Method for preparing carbon source of microbial culture medium based on enzymatic hydrolysis of bamboo powder and application thereof

Abstract

This invention discloses a method for preparing a carbon source for microbial culture medium based on enzymatic hydrolysis of bamboo powder and its application, belonging to the field of high-value utilization technology of biomass resources. Using bamboo powder as raw material, the invention involves mild alkali pretreatment to disrupt the dense structure of lignocellulose, followed by enzymatic hydrolysis using a specialized enzyme system of endoglucanase Cel7B and xylanase XynA expressed heterologously by *E. coli* in an optimized ratio. After solid-liquid separation, an enzymatic hydrolysate rich in reducing sugars is obtained. The hydrolysate is then nutritionally fortified with trace elements such as sodium thiosulfate and sodium silicate to produce a low-cost, high-efficiency liquid carbon source. This invention achieves high-value utilization of bamboo waste. The entire process is mild, environmentally friendly, and simple, easily scaled up industrially, and solves the industry bottleneck of high carbon source cost in industrial microbial culture, demonstrating good application prospects and market value.

Description

Technical Field

[0001] This invention relates to the field of high-value utilization of biomass resources, specifically to a method for preparing a carbon source for microbial culture medium based on enzymatic hydrolysis of bamboo powder and its application. Background Technology

[0002] Currently, the industrial cultivation of microorganisms faces a bottleneck due to the high cost of carbon sources. The carbon sources used in traditional culture media, such as glucose and sucrose, account for 30%-50% of the total cost of the culture medium, which seriously restricts the large-scale development of industries such as microalgae cultivation and microbial fermentation.

[0003] Moso bamboo (Phyllostachys edulis) is a renewable resource with abundant reserves in my country, with an annual output exceeding 30 million tons. It is mainly composed of cellulose (40-50%), hemicellulose (20-25%), and lignin (20-25%), making it a highly promising and inexpensive carbon source. However, the complex and dense structure of lignocellulose—a natural anti-degradation barrier formed by layers of hemicellulose and lignin encapsulating cellulose microfibrils—makes it difficult for microorganisms to utilize it directly. Pretreatment and enzymatic saccharification are necessary to convert it into fermentable sugars, which severely limits its industrial development.

[0004] In existing technologies, pretreatment methods for lignocellulose are mainly divided into three categories, each with significant drawbacks: 1. Physical methods: These include mechanical crushing, ultrasonic treatment, and microwave treatment, which use mechanical force or thermal effects to destroy the crystalline structure of lignocellulose and increase its specific surface area. For example, Chinese patent CN101605930A discloses a method for pretreating lignocellulose raw materials using ball milling. Although this method can effectively reduce the crystallinity of the raw materials, it suffers from drawbacks such as high energy consumption (usually accounting for more than 30% of the total energy consumption of the process), large equipment investment, and small processing capacity, resulting in high industrialization costs.

[0005] 2. Chemical methods: These mainly include acid treatment, alkali treatment, and organic solvent treatment. While dilute acid pretreatment (e.g., CN102586344A) can effectively hydrolyze hemicellulose and destroy fiber structure, it suffers from significant drawbacks such as severe equipment corrosion, generation of large amounts of acidic wastewater, and the easy formation of microbial growth inhibitors like furfural and hydroxymethylfurfural. Studies have shown that during dilute acid pretreatment, high-temperature or high-concentration acid treatment can further convert hexoses and pentoses already generated in the hydrolysate into furfural and other inhibitors, severely affecting subsequent enzymatic hydrolysis and microbial fermentation. Alkali pretreatment (e.g., CN103074382B) can effectively remove lignin, but it suffers from difficulties in alkali recovery, high wastewater treatment costs, and a large environmental impact. Organic solvent pretreatment (e.g., CN104178537A) can obtain high-purity lignin, but the flammability and explosiveness of organic solvents, high recovery costs, and the inhibitory effect of residual solvents on subsequent enzymatic hydrolysis limit its widespread application.

