Preparation method of reed straw lixivium and application of reed straw lixivium in microalgae culture

Abstract

The invention relates to a preparation method of a reed straw leachate. The preparation method comprises the following steps: raw material pretreatment: crushing reed straws; soaking condition optimization: mixing and soaking the pretreated reed straws with water, and analyzing the influence of each factor on the concentration of the reducing sugar in the reed straw leachate under the treatment conditions of different soaking durations, straw particle sizes and solid-liquid ratios by taking the content of the reducing sugar as an evaluation index; after the single factor data is processed, each factor obtains a plurality of experimental levels, an L9 (33) orthogonal experimental method is adopted, an orthogonal experiment is carried out according to the selected experimental factors and experimental levels, and optimized soaking conditions are obtained; the pretreated reed straw and water are mixed and soaked according to optimized soaking conditions, and the leachate is obtained after filtration. The microalgae culture cost is reduced, agricultural waste resource utilization is achieved, and the method has application prospects and economic benefits in the fields of agricultural waste treatment and microalgae industrial culture.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of agricultural waste resource utilization and microalgae cultivation, and particularly relates to a preparation method of reed straw leaching liquor and application thereof in microalgae cultivation. BACKGROUND

[0002] As a photosynthetic autotrophic organism, the large-scale cultivation of microalgae relies on the supplementation of exogenous carbon sources to improve biomass yield. In traditional cultivation, industrial carbon sources such as glucose are often used, but the cost is high, which restricts the application of microalgae in high-value-added fields such as biofuels, feed and medicine. In recent years, the use of agricultural waste (such as straw) hydrolysate as a substitute carbon source has become a research hotspot. Chinese patents with publication numbers CN106148195A, CN107815418A and CN106967609A use alkaline hydrolysis pretreatment and cellulase, hemicellulase enzymatic hydrolysis or cellulose-decomposing bacteria (Saprospira) co-cultivation to prepare straw-derived organic carbon sources for microalgae cultivation. However, the technical processes adopted in the above-mentioned patents include alkaline hydrolysis, enzyme treatment, biological treatment and other processes, which have problems such as complex process, high risk of chemical pollution and high cost. Therefore, developing a low-cost and environmentally friendly straw-derived carbon source acquisition technology is a key problem that needs to be solved in the microalgae industry.

[0003] As a common agricultural waste, reed straw contains rich cellulose, hemicellulose and lignin, and has great resource utilization potential. At present, it is mainly used in papermaking, soil improvement and biochar production, but the overall utilization rate is less than 30%. Burning not only causes CO2 emission, but also leads to resource waste; chemical hydrolysis or enzymatic hydrolysis process has high energy consumption and heavy pollution. Therefore, developing a low-cost and environmentally friendly reed straw resource utilization technology is a key problem that needs to be solved in the agricultural waste resource utilization industry

[0004] Therefore, providing a rapid, pollution-free and low-cost reed straw-derived carbon source acquisition technology and being able to replace glucose for microalgae cultivation will effectively solve the double problems faced by the current microalgae industry and agricultural waste resource utilization industry. SUMMARY

[0005] The purpose of the present application is to provide a preparation method of reed straw leaching liquor and application thereof in microalgae cultivation, which can prepare reed straw leaching liquor at low cost and high efficiency, and apply it to microalgae cultivation to reduce the cultivation cost and realize the resource utilization of agricultural waste;

[0006] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is:

[0007] A preparation method of reed straw leaching liquor and application thereof in microalgae cultivation, comprising the following steps:

[0008] (1) Raw material pretreatment: crush the reed stalks;

[0009] (2) Optimization of soaking conditions: Pretreated reed straw was mixed with deionized water for soaking. The effect of each factor on the reducing sugar concentration in the reed straw leachate was analyzed under different soaking times, straw particle sizes and material-to-liquid ratios. After processing the above single-factor data, multiple experimental levels were obtained for each factor, and L9(3) was used. 3 Orthogonal experimental design involves conducting orthogonal experiments based on selected experimental factors and experimental levels to obtain optimized soaking conditions.

[0010] (3) Preparation of leachate: The pretreated reed straw was mixed with deionized water and soaked according to the optimized soaking conditions in step (2), and the leachate was obtained after filtration.

[0011] Preferably, in step (1), the raw material pretreatment further includes: washing and air-drying the reed stalks, then crushing them, and then drying them in an oven.

