A method for producing bio-based chemicals in an anaerobic fermentation system based on microbial niche regulation

Abstract

The present application belongs to the technical field of anaerobic fermentation, and provides a method for producing bio-based chemicals based on microbial niche regulation of an anaerobic fermentation system. The method comprises the steps of straw crushing, carbon chain extension reaction, short acid production by hydrolysis acidification, biogas production by acid methanation, and discharging. The present application regulates the high-solid-content anaerobic fermentation system through microbial niche to achieve the purpose of directional synthesis of products, enhances the synergistic metabolism mechanism between acid-producing bacteria and methanogenic bacteria, improves the activity of different functional microbial communities, and for the first time realizes the co-production of medium-chain fatty acids, short-chain fatty acids and methane in a high-solid-content anaerobic fermentation system, further widens the energy utilization path of straw, and improves the added value of products.

Description

Technical Field

[0001] This invention relates to the field of anaerobic fermentation technology, and in particular to a method for producing bio-based chemicals based on a microbial niche-regulated anaerobic fermentation system. Background Technology

[0002] It is reported that countless amounts of agricultural waste are generated annually from planting, such as cereal straw (rice straw, wheat straw, corn stalks, etc.), bean straw, potato straw, oilseed straw, and various fruit and vegetable straw. Anaerobic fermentation is one of the technologies for the resource utilization of agricultural waste. High-solids-content anaerobic fermentation devices have the advantage of processing a large amount of raw materials per unit volume. However, because straw contains a large amount of cellulose and hemicellulose, high-solids-content fermentation suffers from slow start-up rates, low material conversion efficiency, and low gas production rates. Moreover, the biogas produced has limited utilization pathways and low added value. Biogas residue and biogas slurry fertilizer have not yet been commercialized. The technical route of anaerobic fermentation for gas and fertilizer co-production is difficult to achieve the effect of producing high-value-added products. Therefore, there is an urgent need to provide a continuous, stable, efficient, and high-value anaerobic fermentation process control method. Summary of the Invention

[0003] To overcome the aforementioned deficiencies in the existing technology, this invention provides a method for producing bio-based chemicals based on a microbial niche-regulated anaerobic fermentation system. This method utilizes the differences in metabolic characteristics of different functional microorganisms during different stages of continuous anaerobic fermentation of high-solids-content straw to design process control methods, thereby altering the interspecies interactions of microorganisms at different fermentation stages, enhancing the directed metabolic rate of microorganisms, and achieving integrated production of bio-based chemicals.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0005] This invention provides a method for producing bio-based chemicals based on a microbial niche-regulated anaerobic fermentation system, comprising the following steps:

[0006] (1) Mix the straw bundles with whey wastewater to obtain a mixture;

[0007] (2) After mixing the mixture with anaerobic granular sludge, add carbon chain elongation functional microorganisms to carry out the first stage of fermentation to obtain the first fermentation product.

[0008] (3) The first fermentation product is mixed with cellulose-decomposing bacteria and fermented in the second stage to obtain the second fermentation product.

[0009] (4) Mix the second fermentation product with cellulase and carry out the third stage of fermentation to obtain the third fermentation product.

[0010] The first fermentation product includes medium-chain fatty acids;

[0011] The second fermentation product includes short-chain fatty acids;

[0012] The third fermentation product includes methane and CO2.

[0013] Preferably, the particle size of the straw tang in step (1) is 1-3 cm, the lactic acid content in the whey wastewater is 3-5 g / L, the ratio of the straw tang to the whey wastewater is 5-10 g: 1 mL, and the solid content of the mixture is 15-25%.

[0014] Preferably, in step (2), the amount of anaerobic granular sludge added is 20-40% of the mass of the mixture, the particle size of the anaerobic granular sludge is 60-70%, the particle diameter of the anaerobic granular sludge is 0.5-4 mm, the moisture content of the anaerobic granular sludge is 85-95%, the inoculation amount of the carbon chain elongation functional microorganisms is 20-35% of the mass of the mixture, and the carbon chain elongation functional microorganisms include Clostridium kluene, Clostridium perfringens and Giant Pleurotus erythrorhizon.

