BE1029281B1 - Process for the biological production of ethanol from aleurite leaves - Google Patents

Abstract

The present invention relates to the technical field of the preparation of ethanol, and more specifically to a process for the biological production of ethanol from aleurite sheets.

Description

Process for the biological production of ethanol from aleurite leaves

Technical area

The present invention relates to the technical field of the preparation of ethanol, and more specifically to a process for the biological production of ethanol from aleurite leaves.

Core technology

China is a major producer and consumer of oilseeds and has a vast area of woody oil crops, mainly oil tea, aleurite and Litsea cubeba fruit. Aleurite is native to China and is a unique economic forest tree whose cultivation dates back over a thousand years. It has a high oil content, a wide range of oil applications, is easy to cultivate and has a long economic life. Aleurite has great economic advantages. In the 1990s, Chinese scholars carried out various research works, such as the classification of germplasm resources, the selection and breeding of good family lines, asexual lines and superior trees, the sexual reproduction of the aleurite, seed introduction trials and analysis of its biological traits. During the growth of aleurite and the production of oil from it, a large amount of biomass waste is generated, such as the leaves and branches of aleurite and its residue after oil production, ete. The use of this biomass is a subject that deserves to be studied.

Ethanol is widely used in all aspects of social production and plays an important role in food preparation, chemical raw materials, medicine and medical care, and fossil fuel substitution. Biological ethanol production has the advantages of a wide range of degradable substrates, simple production processes, and easy product storage and transfer. However, traditional ethanol production mainly uses agricultural crops (rice, corn, sweet potatoes, sorghum, etc.) as raw materials. With the continued and steady increase in China's population and the decline of efficient cropland year after year, the search for a cheap alternative to crop fermentation for ethanol production has become an urgent priority. Based on the above, a lot of research has also been done on the use of biomass, such as cornstalk, for the biological production of ethanol. Johnson et al. investigated the feasibility of replacing corn with corn stover for organic ethanol production and analyzed the effect of mixing corn stover with corn on ethanol yield, fermentation rate, and residual sugar . They found that substituting corn stover for corn for organic ethanol production could yield good results. Li et al. investigated the effect of laccase pretreatment and surfactant addition on the simultaneous saccharification of corn stalk fermented by modified yeasts. The combination of laccase pretreatment and the addition of rhamnolipids increased ethanol production. The et al. used ammonia pretreatment to enhance enzymatic saccharification and biological ethanol production from corn stover, achieving a maximum ethanol concentration of 14.5 g/L. Molaverdi et al. used sodium carbonate pretreatment of cornstalk to improve the fermentation yield of solid ethanol, increasing ethanol concentrations from 24 g/L to 41 g/L under low cellulase loading conditions ( 5

FPU/g of substrate) and sodium carbonate pretreatment. Stenberg et al. investigated the effect of substrate concentration and cellulase on ethanol production by simultaneous saccharification and found that ethanol production increased with increasing cellulase load; the low substrate concentration resulted in the production of lactic acid and the high substrate concentration also inhibited the production of ethanol.

In references on the resource use of woody oilseed crops, Hu et al. used the fruit of Litsea cubeba to extract basic substances for perfumes such as ethanol, isopropyltoluene and peat moss. Zhu et al. treated the tea tree shells in oil with NaOH to obtain the obtained solid and liquid fractions. The solid fraction was hydrolyzed by cellulase and fermented to produce ethanol, while the liquid fraction was oxidized to prepare a vanillin paste with oligosaccharides. 17.35 g/L of ethanol was obtained from the solid fraction containing cellulose and a small amount of xylan at a concentration of 10%. In the study by Yan et al, researchers used ethanol as a pretreatment method, using microwave ethanol to pretreat tea tree seed shells with oil, then enzymatically hydrolyzed them to prepare oligosaccharides. Published studies have mainly used fruit waste from woody oilseed crops, while little research has been conducted on residual biomass such as branches and leaves produced by woody oilseed crops. Existing studies focus on the effects of temperature on photosynthetic effects and internal leaf structure, and no studies were found on the use of good quality aleurite leaves.