[0012] Preferably, in step (2), the analysis of the effect of different soaking times on the reducing sugar concentration in the reed straw leachate includes: soaking the reed straw at a straw particle size of 30 mesh and a material-to-liquid ratio of 1 g: 20 mL for soaking times of 1 h, 6 h, 12 h, 24 h, 36 h, 48 h and 60 h respectively, filtering, detecting the reducing sugar concentration in the leachate, and taking the soaking time of the leachate with the highest reducing sugar concentration as the first soaking time.

[0013] Preferably, the analysis of the effect of different straw particle sizes on the reducing sugar concentration in the reed straw leachate includes: soaking reed straw at a first soaking time and a material-to-liquid ratio of 1 g: 20 mL at straw particle sizes of 10 mesh, 20 mesh, 30 mesh, 40 mesh, 50 mesh, 60 mesh, and 70 mesh, respectively; after filtration, detecting the reducing sugar concentration in the leachate; and taking the straw particle size of the leachate with the highest reducing sugar concentration as the first straw particle size.

[0014] Preferably, the analysis of the effect of different material-liquid ratios on the reducing sugar concentration in the reed straw leachate includes: soaking the reed straw at different ratios of 1 g:30 mL, 1 g:25 mL, 1 g:20 mL, 1 g:15 mL, and 1 g:10 mL according to the first soaking time and the first straw particle size, respectively; filtering; detecting the reducing sugar concentration in the leachate; and taking the straw particle size of the leachate with the highest reducing sugar concentration as the first material-liquid ratio.

[0015] Preferably, in step (2), three experimental levels are obtained for each factor: soaking time: 6 h, 12 h and 24 h, straw particle size: 50 mesh, 60 mesh and 70 mesh, and material-liquid ratio: 1 g: 20 mL, 1 g: 15 mL and 1 g: 10 mL, and an orthogonal experiment is conducted.

[0016] Preferably, in step (3), the filtration is performed by first using gauze for coarse filtration, and then using a 0.22μm aqueous microporous membrane for filtration.

[0017] This invention also provides the application of the reed straw leachate prepared by the above-described preparation method in microalgae cultivation.

[0018] The present invention also provides a method for culturing microalgae, comprising the following steps: mixing the reed straw extract prepared by the preparation method according to any one of claims 1-7 with BG11 culture medium at a volume ratio of 1:1, and inoculating microalgae for cultivation.

[0019] Preferably, the microalgae is Chlorella vulgaris, the initial inoculation concentration is 200 mg / L, the shaking speed is 120 rpm, and the culture conditions are: temperature 25±1℃, light intensity 3000 lux, light-dark ratio 12h:12h, and culture time is 4 days.

[0020] Compared with chemical and biological methods for treating straw, the present invention uses a physical treatment method of simple soaking in deionized water, which is short, low-cost and pollution-free.

[0021] The leachate prepared by the method of this invention can be used to cultivate microalgae without inhibition. The operation process is simple, stable, and highly reproducible, and is independent of the algal species being cultivated.

[0022] The leachate prepared by the method of this invention, when mixed with BG11 medium to culture *Chlorella vulgaris*, exhibits a biomass concentration significantly higher than that of traditional cultures using glucose as a carbon source. This invention reduces the cost of microalgae cultivation, realizes the resource utilization of agricultural waste, and has promising application prospects and economic benefits in the fields of agricultural waste treatment and industrialized microalgae cultivation.

[0023] Since the reducing sugar content in reed stalks varies in different regions and environments, this invention can accurately obtain the optimal soaking conditions for the current reed stalks through orthogonal experimental methods, so that the reed stalk leachate has more reducing sugar, thus facilitating the cultivation of Chlorella vulgaris. Attached Figure Description

[0024] Figure 1 The effects of soaking time (a), straw particle size (b), and material-to-liquid ratio (c) on the concentration of reducing sugars in reed straw leachate;

[0025] Figure 2The growth of *Chlorella vulgaris* in reed straw extract, reed straw extract + BG11, and glucose + BG11 is shown. Detailed Implementation

[0026] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0027] Example 1:

[0028] This invention discloses a method for preparing reed straw leachate, comprising the following steps:

[0029] Step 1: Collect and wash the reed stalks to remove surface dust and other substances. Then, use a shredder to shred the air-dried reed stalks, passing them through sieves of the following sizes in sequence: 10 mesh (0.900 mm-2.000 mm), 20 mesh (0.600 mm-0.900 mm), 30 mesh (0.450 mm-0.600 mm), 40 mesh (0.355 mm-0.450 mm), 50 mesh (0.280 mm-0.355 mm), 60 mesh (0.224 mm-0.280 mm), and 70 mesh (0.180 mm-0.224 mm). Finally, place the collected reed stalk particles in a 90℃ oven to dry for 3 days for later use.