[0015] Preferably, in step (2), the fermentation time of the first stage is 5 to 10 days, the fermentation temperature of the first stage is 35 to 45°C, and the pH value of the first stage fermentation is 5 to 6.

[0016] Preferably, the inoculation amount of the cellulose-decomposing bacteria in step (3) is 10-20% of the mass of the first fermentation product, and the cellulose-decomposing bacteria include Bacteroides var. mongolica and Rumen cellulose-decomposing bacteria.

[0017] Preferably, the fermentation time of the second stage in step (3) is 4 to 6 days, the fermentation temperature of the second stage is 45 to 55°C, and the pH value of the second stage fermentation is 8 to 9.

[0018] Preferably, the amount of cellulase added in step (4) is 1-5% of the mass of the second fermentation product, the enzyme activity of the cellulase is >400U / mg, and the cellulase includes β-1,4-glucan hydrolase, β-glucan cellobiase or β-glucosidase.

[0019] Preferably, the fermentation time of the third stage in step (4) is 8 to 12 days, the temperature of the third stage fermentation is 35 to 45°C, and the pH value of the third stage fermentation is 6 to 8.

[0020] Preferably, the feed rate and discharge rate are the same for the first stage fermentation in step (2), the second stage fermentation in step (3), and the third stage fermentation in step (4).

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] 1. This invention provides a continuous, stable, efficient, and high-value anaerobic fermentation process control method based on microbial niche regulation of a high-solids-content anaerobic fermentation system to simultaneously produce medium-chain fatty acids, short-chain fatty acids, and methane, while simultaneously and efficiently degrading soluble substances, hemicellulose, and cellulose in straw.

[0023] 2. This invention is based on the regulation of microbial ecological niches, regulating the environmental conditions for the survival of strains at each stage, and treating waste in three stages. Different functional microbial communities are added, and cellulase is added when the consumption of usable materials reaches a bottleneck to finally stimulate substrate decomposition, so that straw degradation is more complete and the resource utilization effect of planting waste is achieved.

[0024] 3. This invention achieves the purpose of targeted product synthesis by regulating the microbial ecological niche of a high-solids-content anaerobic fermentation system, enhancing the synergistic metabolic mechanism between acid-producing bacteria and methanogenic bacteria, improving the activity of different functional microbial communities, and for the first time realizing the co-production of medium-chain fatty acids, short-chain fatty acids and methane in a high-solids-content anaerobic fermentation system, further broadening the pathways for straw energy utilization and increasing product added value. Attached Figure Description

[0025] Figure 1 This is a high-solids-content anaerobic fermentation system device according to the method of the present invention;

[0026] Figure 2 This is a reaction flow diagram of the method of the present invention. Detailed Implementation

[0027] This invention provides a method for producing bio-based chemicals based on a microbial niche-regulated anaerobic fermentation system, comprising the following steps:

[0028] (1) Mix the straw bundles with whey wastewater to obtain a mixture;

[0029] (2) After mixing the mixture with anaerobic granular sludge, add carbon chain elongation functional microorganisms to carry out the first stage of fermentation to obtain the first fermentation product.

[0030] (3) The first fermentation product is mixed with cellulose-decomposing bacteria and fermented in the second stage to obtain the second fermentation product.

[0031] (4) Mix the second fermentation product with cellulase and carry out the third stage of fermentation to obtain the third fermentation product.

[0032] The first fermentation product includes medium-chain fatty acids;

[0033] The second fermentation product includes short-chain fatty acids;

[0034] The third fermentation product includes methane and CO2.

[0035] In this invention, the particle size of the straw tang in step (1) is preferably 1-3 cm, more preferably 2 cm; the whey wastewater is preferably milk processing acidified whey wastewater; the lactic acid content in the whey wastewater is preferably 3-5 g / L, more preferably 4 g / L; the ratio of the amount of straw tang to whey wastewater is preferably 5-10 g:1 mL, more preferably 8 g:1 mL; the solid content of the mixture is preferably 15-25%, more preferably 18-22%, and even more preferably 20%.