The biotransformation of biomass waste from aleurite sheets into ethanol allows good quality utilization of the waste. Solid woody oilseed wastes contain polysaccharides, acids and proteins, and these organic components can be recovered. The biotransformation of woody solid waste from oilseeds into ethanol is also a good way to use resources. However, little research has been reported on the biological production of ethanol from solid wastes of woody oilseed crops. The physical and chemical properties of woody oilseed solid waste must be analyzed in detail, and their unique physical and chemical properties (high toxin content, high lignin content, nutrient imbalance, etc.) place higher demands on saccharification and enzymatic degradation, and the law of effect and enhancement mechanism of woody oilseed solid waste on biological ethanol production should be further investigated.

Content of the invention

The present invention aims to provide a method for the biological production of ethanol from aleurite sheets which can increase the production of ethanol.

In order to achieve the aforementioned objective, the present invention proposes the following technical solutions:

Process for the biological production of ethanol from aleurite sheets, characterized in that it comprises the following stages: 1) Grinding: The aleurite sheets are ground into a powder using a grinder and then put next to ; 2) Mixing: We weigh a certain mass of powder of aleurite leaves in step 1) in a prismatic bottle, and pour a certain mass of distilled water into the bottle to mix it with the powder of aleurite leaves in order to obtain a mixture; 3) Finished product: Accurately weigh cellulase according to the ratio of 0.4:1 of cellulase and aleurite leaf powder, then weigh a certain mass of yeast and put them into a prismatic flask, finally shakes the flask, so that the cellulase and yeast can be fully mixed into the mixture to produce ethanol.

Preferably, in the above method for the biological production of ethanol from aleurite leaves, the mass of the aleurite leaf powder is 1-20g, preferably 1g, 5g, 10 g, 15g, 20g.

Preferably, in the above process for the biological production of ethanol from aleurite sheets, the distilled water is 200 mL.

Preferably, in the above method for the biological production of ethanol from aleurite leaves, the yeast is 0.5 g. 5 Preferably, in the above process for the biological production of ethanol from aleurite leaves, the yeast is a high potency dry winemaking yeast.

As apparent from the above technical solutions, compared to existing techniques, the present invention investigates the feasibility of the biological production of ethanol from aleurite leaves using aleurite leaves as a substrate and an active yeast as ethanol-producing microorganism; she mainly studied the rules of change of gas properties, liquid properties, soluble metabolites and kinetic properties during the biological preparation of ethanol from aleurite sheets. The results showed that the gas production rate first increased and then decreased during preparation, and that the maximum gas production rate of 43 mL/h, the maximum accumulated gas production of 1201.5 mL, and the concentration Maximum ethanol of 6.17 g/L was obtained when the substrate concentration was 100 g/L.

Description of the drawings

In order to more clearly illustrate the technical solutions in the examples or the technologies of the present invention, a brief description will now be given of the drawings which are necessary for the description of the examples or the technologies. It will be apparent that the accompanying drawings of the following description are only examples of the present invention, and that other drawings may be obtained from the drawings provided without any creative effort by ordinary skill in the art.

FIG. 1 shows the gas properties during biological ethanol production of the present invention.

FIG. 2 shows the properties of the liquid during the biological ethanol production of the present invention.

FIG. 3 shows the properties of the soluble substances during the biological production of ethanol of the present invention.

specific application

The technical solutions in the examples of the present invention will be clearly and completely described below in connection with the examples of the present invention, and it is clear that the examples described are only a part of the examples of the present invention, and not their totality. Based on the examples of the present invention, all other examples obtained without any creative effort by ordinary technicians in this field fall within the scope of protection of the present invention.

Example 1:

In the experiment, a 200 ml glass flask was taken as a biological ethanol production reactor. 1g of aleurite leaf powder was weighed and added to a previously prepared 200ml prismatic flask, then 200ml of distilled water was measured with a measuring cylinder and poured into the flask to mix with aleurite leaf powder; 0.4 g of cellulase was weighed using an electronic balance and added to the prismatic flask.

