obtained by adding 10g S2 was added. The volatile solid (VS) content in the anaerobic fermentation system was 88.90% ± 0.01, and the total solid (TS) content was 89.33% ± 0.01. The culture was carried out under magnetic stirring. The methane content in the reaction bottle was detected at a fixed time every two days to obtain the daily methane production and total production data. When the daily methane production data was 0mL, it was considered as the fermentation end point, the culture was terminated, and the microbial community analysis was performed.

[0037] The microbial community analysis method is as follows: 50 mL of the mixed solution was taken at the end of the fermentation, centrifuged at 200 g for 10 min, and the supernatant was taken to obtain the microbial community that was not bound to the substrate; the supernatant was taken to obtain the microbial community that was loosely bound to the substrate after centrifugation at 1000 g for 20 min, and the remaining substrate was the microbial community that was tightly bound to the substrate. The total genomic DNA sample was extracted using a DNA extraction kit, and DNA quality control was performed. The bacterial diversity identification region was the 16S V3-V4 region. After PCR amplification and purification, it was sequenced using the Illumina Hiseq 2000 Caramel Sequencing Platform, and bioinformatics analysis was performed using Krona (version 2.6), QIIME (version 1.9.1), and Muscle (version 3.8.31).

[0038] Comparative Example 1

[0039] The method is the same as in Example 1, except that bamboo powder that has not been pretreated with Pleurotus ostreatus is used. The specific steps are as follows:

[0040] S1. Treatment of bamboo waste

[0041] The bamboo waste was crushed, naturally air-dried, passed through a 50-mesh sieve, and dried at 50° C. for 24 h to obtain bamboo powder (BW).

[0042] S2. Anaerobic fermentation

[0043] An anaerobic fermentation reactor (effective volume of 1.6L, inoculum 1L, sterile saline 0.6L) was established. Under nitrogen insufflation at 37°C, 10g of bamboo powder (BW) obtained by S1 was added. The volatile solid (VS) content in the anaerobic fermentation system was 93.53% ± 0.01, and the total solid (TS) content was 96.20% ± 0.01. The culture was carried out under magnetic stirring. The methane content in the reaction bottle was detected at a fixed time every two days to obtain the daily methane production and total production data. When the daily methane production data was 0mL, it was considered the fermentation end point, the culture was terminated, and the microbial community analysis was performed.

[0044] Figure 2 The following are the scanning electron microscope (SEM) images of bamboo powder (BW) and bamboo powder pretreated with Pleurotus ostreatus (FBW), where Figure a is bamboo powder and Figure b is bamboo powder pretreated with Pleurotus ostreatus. The results show that the lignocellulose structure in bamboo powder is relatively flat and tight, many fiber structures are stacked together in sheets, and the internal structure is relatively complex; the lignocellulose structure in bamboo powder pretreated with Pleurotus ostreatus is severely damaged, and the lignocellulose is wrapped by a large number of hyphae, and the hyphae can extend into the structure.

[0045] Figure 3 The Fourier transform infrared spectra of bamboo powder (BW) and bamboo powder pretreated with Pleurotus ostreatus (FBW) show that after Pleurotus ostreatus pretreatment, the -1 There is no obvious change in the absorption peak at 1604cm -1 and 1508cm -1 It is the characteristic absorption peak of the aromatic ring in lignin. The absorption value decreases significantly, indicating that the lignin is degraded; 900cm -1 The absorption peak at the vibrating point is the β-glycosidic bond in cellulose, and the absorption value decreases slightly, which indicates that the cellulose content does not decrease significantly.

[0046] Figure 4The X-ray spectra of bamboo powder (BW) and bamboo powder pretreated with Pleurotus ostreatus (FBW) are shown in Figure 1. The CC content of the bamboo powder pretreated with Pleurotus ostreatus in the C1s spectrum is greatly reduced, the CO content is basically unchanged, and the C=O / OCO content is slightly increased, indicating that the lignin content of the bamboo powder lignocellulose is greatly reduced, the cellulose and hemicellulose contents are basically unchanged, and the increase in the C=O content may be due to the fungus decomposing lignin to expose more ketone and aldehyde groups. The peak value in the N1s spectrum does not change significantly, indicating that the N content in the bamboo powder is low and the functional groups are almost unchanged before and after pretreatment. The C=O content in the O1s spectrum does not change significantly, and the CO content increases significantly, which may be due to the fungus destroying the structure of lignocellulose, so that cellulose and hemicellulose are fully exposed.