[0036] In this invention, the amount of anaerobic granular sludge added in step (2) is preferably 20-40% of the mass of the mixed materials, more preferably 25-35% of the mass of the mixed materials, and even more preferably 30% of the mass of the mixed materials; the particle size of the anaerobic granular sludge is preferably 60-70%, more preferably 65%; the particle diameter of the anaerobic granular sludge is preferably 0.5-4 mm, more preferably 2 mm; the moisture content of the anaerobic granular sludge is preferably 85-95%, more preferably 90%; the inoculation amount of the carbon chain elongation functional microorganisms is preferably 20-35% of the mass of the mixed materials, more preferably 28-32% of the mass of the mixed materials, and even more preferably 30% of the mass of the mixed materials; the carbon chain elongation functional microorganisms preferably include Clostridium kluylate, Clostridium perfringens, and Giant Pleurotus erythrorhizon.

[0037] In this invention, the fermentation time of the first stage in step (2) is preferably 5-10 days, more preferably 9 days; the fermentation temperature of the first stage is preferably 35-45℃, more preferably 38-42℃, and even more preferably 40℃; the pH value of the first stage fermentation is preferably 5-6, more preferably 5.2-5.8, and even more preferably 5.5. The purpose of the first stage fermentation is to improve the production of lactic acid and acetic acid by lactic acid bacteria using soluble components of straw, and to enable carbon chain elongation functional bacteria to rapidly synthesize medium-chain fatty acids using lactic acid and acetic acid as substrates. This results in functional complementarity among the bacterial species. Methanogenic bacteria in granular sludge cannot produce methane in a low pH environment and are in a dormant state. Furthermore, the addition of carbon chain elongation functional bacteria strengthens the reverse β-oxidation process in the carbon chain elongation metabolic pathway, promoting the expression of key enzymes such as acetyl-CoA and butyryl-CoA.

[0038] In this invention, the inoculation amount of the cellulose-decomposing bacteria in step (3) is preferably 10-20% of the mass of the first fermentation product, more preferably 13-17% of the mass of the first fermentation product, and even more preferably 15% of the mass of the first fermentation product; the cellulose-decomposing bacteria preferably include Bacteroides var. sarcodactylis and Rumenidae cellulose-decomposing bacteria.

[0039] In this invention, the fermentation time of the second stage in step (3) is preferably 4-6 days, more preferably 5 days; the fermentation temperature of the second stage is preferably 45-55℃, more preferably 50℃; the pH value of the fermentation of the second stage is preferably 8-9, more preferably 8.5. The purpose of the second stage fermentation is to utilize the remaining soluble components, hemicellulose and relatively loosely structured cellulose of the raw materials to produce short-chain fatty acids. During the second stage fermentation, biogas slurry is refluxed daily. The biogas slurry comes from the liquid after solid-liquid separation at the discharge port. The reflux ratio is set by sampling and monitoring the accumulation of volatile acids in the second stage fermentation. When the volatile acid concentration is <2g / L, the reflux ratio in the methanogenesis stage is maintained at 30-40%, more preferably 35%. When the volatile acid concentration is >2g / L, the reflux ratio is increased to 40-50%, and further increased to 45%.

[0040] In this invention, the amount of cellulase added in step (4) is preferably 1-5% of the mass of the second fermentation product, more preferably 2-4%, and even more preferably 3%; the enzyme activity of the cellulase is preferably >400U / mg; the cellulase preferably includes β-1,4-glucan hydrolase, β-glucan cellobiase or β-glucosidase, and more preferably β-1,4-glucan hydrolase.

[0041] In this invention, the fermentation time of the third stage in step (4) is preferably 8-12 days, more preferably 10 days; the fermentation temperature of the third stage is preferably 35-45℃, more preferably 38-42℃, and even more preferably 40℃; the pH value of the fermentation of the third stage is preferably 6-8, more preferably 7. The purpose of the third stage fermentation is to utilize the recalcitrant hemicellulose, cellulose, and a small amount of lignin in the raw materials, and to fully release the methanogenic potential of the raw materials through post-fermentation treatment under the action of cellulase.