After adding the cellulase, 0.2 g of high potency dry wine yeast was separately weighed using the balance and added to the flask, and finally the mixture was shaken well so that the enzyme and yeast are fully mixed with the reaction solution to improve the ethanol yield. The temperature of the incubator was set at 30°C and the experimental data was tested according to the preset time.

Example 2:

In the experiment, a 200 ml glass flask was taken as a biological ethanol production reactor. 5g of aleurite leaf powder was weighed and added to a previously prepared 200ml prismatic flask, then 200ml of distilled water was measured with a measuring cylinder and poured into the flask to mix with aleurite leaf powder; 2 g of cellulase was weighed using an electronic balance and added to the prismatic flask.

After adding the cellulase, 0.2 g of high potency dry wine yeast was separately weighed using the balance and added to the flask, and finally the mixture was shaken well so that the enzyme and yeast are fully mixed with the reaction solution to improve the ethanol yield. The temperature of the incubator was set at 30°C and the experimental data was tested according to the preset time.

Example 3:

In the experiment, a 200 ml glass flask was taken as a biological ethanol production reactor. 10g of aleurite leaf powder was weighed and added to a previously prepared 200ml prismatic flask, then 200ml of distilled water was measured with a measuring cylinder and poured into the flask to mix with aleurite leaf powder; 4 g of cellulase was weighed using an electronic balance and added to the prismatic flask.

After adding the cellulase, 0.2 g of high potency dry wine yeast was separately weighed using the balance and added to the flask, and finally the mixture was shaken well so that the enzyme and yeast are fully mixed with the reaction solution to improve the ethanol yield. The temperature of the incubator was set at 30°C and the experimental data was tested according to the preset time.

Example 4:

In the experiment, a 200 ml glass flask was taken as a biological ethanol production reactor. 15g of aleurite leaf powder was weighed and added to a previously prepared 200ml prismatic flask, then 200ml of distilled water was measured with a measuring cylinder and poured into the flask to mix with aleurite leaf powder; 6 g of cellulase was weighed using an electronic balance and added to the prismatic flask.

After adding the cellulase, 0.2 g of high potency dry wine yeast was separately weighed using the balance and added to the flask, and finally the mixture was shaken well so that the enzyme and yeast are fully mixed with the reaction solution to improve the ethanol yield. The temperature of the incubator was set at 30°C and the experimental data was tested according to the preset time.

Example 5:

In the experiment, a 200 ml glass flask was taken as a biological ethanol production reactor. 20 g of aleurite leaf powder was weighed and added to a previously prepared 200 ml prismatic flask, then 200 ml of distilled water was measured with a measuring cylinder and poured into the flask to mix with aleurite leaf powder; 8 g of cellulase was weighed using an electronic balance and added to the prismatic flask.

After adding the cellulase, 0.2 g of high potency dry wine yeast was separately weighed using the balance and added to the flask, and finally the mixture was shaken well so that the enzyme and yeast are fully mixed with the reaction solution to improve the ethanol yield. The temperature of the incubator was set at 30°C and the experimental data was tested according to the preset time.

Materials and method

Materials

Aleurite sheets are crushed with a grinder to destroy the crystal structure of cellulose and reduce crystallinity. The crushed cellulose powder does not swell and is small in size, which increases the concentration of substrate and improves the rate of saccharification.

Cellulase: (51 FPU/mL, Novozymes Biotechnology Co., Ltd, Denmark).

Yeast: A high potency dry winemaking yeast from Anchor was used in this experiment;

Test method

Ethanol concentration was determined using an Agilent gas chromatograph (7090B) with a column oven temperature of 40°C, sample inlet and detector temperatures of 250°C and 300°C respectively, a pressure of 10 psi, a chromatographic column flow of 2.396 mL/min and a valve oven temperature of 44.1°C. The pH of the reaction solution was measured using a pH meter (PHSJ-6L, Shanghai

INESA Scientific Instruments Co, LTD., China), the redox potential of the reaction solution was measured using a redox potential meter (SX712,

Shanghai San-Xin Instrumentation, Inc, China) and the concentration of reducing sugars was measured using a visible spectrophotometer (721, Shanghai

Metash Instruments Co., Ltd, China) at OD540 nm.