[0047] The methane production of Example 1 and Comparative Example 1 was analyzed, and the results were as follows: Figure 5 As shown. Figure a shows the cumulative methane production curve of bamboo powder (BW) and bamboo powder pretreated with Pleurotus ostreatus (FBW) during 90 days of anaerobic fermentation. It can be seen that the cumulative methane production of bamboo powder fermented with fungi pretreatment is always higher than that of bamboo powder fermentation, and the final production increased by 16.2%. Figure b shows the daily methane production curve of bamboo powder (BW) and bamboo powder pretreated with Pleurotus ostreatus (FBW) during anaerobic fermentation. In the first 10 days, the methane production of bamboo powder pretreated with Pleurotus ostreatus increased greatly, and basically maintained a high methane production state from the 10th day to the 28th day. After the 28th day, the production gradually decreased; while the daily methane production of bamboo powder fermentation was generally unstable, and the daily production decreased significantly after the 55th day.

[0048] The microbial community analysis was performed on Example 1 and Comparative Example 1 after 90 days of anaerobic fermentation. The results are as follows: Figure 6 As shown, a: phylum level, b: genus level. The results show that Firmicutes has the highest richness in the microbial community. Compared with bamboo powder fermentation, the richness of Bacteroides in the microbial community of bamboo powder fermentation after Pleurotus ostreatus pretreatment increased significantly by 49%, Firmicutes slightly decreased, and the richness of other bacterial communities remained basically unchanged. Combined with the methane production data, the reason why bamboo powder pretreated with Pleurotus ostreatus achieves high methane production may be that the fermentation environment of bamboo powder after fungal pretreatment is more suitable for the survival of Bacteroidetes, thereby greatly increasing its richness, thereby optimizing the hydrolysis and acidification links in the anaerobic fermentation process, improving the utilization rate of the substrate, and making methane production more efficient.

[0049] Figure 7The difference in the distribution of microbial communities in different spaces between the bamboo powder fermentation after pretreatment of Pleurotus ostreatus in Example 1 and the bamboo powder fermentation of Comparative Example 1, wherein T represents a tightly bound space, L represents a loosely bound space, and S represents a suspended bound space. It can be seen that the Bacteroides phylum clearly occupies an advantage in the tightly bound and suspended bound space niches, which may be due to the fact that the pretreated substrate is easier for the Bacteroides phylum to bind, and the optimization of the fermentation environment also allows it to be distributed in large quantities in the suspended space. The Halobacterota phylum, which mainly plays a role in methanogenesis, is slightly reduced in the tightly bound space, but increases in the loosely bound space. It is inferred that substrate-degrading microorganisms have an advantage in the tightly bound space distribution, and methanogenic microorganisms have an advantage in the loosely bound space distribution, which can be more beneficial to the effective utilization of substrates and the increase in methane production.

[0050] Example 2

[0051] This embodiment is a method for increasing methane production by biological delignification, comprising the following steps:

[0052] S1. Bamboo waste treatment

[0053] The bamboo waste was crushed, naturally air-dried, and then passed through a 50-mesh sieve to obtain a bamboo powder raw material. The bamboo powder raw material was dried at 55° C. for 22 hours, and then water was added to a moisture content of 55% to obtain a cultivation material. 900 g of the cultivation material was put into a mushroom bag, which was then sterilized to obtain a mushroom bag.

[0054] S2. Inoculation of Pleurotus ostreatus

[0055] 25 mL of the liquid spawn of Pleurotus ostreatus with vigorous growth was inoculated into the fungus bag obtained in S1, and cultured in the dark at 27° C. for 30 days until the mycelium filled the fungus bag and buds appeared. The fungus bag was opened and crushed to obtain bamboo powder (FBW) pretreated with Pleurotus ostreatus.