[0030] Step two: With the straw particle size fixed at 30 mesh and the material-to-liquid ratio at 1:20 (g:mL), the effects of soaking times of 1 h, 6 h, 12 h, 24 h, 36 h, 48 h, and 60 h on the concentration of reducing sugars in the leachate were investigated. Figure 1 As shown, when the soaking time was 12 hours, the concentration of reducing sugar in the reed straw leachate reached its maximum value of 854.17 mg / L.

[0031] Step 3: With a fixed soaking time of 12 hours and a material-to-liquid ratio of 1:20 (g:mL), the effects of seven straw particle sizes (10 mesh, 20 mesh, 30 mesh, 40 mesh, 50 mesh, 60 mesh, and 70 mesh) on the concentration of reducing sugars in the leachate were investigated. Figure 1 As shown, when the particle size of reed straw is 60 mesh, the concentration of reducing sugar in the reed straw leachate reaches its maximum value of 1068.72 mg / L;

[0032] Step four: With a fixed soaking time of 12 hours and a straw particle size of 60 mesh, the effect of five material-to-liquid ratios (1:30, 1:25, 1:20, 1:15, and 1:10) on the concentration of reducing sugars in the leachate was investigated; Figure 1 As shown, when the material-to-liquid ratio is 1:10, the reducing sugar concentration in the reed straw leachate is 1545.76 mg / L.

[0033] Step 5: Based on the results of the single-factor experiments, L9 (3)3 An orthogonal experimental design was used to investigate the three factors affecting the reducing sugar concentration in the leachate: soaking time, straw particle size, and material-to-liquid ratio, denoted as A, B, and C, respectively. Each factor had three levels. An orthogonal experiment was conducted based on the selected experimental factors and levels. The orthogonal experimental design and the levels of influencing factors in the deionized water soaking of reed straw are shown in Tables 1 and 2. As shown in Table 3, according to the orthogonal experimental results, the influence of the factors on the reducing sugar concentration in the reed straw leachate, from largest to smallest, was: material-to-liquid ratio > soaking time > straw particle size. The optimal level combination was a soaking time of 24 h, a straw particle size of 60 mesh, and a material-to-liquid ratio of 1:10 (g:mL).

[0034] Table 1. Factor Levels in Orthogonal Experiments

[0035]

[0036] Table 2 L9 (3) 3 Orthogonal experimental design table

[0037] Experiment No. Soaking time (A) Straw particle (B) Material liquid ratio (C) 1 1 1 1 2 1 2 2 3 1 3 3 4 2 1 2 5 2 2 3 6 2 3 1 7 3 1 3 8 3 2 1 9 3 3 2

[0038] Table 3. Results of the orthogonal experiment

[0039]

[0040] Step 6: Based on the optimal combination obtained from the orthogonal experiment, six verification experiments were conducted to determine the reducing sugar concentration in the leachate, which were 1063.85 mg / L, 1067.20 mg / L, 1064.97 mg / L, 1070.56 mg / L, 1068.32 mg / L, and 1069.44 mg / L, with an average reducing sugar concentration of 1067.39 mg / L.

[0041] Step 7: The *Chlorella vulgaris* was cultured in the reed straw extract prepared under the optimized conditions described above. The initial inoculum concentration of *Chlorella vulgaris* was 200 mg / L, the culture temperature was 25±1℃, the light intensity was 3000 lux, and the light-dark cycle was 12 h.

[0042] 12 hours, shaker speed 120 rpm, culture for 4 days; Figure 2 As shown, after 4 days of cultivation, the biomass concentration of Chlorella reached 807 mg / L.

[0043] Example 2:

[0044] The only difference between Example 2 and Example 1 is that the leachate was mixed with BG11 culture medium at a volume ratio of 1:1; all other aspects are the same as in Example 1. Figure 2 As shown, after 4 days of cultivation, the biomass concentration of Chlorella reached 1027 mg / L.

[0045] Comparative example:

[0046] The only difference between the comparative example and Example 2 is that glucose was used instead of the extract in the BG11 culture mixture, based on the reducing sugar concentration; all other aspects are the same as in Example 2. Figure 2 As shown, after 4 days of cultivation, the biomass concentration of Chlorella reached 853 mg / L.