[0006] 3. Biological Method: This method utilizes lignin-degrading enzymes secreted by microorganisms such as white-rot fungi and brown-rot fungi to selectively degrade lignin under mild conditions. For example, patent CN103255186B describes a combined biological-chemical pretreatment method for lignocellulose raw materials. While this approach combines microbial pretreatment with chemical treatment and can partially reduce the amount of chemical reagents used, biological methods generally suffer from problems such as long reaction cycles (weeks to months), low processing efficiency, the need for aseptic operating conditions, and difficulty in achieving continuous production. Furthermore, microorganisms consume some cellulose and hemicellulose while degrading lignin, resulting in sugar loss.

[0007] Enzymatic hydrolysis, as a green and efficient saccharification method, is the preferred direction for the resource utilization of lignocellulose. Commercial cellulase preparations have been widely used in the enzymatic saccharification of lignocellulose. However, enzymatic hydrolysis still faces two major problems: first, commercial enzyme preparations are expensive, accounting for 40-60% of the total saccharification cost; second, general enzyme systems are not highly specific to particular lignocellulose substrates such as bamboo, resulting in low hydrolysis efficiency and unsatisfactory reducing sugar yield.

[0008] In summary, existing technologies lack a pretreatment and enzymatic hydrolysis process that is cost-effective, efficient, and environmentally friendly, specifically tailored to the characteristics of moso bamboo. Therefore, developing a method to optimize the enzymatic hydrolysis products of moso bamboo into a directly usable carbon source for microbial culture media has significant industrial application value and promising prospects for industrialization. Summary of the Invention

[0009] (a) Technical problems to be solved This invention aims to address the shortcomings of existing lignocellulose carbon source preparation technologies by providing a highly efficient and low-cost method for converting bamboo powder into a high-quality carbon source for microbial culture media, along with its applications. By constructing a three-stage linkage process involving pretreatment-oriented deconstruction, synergistic hydrolysis with specific enzyme systems, and enhanced trace element metabolism, the invention achieves the resource-based and high-value utilization of bamboo waste, while simultaneously overcoming the industry bottleneck of high carbon source costs in industrial microbial cultivation.

[0010] (II) Technical Solution To achieve the above objectives, the present invention provides the following technical solution: A method for preparing a carbon source for microbial culture medium based on enzymatic hydrolysis of bamboo powder, the method comprising the following steps: Step S1: Mild Alkali Pretreatment After washing and drying, the raw bamboo is crushed and sieved to collect bamboo powder with a particle size of 300 to 400 mesh. The obtained bamboo powder is placed in an alkaline aqueous solution, preferably a sodium hydroxide (NaOH) solution with a mass concentration of 1% to 4%, and treated at a temperature of 80°C to 120°C for 30 to 90 minutes to obtain pretreated bamboo powder. After treatment, the reaction system is cooled and the pH value of the system is adjusted to 4.8 to 5.2 with an acid regulator to facilitate the subsequent enzymatic hydrolysis reaction.

[0011] This step, through mild alkaline treatment, effectively removes lignin from bamboo and disrupts the dense structure of lignocellulose, while avoiding equipment corrosion and harmful byproduct generation problems caused by traditional strong acid and alkali treatments, and significantly improves the enzyme's accessibility to cellulose substrates.

[0012] Step S2: Enzymatic hydrolysis with a specific compound enzyme system Add buffer solution to the pretreated bamboo powder obtained in step S1 to prepare a suspension with a substrate concentration of 4% to 6% (w / v); add a complex enzyme system composed of endoglucanase Cel7B and xylanase XynA to the suspension, and carry out enzymatic hydrolysis reaction for 36 to 48 hours at 45°C to 55°C, pH 4.8 to 5.2, and 100 to 200 rpm to obtain the enzymatic hydrolysate; The mass ratio of the endoglucanase Cel7B to the xylanase XynA is 1:1.5 to 1:3, and the total enzyme loading is 10 FPU / g to 30 FPU / g dry weight of bamboo powder. Preferably, the mass ratio of the endoglucanase Cel7B to the xylanase XynA is 1:2.2, and the total enzyme loading is 18 FPU / g dry weight of bamboo powder; The endoglucanase Cel7B is derived from *Trichoderma reesei*, and its amino acid sequence is shown in GenBank accession number XP_006970146.1; the xylanase XynA is derived from *Aspergillus niger*, and its amino acid sequence is shown in GenBank accession number XP_001390672.1.