[0042] In this invention, the feed and discharge rates are the same for the first stage fermentation in step (2), the second stage fermentation in step (3), and the third stage fermentation in step (4). The first stage fermentation, the second stage fermentation, and the third stage fermentation belong to three stages of continuous fermentation, each stage consisting of three phases located at... Figure 1 Different strains of bacteria are introduced into the feeding ports (1), (2), and (3) of the device. The mixing of materials in each stage is carried out by the stirring device inside the device, and the material as a whole moves evenly from the feeding end to the discharging end.

[0043] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0044] The carbon chain elongation functional microorganisms in the following examples were obtained from a continuous fermentation acid-producing reactor using straw as a substrate. High-throughput sequencing revealed the following composition: *Clostridium kluenes* 25%–35%, *Clostridium* 15%–20%, *Gastrococcus echocerata* 15%–20%, *Vibrio succinate* 10%–15%, *Bifidobacterium* 5%–10%, *Lactobacillus* 5%–10%, *Bacillus* 2%–5%, *Acetobacter* 2%–5%, and others 10%–15%. Cellulose-decomposing bacteria were obtained from high-temperature (55°C) straw compost. High-throughput sequencing revealed the following composition: *Bacteroides vulgaris* 25%–35%, *Ruminococcus rumeniformis* 15%–25%, *Bacillus acidophilus* 10%–20%, *Cibotium* 5%–10%, *Vibrio fibrocarpus* 5%–10%, and others 10%–15%. Cellulase was purchased from Beijing Boao Tuoda Technology Co., Ltd., CAS number 9012-54-8.

[0045] Example 1

[0046] (1) The straw is shredded into winding balls with a particle size of 2cm, and milk processing whey wastewater is added to pre-soak the straw (the ratio of the straw winding balls to the milk processing whey wastewater is 8g:1mL, and the lactic acid content in the milk processing whey wastewater is 4g / L). The straw is then fed into the whey wastewater until the solid content of the straw is 20%. The pH of the uniformly mixed material is 5.5.

[0047] (2) Set the hydraulic retention time of the high solids content fermentation system to 25 days. Add the straw processed in step (1) and anaerobic granular sludge with an inoculation ratio of 30% simultaneously from the feed inlet to the high solids content continuous fermentation device with a working volume of 150L. Add 6kg of material to the feed inlet every day to maintain the normal operation of the high solids content continuous fermentation system.

[0048] (3) The first stage of fermentation lasts for 9 days. The fermentation temperature is maintained at 40℃ by the heat preservation device, and the pH value is controlled at 5.5. 30% of carbon chain elongation functional microorganisms (Clostridium klureum: Clostridium spp.: Giant cocci elutiliforme = 1:1:1) are added to the feeding port (1) above. At the same time, liquid samples containing medium chain fatty acids are collected every day at the sampling port below the device, and the yield of hexanoic acid reaches 10g / L.

[0049] (4) The second stage of fermentation is the 10th to 15th day of the hydraulic retention time. The fermentation temperature is maintained at 50℃ by the heat preservation device, and the fermentation time is 6 days. When the material is moved to the second stage, add cellulose-decomposing bacteria (Bacteroides var. rubella: Ruminosa cellulolytic bacteria = 1:1) to the feeding port (2) above. The inoculation amount is 15%, and the pH value is adjusted to 8.5.

[0050] (5) During the second stage of fermentation, biogas slurry is refluxed daily. The biogas slurry comes from the liquid after solid-liquid separation at the discharge port. The reflux ratio is set by sampling and monitoring the cumulative amount of volatile acids in the second stage. When the concentration of volatile acids is <2 g / L, the reflux ratio in the methanogenesis stage is maintained at 35%. When the concentration of volatile acids is >2 g / L, the reflux ratio is increased to 45%.

[0051] (6) The third stage of fermentation is the 16th to 25th day of the residence time. The fermentation temperature is maintained at 40℃ by the heat preservation device. The fermentation time is 10 days. After the methanogenic production is stable, the pH value is not adjusted in this stage and the pH can be kept neutral. When the material runs to the third stage, cellulase (β-1,4-glucan hydrolase) is added at the upper feeding port (3). The amount of cellulase added is 3% (500U / mg) to decompose the remaining cellulose and produce methane.

[0052] (7) After 25 days of fermentation, the biogas produced reached 450m³. 3 / tTS, the number of functional microorganisms at each stage increased by 120%, and cellulase activity increased by 50%.