Analysis of kinetic properties

The kinetics of gas production was analyzed using the equation of

Gompertz. The Gompertz equation was used for gas production analysis and data presentation so that the maximum cumulative potential gas production P, the maximum gas production rate Rmax and the lag period λ are expressed in encrypted form:

H(t) =P x exp (-exp [Rmax Xe (at-t) + 1]) (1)

Equation (2) is the derivative of the Gompertz equation and R(t) represents the gas production rate.

R(t) = Bax Xe x exp (max xe (A-f) + 1) x H(t) (2)

The average gas production rate characterizes the average gas production rate over the entire gas production period.

Roverall= P 1 3) (P/Rmax)+ 2 V

The interpretation of the equation symbols is shown in Table 1.

Table 1 Symbolic annotation of equation

Symbol Notes

HO Cumulative photo-fermentation hydrogen production (mL)

P Maximum potential hydrogen production (mL)

Rmax Maximum hydrogen production rate (mL/h)

À Lag phase (h) í Time (h) e 2.718

R() Time-dependent hydrogen production rate (mL/h)

Roverall Average hydrogen production rate (mL/h)

V Working volume of the bioreactor (mL)

Results and discussion :

As shown in Figure 1-3: Effect of substrate concentration on gas properties during biological ethanol production.

The biological production of ethanol has been accompanied by the production of large amounts of carbon dioxide gas. Figure la shows that the rate of gas production tended to increase and then decrease over time, with a peak around 12 p.m., indicating that the biological production of ethanol was at its peak in this period, which is also evidenced by the variation in ethanol concentration in Figure 3. With increasing substrate concentration, the production rate tended to increase. The maximum gas production rate of 38.5 mL/h (12 h) was reached at a substrate concentration of 100 g/L, which is closely related to the substrate concentration.

As shown in Figure 1b, the cumulative gas production tended to continuously increase with time, and with increasing substrate concentration. The maximum cumulative gas production of 1201.5 ml occurred at a substrate concentration of 100 g/L. This trend is very similar to the photosynthetic production of hydrogen from corn stover, where microorganisms degrade the biomass into sugars, which are then converted into the target product (hydrogen or ethanol).

As shown in Figure 3: Effect of substrate concentration on liquid properties during biological ethanol production:

Figure 2a shows the evolution of pH during the biological production of ethanol from aleurite sheets. The pH tended essentially to fall over time, due to the conversion of sugars converted from cellulase to acids by the yeast and the simultaneous production of ethanol. As the concentration of aleurite sheets increased, the pH tended to decrease, indicating that the higher substrate concentration produced more fatty acid volatiles (FIG. 3). Similar to photosynthetic hydrogen production, the pH of biological ethanol production exhibited a downward trend over time, but the pH of photosynthetic hydrogen production rose slowly in the later stages of ethanol production. hydrogen due to the conversion of photosynthetic bacteria into hydrogen by using volatile fatty acids in the later stages.

Figure 2b shows the rules for changing redox potential and substrate concentration with time during the biological production of ethanol from aleurite sheets. The redox potential trended down and then up with time, and trended up with increasing substrate concentration. At the peak of microbial metabolism, the redox potential tended to fall rapidly.

Figure 2c shows the trend of reducing sugar concentration during the biological production of ethanol from aleurite sheets, the reducing sugar concentration tended to increase with time and then decrease, and tended rising steadily with increasing concentration. At the start of biological ethanol production, the rate of conversion of cellulase to reducing sugars was greater than the rate of degradation of reducing sugars by yeast, thus the concentration of reducing sugars tended to increase. This indicates that microbial activity exhibits a similar change rule during biochemical conversion.