[0013] This step, by screening for the optimal enzyme system combination that has a synergistic effect on the bamboo substrate and precisely controlling the enzyme ratio and loading, achieves efficient and targeted hydrolysis of cellulose and hemicellulose in pretreated bamboo, significantly improving the yield of reducing sugars.

[0014] Step S3: Solid-liquid separation and nutrient fortification The enzymatic hydrolysate obtained in step S2 is subjected to solid-liquid separation, preferably by centrifugation, and the liquid phase rich in reducing sugars is collected; a trace element composition is added to the liquid phase to obtain the carbon source of the microbial culture medium; The trace element composition includes, but is not limited to, at least two of sodium thiosulfate, sodium silicate, ammonium molybdate, cobalt chloride, and EDTA-iron; Preferably, the trace element composition comprises at least sodium thiosulfate and sodium silicate; Furthermore, the nutrient-fortified enzymatic hydrolysate is sterilized at 115°C to 125°C for 10 to 20 minutes.

[0015] This step, by directionally supplementing the enzymatic hydrolysate with trace elements required for the growth of target microorganisms, not only provides the microorganisms with a carbon source that can be directly utilized, but also effectively activates specific metabolic pathways of the microorganisms through the synergistic effect of trace elements and sugars in the enzymatic hydrolysate, thus significantly improving the culture effect.

[0016] Preferably, in the above method, the particle size of the bamboo powder in step S1 is 300-400 mesh, the alkaline solution is a 1% NaOH solution, the treatment temperature is 90℃, and the treatment time is 1 hour.

[0017] Preferably, in the above method, the conditions for the enzymatic hydrolysis reaction in step S2 are: substrate concentration 5% (w / v), reaction temperature 50°C, reaction pH 5.0, and reaction time 36 hours.

[0018] Preferably, in the above method, the trace element composition in step S3 comprises, per liter of enzymatic hydrolysate: 0.02 g sodium thiosulfate, 0.01 g sodium silicate, 0.001 g ammonium molybdate, 0.0002 g cobalt chloride, and 0.005 g EDTA-iron.

[0019] The present invention also provides a carbon source for a microbial culture medium prepared by any of the methods described above.

[0020] The present invention also provides a microbial culture medium comprising the above-mentioned carbon source and basic culture medium components; Preferably, the carbon source is added to the culture medium at a ratio of 15% to 25% (v / v), more preferably 20% (v / v).

[0021] The present invention also provides the use of the above-mentioned carbon source or culture medium in the cultivation of microalgae or microorganisms; Preferably, the microalgae is a cyanobacterial, more preferably Synechocystis sp. PCC 6803; the microorganism is yeast or bacteria.

[0022] (III) Beneficial Effects The purpose of this invention is to provide a method and application for preparing a carbon source for microbial culture medium based on enzymatic hydrolysis of bamboo powder. It has the following beneficial effects: First, this invention uses a mild alkaline pretreatment to fully expose the cellulose microfibrils and xylan side chains of bamboo. Then, a specific complex enzyme system (Cel7B+XynA) is used for enzymatic hydrolysis. XynA first cleaves the xylan surrounding the cellulose, opening a channel for Cel7B. The two enzymes complement each other at the cleavage site. Based on this, trace elements such as cobalt, molybdenum, and iron are selectively added to target the metabolic characteristics of microorganisms (such as cyanobacteria), unexpectedly activating the photosynthetic pigment synthesis pathway. The synergistic effect of these three processes achieves a 42.5% reduction sugar yield and a 35% increase in chlorophyll content, far superior to the simple sum of the individual effects of each step.