[0053] In summary, this invention utilizes the differences in the metabolism of functional microorganisms at different fermentation stages to induce changes in the microbial ecological niche, thereby altering the dominant biochemical reaction pathways to produce the target product. The designed process control conditions are suitable for the growth of microorganisms in all three stages and are conducive to the coordination and mutual promotion among functional microorganisms, significantly improving the yield of single-chain fatty acids, short-chain fatty acids, and methane in high-solids-content anaerobic fermentation.

[0054] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for regulating the production of bio-based chemicals by an anaerobic fermentation system based on microbial niches, characterized in that, Includes the following steps: (1) Mix the straw bundles with whey wastewater to obtain a mixture; (2) After mixing the mixture with anaerobic granular sludge, add carbon chain elongation functional microorganisms to carry out the first stage of fermentation to obtain the first fermentation product; (3) The first fermentation product is mixed with cellulose-decomposing bacteria and fermented in the second stage to obtain the second fermentation product; (4) The second fermentation product is mixed with cellulase and fermented in the third stage to obtain the third fermentation product; The carbon chain elongation functional microorganisms include Clostridium kluene, Clostridium perfringens, and Giant cocci. The cellulolytic bacteria include Bacteroides commonis and Rumen cellulolytic bacteria; The first fermentation product includes medium-chain fatty acids; The second fermentation product includes short-chain fatty acids; The third fermentation product includes methane and CO2.

2. The method for producing bio-based chemicals by regulating anaerobic fermentation system based on microbial niche according to claim 1, characterized in that, The straw tangling pellets in step (1) have a particle size of 1-3 cm, the lactic acid content in the whey wastewater is 3-5 g / L, the ratio of straw tangling pellets to whey wastewater is 5-10 g: 1 mL, and the solid content of the mixture is 15-25%.

3. The method for producing bio-based chemicals based on microbial niche regulation of anaerobic fermentation system according to claim 1 or 2, characterized in that, In step (2), the amount of anaerobic granular sludge added is 20-40% of the mass of the mixture, the particle size of the anaerobic granular sludge is 60-70%, the particle diameter of the anaerobic granular sludge is 0.5-4 mm, the moisture content of the anaerobic granular sludge is 85-95%, and the inoculation amount of the carbon chain elongation functional microorganisms is 20-35% of the mass of the mixture.

4. The method for producing bio-based chemicals by regulating anaerobic fermentation system based on microbial niche according to claim 3, characterized in that, In step (2), the fermentation time of the first stage is 5 to 10 days, the fermentation temperature of the first stage is 35 to 45°C, and the pH value of the first stage fermentation is 5 to 6.

5. The method for regulating the production of bio-based chemicals by an anaerobic fermentation system based on microbial niches according to claim 4, characterized in that, The amount of cellulose-decomposing bacteria in step (3) is 10-20% of the mass of the first fermentation product.

6. The method for producing bio-based chemicals by regulating anaerobic fermentation system based on microbial niche according to claim 5, characterized in that, In step (3), the fermentation time of the second stage is 4 to 6 days, the fermentation temperature of the second stage is 45 to 55°C, and the pH value of the second stage fermentation is 8 to 9.

7. The method for producing bio-based chemicals by regulating anaerobic fermentation system based on microbial niche according to claim 6, characterized in that, The amount of cellulase added in step (4) is 1-5% of the mass of the second fermentation product. The cellulase has an enzyme activity >400 U / mg and includes β-1,4-glucan hydrolase, β-glucan cellobiase or β-glucosidase.

8. The method for producing bio-based chemicals by regulating anaerobic fermentation system based on microbial niche according to claim 7, characterized in that, The fermentation time in the third stage in step (4) is 8 to 12 days, the temperature of the third stage fermentation is 35 to 45°C, and the pH value of the third stage fermentation is 6 to 8.

9. The method for producing bio-based chemicals by regulating anaerobic fermentation system based on microbial niche according to claim 7, characterized in that, The feed and discharge amounts are the same for the first stage fermentation in step (2), the second stage fermentation in step (3), and the third stage fermentation in step (4).

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