As shown in Figure 3: Effect of substrate concentration on soluble substances during biological ethanol production

Figure 3 shows the evolution of soluble substances during the biological production of ethanol from aleurite sheets. The main component of the soluble metabolites was ethanol, with small amounts of acetic acid and butyric acid. Ethanol concentration tended to continuously increase with time and substrate concentration, with a maximum ethanol yield of 6.18 g/L obtained at a substrate concentration of 100 g/L. In the study by Li et al. the ethanol concentration tended to increase continuously over time, up to 0.73 g/L. In the study by Le et al. the highest ethanol concentration of 14.5 g/L was achieved using ammonia pretreatment to enhance enzymatic saccharification and biological ethanol production from corn stover. The different concentrations of ethanol were determined by different substrates, microorganisms and processing conditions.

From the analysis of the concentration of ethanol converted from aleurite sheets per unit, the concentration of ethanol obtained from substrate per unit tended to first increase and then decrease with increasing of the substrate concentration of aleurite sheets, and the maximum yield obtained at g/L was: 0.35 g of ethanol per g of powder of aleurite sheets.

Effect of substrate concentration on the kinetic properties of biological ethanol production (as shown in Table 2):

Table 2 Kinetic parameters for biological ethanol production

Concentration (g/L) Rım(mL/h) AB) RE Pr (mL) 6 0.966 0035.51 25 7.78 0.98 0.99303 45.90 50 20.98 5.01 0.99808 78.64 75 26.27 5.29 0.99614 477.85 100 36.15 3.56 0.99513 1208.27

The kinetic equation is used to calculate the rate of gas production and the delay period, etc. for biological ethanol production and predicts maximum cumulative gas production, which is important for the scientific understanding of biological ethanol production. Table 2 shows the kinetic parameters of biological ethanol production. With increasing substrate concentration of aleurite sheets, the rate of gas production tended to increase. With increasing substrate concentration, the maximum gas production tended to continuously increase and the maximum accumulated gas production was obtained at a substrate concentration of 100 g/L. The correlation coefficients for all experimental groups were greater than 0.9, indicating that the equation for

Gompertz is well suited for the biological production of ethanol.

In summary, it can be seen that the biological production of ethanol from aleurite leaves has good prospects. Bioethanol concentration increased with aleurite sheet substrate concentration, but specific yield tended to increase and then decrease. The maximum yield of 0.35 g/L was obtained when the concentration of aleurite sheets was 50 g/L. Along with the biological production of ethanol, the pH of the fermentation broth trended continuously lower, the redox potential trended lower then higher, and the concentration of reducing sugar trended higher then higher. on the decline. The experimental results provide a theoretical and technical basis for the biotransformation of aleurite sheets.

Each example in this manual is described step by step, each example focuses on differences from other examples, and it is sufficient to refer to the same and similar parts of each example. For the device disclosed in the examples, the description is simpler because it corresponds to the method disclosed in the examples, and the relevant parts are described in the method section.

The above description of the disclosed examples enables skilled technicians to practice or use the present invention. A variety of modifications of these examples will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other examples without departing from the spirit or scope of the present invention. Accordingly, the present invention shall not be limited to the examples presented herein, but shall be subject to the broadest scope consistent with the principles and novel features disclosed herein.

Claims (5)

1. Process for the biological production of ethanol from aleurite sheets, characterized in that it comprises the following steps: 1) Grinding: The aleurite sheets are ground into a powder using a grinder then are set aside; 2) Mixing: We weigh a certain mass of powder of aleurite leaves in step 1) in a prismatic bottle, and pour a certain volume of distilled water into the bottle to mix it with the powder of aleurite leaves in order to obtain a mixture; 3) Finished product: Accurately weigh cellulase according to the 0.4:1 ratio of cellulase and aleurite leaf powder, then weigh a certain mass of yeast and put them into a prismatic flask, finally shakes the flask, so that the cellulase and yeast can be fully mixed into the mixture to produce ethanol. 2. Method according to claim 1, characterized in that the aleurite leaf powder has a mass of 1-20 g, preferably 1 g, 5 g, 10 g, 15 g, 20 g. 3. Method according to claim 1, characterized in that the volume of distilled water is 200 mL. 4. Method according to claim 1, characterized in that the mass of yeast is 0.5 g. 5. Method according to claim 4, characterized in that the yeast is a high-activity dry winemaking yeast.