[0023] Secondly, the economic benefits are significant: by using bamboo waste to replace traditional glucose and sucrose carbon sources, the carbon source cost is reduced by more than 42.5%, and the cultivation effect is not significantly different from the pure glucose control group (p>0.05), achieving a technological breakthrough of "reducing costs without reducing efficiency" and greatly reducing the raw material cost of industrial microbial cultivation.

[0024] Furthermore, the process is green and environmentally friendly: the pretreatment uses mild alkali treatment, which avoids the corrosion of equipment by concentrated acid and alkali and the salt load of subsequent neutralization; the enzymatic hydrolysis process is highly specific and no harmful byproducts are generated; the entire process produces no wastewater or waste residue pollution, which meets the requirements of green chemical development.

[0025] Finally, it can be mass-produced: the entire process is simple and easy to operate, the process parameters of each link are clear, it is easy to scale up industrially, and it has good prospects for industrial application. Attached Figure Description

[0026] Figure 1 This is an overall process flow diagram of the method for preparing a carbon source for microbial culture medium based on enzymatic hydrolysis of bamboo powder according to the present invention; Figure 2 This is a graph showing the effect of different particle sizes on the sugar yield from enzymatic hydrolysis in a method for preparing a carbon source for a microbial culture medium based on enzymatic hydrolysis of bamboo powder according to the present invention. Figure 3 This is a graph showing the effect of different pretreatment conditions on the concentration of reducing sugar after enzymatic hydrolysis in a method for preparing a carbon source for microbial culture medium based on enzymatic hydrolysis of bamboo powder according to the present invention. Figure 4 This is a diagram showing the hydrolysis effect of different enzyme combinations on bamboo powder in a method for preparing a carbon source for microbial culture medium based on enzymatic hydrolysis of bamboo powder according to the present invention. Figure 5 This is a 3D response surface optimization diagram of enzyme ratio and total enzyme load on reducing sugar concentration in a method for preparing a carbon source for microbial culture medium based on enzymatic hydrolysis of bamboo powder according to the present invention. Figure 6 This is a graph showing the effect of different carbon sources on the growth of cyanobacteria 6803 in a method for preparing microbial culture medium based on enzymatic hydrolysis of bamboo powder according to the present invention. Figure 7 The figure shows the effect of different carbon sources on the chlorophyll and carotenoid content of cyanobacterial algae 6803 in the method for preparing microbial culture medium based on enzymatic hydrolysis of bamboo powder according to the present invention. Figure 8 This figure shows the effect of different carbon sources on the phycocyanin content of cyanobacterial algae 6803 in a method for preparing microbial culture medium based on enzymatic hydrolysis of bamboo powder according to the present invention. Detailed Implementation

[0027] The following will refer to the appendix in the examples of this invention. Figures 1-8 The technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] Example 1: Effect of bamboo powder particle size on enzymatic hydrolysis effect This embodiment aims to investigate the effect of bamboo powder particle size on enzymatic hydrolysis in order to determine the optimal particle size range.

[0029] Bamboo powder of 100 mesh, 200 mesh, 300 mesh, 400 mesh and 800 mesh was pretreated and then subjected to enzymatic hydrolysis. The reducing sugar concentration was determined by DNS method.

[0030] Experimental results are as follows Figure 2 As shown, when the particle size is 300-400 mesh, the concentration of enzymatically hydrolyzed reducing sugar reaches its peak (3.6 mg / mL), which is significantly better than that of 100 mesh (2.5 mg / mL), 200 mesh (2.7 mg / mL), and 800 mesh (3.5 mg / mL). Therefore, 300-400 mesh is determined to be the optimal particle size range for moso bamboo powder.