[0047] Based on the above specific implementation, the main conclusions are as follows:

[0048] (1) The physical treatment method of simply soaking in deionized water can quickly prepare carbon-rich reed straw leachate;

[0049] (2) The leachate prepared by this method can be used to cultivate microalgae without inhibition.

[0050] (3) Supplementing with BG11 can enhance the utilization of nutrients in the leachate by microalgae;

[0051] (4) Reed straw extract can be used as an alternative carbon source for glucose in microalgae culture.

Claims

1. A method for preparing reed straw leachate, characterized in that, Includes the following steps: (1) Raw material pretreatment: crush the reed stalks; (2) Optimization of soaking conditions: The pretreated reed straw was mixed with water and soaked. The effect of each factor on the reducing sugar concentration in the reed straw leachate was analyzed under different soaking times, straw particle sizes and material-to-liquid ratios. After processing the above single-factor data, multiple experimental levels were obtained for each factor, and L9(3) was used. 3 Orthogonal experimental design involves conducting orthogonal experiments based on selected experimental factors and levels to obtain optimized soaking conditions. (3) Preparation of leachate: The pretreated reed straw was mixed with water and soaked according to the optimized soaking conditions in step (2), and the leachate was obtained after filtration.

2. The preparation method according to claim 1, characterized in that, In step (1), the raw material pretreatment also includes: washing and air-drying the reed stalks, crushing them, and then drying them in an oven.

3. The preparation method according to claim 1, characterized in that, In step (2), the effect of different soaking times on the reducing sugar concentration in the leachate of reed straw is analyzed as follows: the reed straw is soaked at a straw particle size of 30 mesh and a material-to-liquid ratio of 1 g: 20 mL for soaking times of 1 h, 6 h, 12 h, 24 h, 36 h, 48 h and 60 h respectively. After filtration, the reducing sugar concentration in the leachate is measured, and the soaking time of the leachate with the highest reducing sugar concentration is taken as the first soaking time.

4. The preparation method according to claim 3, characterized in that, The effect of different straw particle sizes on the reducing sugar concentration in reed straw leachate was analyzed as follows: reed straw was soaked at a material-to-liquid ratio of 1 g: 20 mL for a first soaking time, and at straw particle sizes of 10 mesh, 20 mesh, 30 mesh, 40 mesh, 50 mesh, 60 mesh and 70 mesh respectively. After filtration, the reducing sugar concentration in the leachate was measured, and the straw particle size of the leachate with the highest reducing sugar concentration was taken as the first straw particle size.

5. The preparation method according to claim 4, characterized in that, The effect of different material-to-liquid ratios on the reducing sugar concentration in reed straw leachate was analyzed as follows: reed straw was soaked at different ratios of straw particle size, namely 1 g: 30 mL, 1 g: 25 mL, 1 g: 20 mL, 1 g: 15 mL, and 1 g: 10 mL, according to the first soaking time and the first straw particle size. After filtration, the reducing sugar concentration in the leachate was measured, and the straw particle size of the leachate with the highest reducing sugar concentration was taken as the first material-to-liquid ratio.

6. The preparation method according to claim 1, characterized in that, In step (2), three experimental levels were obtained for each factor: soaking time: 6 h, 12 h and 24 h; straw particle size: 50 mesh, 60 mesh and 70 mesh; and material-liquid ratio: 1 g: 20 mL, 1 g: 15 mL and 1 g: 10 mL. Orthogonal experiments were conducted.

7. The preparation method according to claim 1, characterized in that, In step (3), the filtration process involves first coarse filtration with gauze, and then filtration with a 0.22μm aqueous microporous membrane.

8. The application of the reed straw extract prepared by any one of claims 1-7 in microalgae cultivation.

9. A method for culturing microalgae, characterized in that, The process includes the following steps: mixing the reed straw extract prepared by the preparation method according to any one of claims 1-7 with BG11 culture medium at a volume ratio of 1:1, and then inoculating microalgae for cultivation.

10. The cultivation method according to claim 9, characterized in that, The microalgae used was Chlorella vulgaris, with an initial inoculation concentration of 200 mg / L, a shaking speed of 120 rpm, and culture conditions of 25±1℃, 3000 lux light intensity, 12h:12h light-dark ratio, and a culture time of 4 days.

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