[0056] S3. Anaerobic fermentation culture

[0057] An anaerobic fermentation reactor (effective volume of 3.2 L, inoculum 2 L, sterile saline 1.2 L) was established. Under nitrogen gas conditions at 37°C, 20 g of bamboo powder (FBW) pretreated with Pleurotus ostreatus obtained by S2 was added, and culture was carried out under stirring conditions to detect methane production.

[0058] Example 3

[0059] This embodiment is a method for increasing methane production by biological delignification, comprising the following steps:

[0060] S1. Bamboo waste treatment

[0061] The bamboo waste was crushed, naturally air-dried, and then passed through a 50-mesh sieve to obtain a bamboo powder raw material. The bamboo powder raw material was dried at 52° C. for 24 hours, and then water was added to a moisture content of 55% to obtain a cultivation material. 900 g of the cultivation material was put into a mushroom bag, which was then sterilized to obtain a mushroom bag.

[0062] S2. Inoculation of Pleurotus ostreatus

[0063] 20 mL of the liquid culture of Pleurotus ostreatus with vigorous growth was inoculated into the fungus bag obtained in S1, and cultured in the dark at 26° C. for 30 days until the mycelium filled the fungus bag and buds appeared. The fungus bag was opened and crushed to obtain bamboo powder (FBW) pretreated with Pleurotus ostreatus.

[0064] S3. Anaerobic fermentation culture

[0065] An anaerobic fermentation reactor (effective volume of 2.4 L, inoculum 1.5 L, sterile saline 0.9 L) was established. Under nitrogen gas conditions at 37°C, 15 g of bamboo powder (FBW) pretreated with Pleurotus ostreatus obtained by S2 was added, and culture was carried out under stirring conditions to detect methane production.

[0066] The method of increasing methane production by biological delignification of the present invention provides ideas and options for further resource utilization of bamboo waste, realizes the harmlessness, reduction and resource utilization of agricultural and forestry by-products, has good economic and ecological benefits, and also provides an important basis for the resource utilization of other lignocellulose raw materials. This method uses Pleurotus ostreatus as the test strain, explores its decomposition effect on lignocellulose at various developmental stages, and provides a basis for controlling the fungal pretreatment time and the decomposition of lignocellulose by other fungi.

[0067] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for increasing methane production by biological delignification, characterized in that: The method comprises the following steps: S1, crushing bamboo waste, air-drying, and sieving to obtain bamboo powder raw materials, drying the bamboo powder raw materials at 50° C.-55° C. for 22-24 hours, and then adding water to a moisture content of 55% to obtain a cultivation material, putting the cultivation material into a fungus bag, and then sterilizing to obtain a fungus bag; S2, inoculating the vigorously growing Pleurotus ostreatus liquid spawn into the fungus bag obtained in S1, culturing in the dark at 25° C. to 27° C. for 30 days until the mycelium fills the fungus bag and buds appear, taking out the bamboo powder in the fungus bag, crushing it, and obtaining the Pleurotus ostreatus pretreated bamboo powder; S3. Establish an anaerobic fermentation reactor, add bamboo powder pretreated with Pleurotus ostreatus obtained in S2 at 37°C under nitrogen gas condition, and detect methane production; when the daily methane production is 0, it is regarded as the fermentation end point, and the culture is terminated, and microbial community analysis is performed.

2. The method according to claim 1, characterized in that: The bamboo waste described in S1 comes from waste from bamboo furniture processing factories.

3. The method according to claim 1, characterized in that The sieving in S1 is sieving through a 50-mesh sieve.

4. The method according to claim 1, characterized in that The mycelium biomass in the Pleurotus ostreatus liquid spawn in S2 is 25-30 g / mL, and the ratio of the Pleurotus ostreatus liquid spawn to the culture medium in the spawn bag is 20-25 mL:900 g.

5. The method according to claim 1, characterized in that: The anaerobic fermentation reactor in S3 is composed of inoculation solution and sterile saline, the volume ratio of the inoculation solution to the sterile saline is 1:0.6; the ratio of the inoculation solution to the bamboo powder pretreated with Pleurotus ostreatus is 1L:10g.

6. The method according to claim 5, characterized in that The inoculation solution is anaerobic sludge.

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