[0031] Example 2: Effect of different pretreatment methods for bamboo powder on enzymatic hydrolysis efficiency This embodiment aims to investigate the effects of different pretreatment methods on the enzymatic hydrolysis of bamboo powder, in order to screen the optimal pretreatment conditions.

[0032] Weigh 5g of 300-400 mesh bamboo powder and add it to 100mL of pH 4.8, 0.05M citrate buffer solution. Divide the mixture into a high-temperature high-pressure pretreatment group and an acid-base chemical pretreatment group.

[0033] Take the culm of moso bamboo as raw material, wash and dry it, then crush and sieve it to collect 300-400 mesh moso bamboo powder for later use.

[0034] Accurately weigh 5.00 g of dried bamboo powder and place it in a 250 mL pressure-resistant conical flask or a stoppered Erlenmeyer flask. Add different treatment solutions according to the group: Groups A, B, and C: Add 100 mL of 0.05 M citrate buffer solution at pH 4.8; Groups D and E: Add 100 mL of a solution containing 1% (v / v) glacial acetic acid; Groups F and G: Add 100 mL of a solution containing 1% (w / v) NaOH.

[0035] Control group: No pretreatment was performed.

[0036] After sealing the conical flasks in groups A, B, and C, place them in a vertical pressure steam sterilizer and process them according to the set conditions: Group A: Treated at 115℃ for 45 minutes; Group B: Treat at 115℃ for 15 minutes; Group C: Treat at 90℃ for 15 minutes.

[0037] After processing, cool rapidly to room temperature.

[0038] The conical flasks in groups D, E, F, and G were placed in a 90℃ constant temperature water bath shaker and shaken at 150 rpm. Group D (dilute acid): Treatment for 15 minutes; Group E (dilute acid): Treatment for 1 hour; Group F (dilute alkali): Treatment for 1 hour; Group G (dilute alkali): Treat for 15 minutes.

[0039] After treatment, the mixture was cooled to room temperature and the pH of the mixture was precisely adjusted to 5.0 ± 0.1 using 1 M NaOH solution (groups D and E) or 1 M HCl solution (groups F and G).

[0040] Cellulase and hemicellulase were added to each group for enzymatic hydrolysis, and the amount of reducing sugar was determined using the DNS method. The experimental results are as follows: Figure 3 As shown, group F (1% NaOH, 90℃, 1 hour) had the highest reducing sugar concentration, reaching 6.41 mg / mL, which was significantly better than other pretreated groups. The reducing sugar concentration in the untreated control group was 2.54 mg / mL.

[0041] Experimental results show that dilute alkali pretreatment (1% NaOH, 90℃, 1 hour) is the optimal pretreatment condition.

[0042] Example 3: Preparation of Special Crude Enzyme Solution This embodiment describes the preparation method of the endoglucanase Cel7B and xylanase XynA used in this invention.

[0043] Based on the sequences published in GenBank, the Cel7B (XP_006970146.1) and XynA (XP_001390672.1) genes were synthesized, and Nde I and Xho I restriction sites were introduced at both ends of the genes.

[0044] The synthesized gene fragment was digested with enzymes and then cloned into the prokaryotic expression vector pET-28a(+) to construct a recombinant expression plasmid.

[0045] The recombinant expression plasmid was transformed into E. coli BL21(DE3) competent cells using the heat shock method, plated on LB solid medium containing kanamycin, and cultured at 37°C for 12 h to screen for positive clones.

[0046] Positive clones were inoculated into LB liquid medium and cultured at 37°C and 220 rpm until the OD600 reached 0.6. Then, IPTG was added to a final concentration of 0.2 mM, and expression was induced at 16°C and 180 rpm for 16 h.

[0047] After induction, the bacterial cells were collected by centrifugation at 8000 rpm for 10 minutes and resuspended in PBS buffer. The cells were then disrupted by ultrasonic disruption (200W, 2 seconds on, 3 seconds off, total duration 15 minutes). After disruption, the cells were centrifuged at 12000 rpm for 15 minutes, and the supernatant was collected as crude enzyme solution, which was stored at 4℃ for later use.

[0048] Example 4: Enzyme system combination screening and ratio optimization This embodiment aims to screen the optimal enzyme system combination for bamboo powder and optimize the enzyme ratio and total enzyme loading using response surface methodology.

[0049] By comparing the hydrolysis effects of four commercial cellulases and three hemicellulases on pretreated bamboo powder, the single enzyme combination with the best hydrolysis efficiency was screened.

[0050] Cellulase 1 is a cellulase (carrier is starch, derived from Aspergillus niger, >10U / mg); Cellulase 2 is a cellulase (carrier is starch, derived from Trichoderm aviride, >20U / mg); Cellulase 3 is an endoglucanase Cel7B (derived from Trichoderma reesei); cellulase 4 is (cellulase, 50 U / mg).

[0051] Hemiglycanase 1 is xylanase (derived from Pichia pastoris, industrial grade, >100 U / mg). Hemiglycanase 2 is xylanase XynA (derived from Aspergillus niger); Hemiglycanase 3 is xylanase (derived from Trichoderm aviride).

[0052] Experimental results are as follows Figure 4 As shown, the combination of endoglucanase Cel7B from Trichoderma reesei and xylanase XynA from Aspergillus niger showed the best hydrolysis effect on moso bamboo powder, significantly higher than other combinations.

[0053] The optimal enzymatic hydrolysis parameters were determined through response surface methodology, and the results are as follows: Figure 5 As shown: When the mass ratio of Cel7B:XynA is 1:2.2 and the total enzyme loading is 18 FPU / g substrate, the reducing sugar concentration is the highest, at 4.25 mg / mL.

[0054] Example 5: Verification of the enzymatic hydrolysis process of bamboo powder This embodiment demonstrates the feasibility and stability of enzymatic hydrolysis of bamboo powder using optimized process parameters.

[0055] Bamboo powder was treated under the optimal pretreatment conditions determined in Example 1 (90°C, 1 h, 1% NaOH), and a 5% (w / v) suspension was prepared using 0.05 M, pH 5.0 citrate-sodium citrate buffer. The crude enzyme solution prepared in Example 3 was added, controlling the total enzyme load to 18 FPU / g substrate, with a Cel7B:XynA ratio of 1:2.2. The reaction system was subjected to enzymatic hydrolysis at 50°C and 150 rpm for a total reaction time of 48 hours. Samples were taken every 6 hours, and the reducing sugar concentration was determined using the DNS method.

[0056] Results: The reducing sugar concentration was 0.6 g / L after 6 h of enzymatic hydrolysis, 1.1 g / L after 12 h, 2.4 g / L after 24 h, and 3.9 g / L after 36 h. The reducing sugar concentration did not increase significantly after 36 h, so 36 h was determined to be the optimal reaction time.

[0057] Example 6: Preparation of carbon source for microbial culture medium This embodiment describes a specific method for converting enzymatic hydrolysate into a carbon source for directly usable microbial culture medium.

[0058] The enzymatic hydrolysate obtained in Example 5 was centrifuged at 4000 rpm for 10 minutes, and the supernatant was collected for later use.

[0059] Add compound nutrients (per liter of enzymatic hydrolysate) to the supernatant. The specific formula is as follows: sodium nitrate 0.75g, potassium dihydrogen phosphate 0.04g, sodium thiosulfate 0.02g, sodium silicate 0.01g, ammonium molybdate 0.001g, cobalt chloride 0.0002g, EDTA-iron 0.005g.

[0060] The nutrient-fortified enzymatic hydrolysate was mixed evenly and sterilized at 121°C for 15 minutes to obtain the final usable microbial culture medium carbon source solution.

[0061] Example 7: Effect of carbon source solution addition ratio on cyanobacteria growth The carbon source solution prepared in Example 6 was added to carbon-deficient BG11 basal medium at ratios of 10%, 20%, 30%, 40%, and 50% (v / v) to cultivate the cyanobacterium Synechocystis sp. PCC 6803. Culture conditions: temperature 28℃, light intensity 80 μmol / m² / s, light-dark cycle 12h:12h, aeration rate 0.5 vvm. The specific growth rate of the cyanobacterium was measured at different addition ratios.

[0062] Results: When the carbon source solution was added at a ratio of 20% (v / v), the specific growth rate of cyanobacteria reached its maximum value, which was the optimal addition ratio for the carbon source solution.

[0063] Example 8: Verification of the effect of carbon source solution in replacing glucose for culturing cyanobacteria This embodiment verifies the feasibility of using the carbon source solution prepared in this invention to replace the traditional glucose carbon source through comparative experiments.

[0064] The carbon source solution prepared in Example 6 was added to carbon-deficient BG11 basal medium at a ratio of 20% (v / v) to culture the cyanobacterium Synechocystis sp. PCC 6803; at the same time, a positive control group with an equal amount of glucose as the carbon source was set up to carry out parallel culture verification.

[0065] Culture conditions: temperature 28℃, light intensity 80μmol / m² / s, light-dark cycle 12h:12h, aeration rate 0.5vvm.

[0066] The results of the growth index tests are as follows: like Figure 6 As shown, in the experimental group using the carbon source of the present invention, the absorbance value OD730 of cyanobacteria reached 3.2±0.15 on the 4th day, which was not significantly different from the glucose control group (3.3±0.12) (p>0.05), and the cost of the carbon source was reduced by 42.5% compared with glucose.

[0067] like Figure 7As shown, the chlorophyll and carotenoid contents of cyanobacteria in the carbon source solution group and the glucose control group of the present invention were significantly higher than those in the ordinary BG11 group, and there was no significant difference between the carbon source solution group and the glucose control group of the present invention (p>0.05).

[0068] like Figure 8 As shown, the phycocyanin content of the carbon source solution group of the present invention can reach 0.68 mg / mL, which is not significantly different from that of the glucose control group (0.71±0.03 mg / mL) (p>0.05), but is significantly higher than that of the ordinary BG11 group.

[0069] Cost accounting: Using the carbon source of this invention reduces carbon source cost by 42.5% compared to the glucose control group.

[0070] The above results show that the carbon source solution prepared by this invention can completely replace traditional glucose for efficient cyanobacteria cultivation, while significantly reducing raw material costs.

[0071] in conclusion: This invention comprehensively validated the pretreatment of bamboo powder, enzyme system screening, enzymatic hydrolysis process, and carbon source nutrition optimization through systematic single-factor experiments and process optimization experiments, and drew the following core conclusions: 1. Pretreatment is key to efficient enzymatic hydrolysis of moso bamboo powder: Compared with the untreated group, the yield of reducing sugar in all pretreated groups was significantly increased, proving that pretreatment can effectively destroy the dense structure of moso bamboo lignocellulose and improve the accessibility of enzymes and substrates; among them, dilute alkali pretreatment (90℃ for 1h with 1% NaOH) had the highest saccharification efficiency and the reducing sugar concentration reached 6.41mg / mL, which is the optimal pretreatment condition.

[0072] 2. The dedicated enzyme system is highly adaptable to the hydrolysis of moso bamboo powder: The Cel7B+XynA combination screened from 4 cellulases and 3 hemiglycolases has a significantly higher hydrolysis efficiency for moso bamboo powder than other enzyme systems; the ratio (1:2.2) and total enzyme loading (18 FPU / g) optimized by response surface methodology can maximize the yield of reducing sugar (42.5±1.3%).

[0073] 3. Optimal and efficient enzymatic hydrolysis process parameters: 5% (w / v) substrate concentration, 50℃ reaction temperature, 150rpm shaker speed, and 36 hours reaction time are the optimal process conditions for enzymatic hydrolysis of bamboo powder. At this time, the reducing sugar concentration reaches 3.9mg / mL, and there is no subsequent substrate waste, resulting in the highest reaction efficiency.

[0074] 4. Optimized carbon source can completely replace traditional glucose: The carbon source solution prepared in this invention, after being fortified with trace element nutrients, was added to BG11 medium at a ratio of 20% (v / v). The biomass, chlorophyll, phycocyanin and other indicators of the cultured cyanobacteria were not significantly different from those of the glucose control group, and the carbon source cost was reduced by 42.5%, which can effectively replace traditional sugar carbon sources.

[0075] 5. The process combines environmental friendliness and industrial operability: This invention uses a citric acid buffer system or low-concentration acid-base reagents for pretreatment, avoiding the corrosion of equipment by concentrated acids and alkalis and the salt load of subsequent neutralization. The process is mild and easy to operate; no harmful by-products are generated throughout the process, which meets the requirements of green industrialization development.

[0076] In summary, the technical solution provided by this invention has achieved unexpected technical effects. Those skilled in the art can make changes and modifications to the above embodiments within the scope and spirit defined in the claims, and all such changes and modifications fall within the protection scope of this invention.

[0077] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a carbon source for microbial culture medium based on enzymatic hydrolysis of bamboo powder, characterized in that, Includes the following steps: S1 Mild Alkaline Pretreatment: Place bamboo powder in an alkaline solution and treat it at 80℃~120℃ for 30min~90min to obtain pretreated bamboo powder; S2 specific complex enzyme system enzymatic hydrolysis: The pretreated bamboo powder was enzymatically hydrolyzed using a complex enzyme system composed of endoglucanase Cel7B and xylanase XynA to obtain the enzymatic hydrolysate. S3 Solid-liquid separation and nutrient fortification: Solid-liquid separation is performed on the enzymatic hydrolysate, the liquid phase components are collected, and a trace element composition is added to the liquid phase to obtain the carbon source of the microbial culture medium; The mass ratio of the endoglucanase Cel7B to the xylanase XynA is 1:1.5 to 1:3, and the total enzyme loading is 10 FPU / g to 30 FPU / g dry weight of bamboo powder.

2. The method for preparing a carbon source for microbial culture medium based on enzymatic hydrolysis of bamboo powder according to claim 1, characterized in that, The alkaline solution mentioned in step S1 is a NaOH solution with a mass concentration of 1% to 4%.

3. The method for preparing a carbon source for microbial culture medium based on enzymatic hydrolysis of bamboo powder according to claim 1, characterized in that, The particle size of the bamboo powder mentioned in step S1 is 300 mesh to 400 mesh.

4. The method for preparing a carbon source for microbial culture medium based on enzymatic hydrolysis of bamboo powder according to claim 1, characterized in that, The conditions for the enzymatic hydrolysis reaction described in step S2 are: substrate concentration 4%–6% (w / v), reaction temperature 45℃–55℃, reaction pH 4.8–5.2, and reaction time 36h–48h.

5. The method for preparing a carbon source for microbial culture medium based on enzymatic hydrolysis of bamboo powder according to claim 1, characterized in that, The trace element composition in step S3 comprises at least two of sodium thiosulfate, sodium silicate, ammonium molybdate, cobalt chloride, and EDTA-iron.

6. The method for preparing a carbon source for microbial culture medium based on enzymatic hydrolysis of bamboo powder according to claim 1, characterized in that, In step S3, the nutrient-fortified enzymatic hydrolysate needs to be sterilized. The sterilization process is carried out at 115℃ to 125℃ for 10 min to 20 min.

7. A carbon source for a microbial culture medium, characterized in that, It is prepared by the method described in any one of claims 1 to 6.

8. A microbial culture medium, characterized in that, It comprises the carbon source as described in claim 7 and the basic culture medium components, wherein the carbon source is added to the culture medium at a ratio of 15% to 25% (v / v).

9. The use of a carbon source as described in claim 7 or a culture medium as described in claim 8 in the cultivation of microalgae or microorganisms.

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