Use of a soluble lignin as an enhancer of ethanol fermentation and / or to increase the stress resistance of yeast under fermentation environmental stress conditions
By preparing soluble lignin and adding it to the ethanol fermentation system, the problems of pH reduction, reactive oxygen species and heavy metal ion inhibition during fermentation were solved, the stress resistance of yeast and the efficiency of ethanol fermentation were improved, the process was simplified and the cost was reduced.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-25
- Publication Date
- 2026-03-24
AI Technical Summary
In existing technologies, the ethanol fermentation process suffers from problems such as easy decrease in the pH value of the fermentation system, surge in reactive oxygen species, and inhibition of enzyme activity by heavy metal ions, leading to loss of yeast activity. Existing solutions are characterized by high cost, inconsistent effects, or the introduction of impurities.
High molecular weight lignin was prepared by alkali dissolution, ultrasonication and dialysis of soluble lignin, retaining phenolic hydroxyl and carboxyl groups. It was then added to the ethanol fermentation system to buffer pH, resist oxidation and chelate heavy metals.
It improves ethanol fermentation efficiency, enhances yeast resistance, reduces yeast cytotoxicity, simplifies the process, reduces costs, and is applicable to a variety of fermentation raw materials and regions.
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Figure CN120923814B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of biological ethanol fermentation, in particular, to application of soluble lignin as an efficiency improver for ethanol fermentation and / or for improving stress resistance of yeast under fermentation environmental stress conditions. BACKGROUND
[0002] As a clean and renewable energy, biological fermentation production of ethanol relies on metabolic conversion of carbon sources such as glucose by yeast. However, there are three major problems in the fermentation process that restrict efficiency: first, the pH value of the fermentation system is easily reduced to below 3.0 due to accumulation of organic acids, resulting in loss of enzyme activity of yeast; second, high temperature (such as summer fermentation or insufficient heat dissipation of industrial tanks) causes excessive reactive oxygen species (ROS) to destroy the integrity of the yeast cell membrane; and third, heavy metal ions (such as Cu 2+ , Cd 2+ ) brought in by fermentation raw materials can chelate enzyme active centers and inhibit metabolic pathways.
[0003] Existing solutions have obvious limitations, such as introduction of impurities by artificial titration to adjust pH, and inability to respond dynamically; antioxidant additives (such as vitamin C) are easily metabolized and consumed, with poor sustained effect; heavy metal adsorbents (such as activated carbon) require additional separation steps, increasing cost.
[0004] Lignocellulose contains cellulose (45-55%), hemicellulose (25-35%) and lignin (15-25%), which interact through covalent and non-covalent bonds to form lignin-carbohydrate complexes. In the production of cellulosic fuel ethanol, lignin cannot be bioconverted into fuel ethanol, but is combined with undissolved cellulose and hemicellulose impurities in the form of solid residues. In the pretreatment process of lignocellulose, lignin structure is inevitably degraded, producing a large amount of phenolic inhibitors. When lignin is precipitated and recovered from pretreatment liquid or fermentation broth, not only high molecular weight lignin will precipitate, but also many small molecule phenolic compounds dissolved in the liquid will be brought down together due to reduced solubility (pH change, ion strength change) or co-precipitation / adsorption with precipitated lignin, which makes the recovered lignin product "containing" a large amount of small molecule phenolic impurities. These small molecule phenolic compounds can affect the permeability of the cell membrane, so lignin is generally considered to inhibit the activity of fermentation microorganisms, and no research has been found to apply lignin to the ethanol fermentation system. SUMMARY
[0005] To overcome the above-mentioned defects and deficiencies in the prior art, the present application provides application of soluble lignin as an efficiency improver for ethanol fermentation and / or for improving stress resistance of yeast under fermentation environmental stress conditions.
[0006] The first objective of this invention is to provide a soluble lignin.
[0007] A second objective of this invention is to provide the application of the aforementioned soluble lignin as an enhancer in ethanol fermentation.
[0008] A third objective of this invention is to provide the application of the aforementioned soluble lignin in improving the stress resistance of yeast under fermentation environmental stress conditions.
[0009] The fourth objective of this invention is to provide a method for improving the efficiency of ethanol fermentation.
[0010] The fifth objective of this invention is to provide a method for improving the stress resistance of yeast under fermentation environmental stress conditions.
[0011] This invention claims protection for the following:
[0012] A soluble lignin is prepared by alkali dissolution and sonication of a lignin solution, separation of the liquid after sonication, dialyzing of the liquid, and removal of the solvent after dialysis.
[0013] The lignin includes enzymatically hydrolyzed lignin, alkali lignin, sulfate lignin, lignin extracted by eutectic solvents, or ground wood lignin.
[0014] Preferably, the lignin is enzymatically hydrolyzed lignin.
[0015] Preferably, the alkali dissolution involves adjusting the pH of the lignin solution to 10-13.
[0016] Preferably, the ultrasonic power is 200–400 W and the frequency is 30–50 kHz.
[0017] More preferably, the ultrasonic power is 300 W and the frequency is 40 kHz.
[0018] Preferably, the ultrasound duration is 0.5 to 2 hours.
[0019] More preferably, the duration of the ultrasound is 1 hour.
[0020] Preferably, the molecular weight cutoff of the dialysis bag used for dialysis is 500 to 5000 Da.
[0021] Soluble lignin treated with alkali dissolution, ultrasound, and dialysis retains a large number of phenolic hydroxyl groups (≥2.5 mmol / g) and carboxyl groups (≥1.8 mmol / g). It can buffer pH through proton transfer, scavenge ROS with hydrogen donation, and coordinate chelate heavy metals. Furthermore, its molecular weight is concentrated in the range of 500–5000 Da, avoiding penetration of yeast cell membranes and thus preventing toxicity. Specifically:
[0022] pH buffer: The carboxyl group (pKa≈4.5) is protonated (-COOH) at pH < 4.0 and deprotonated (-COO) at pH > 5.0. - ), to alleviate the decrease in system pH;
[0023] Antioxidant: Phenolic hydroxyl groups (-OH) terminate the ROS chain reaction by providing hydrogen atoms, and the DPPH free radical scavenging rate reaches 85%±3%, reducing the degree of cell membrane lipid peroxidation;
[0024] Heavy metal chelation: Phenolic hydroxyl groups and carboxyl groups bind to metal ions (such as Cu) via the lone pair electrons of the O atom. 2+ Cd 2+ It forms a stable five-membered ring chelate, reducing the concentration of free ions to a safe threshold (<0.1 mg / L).
[0025] Preferably, the method for removing the solvent is rotary evaporation.
[0026] More preferably, the temperature of the rotary evaporation is 45–60°C.
[0027] Lignin is a complex three-dimensional network formed by phenylpropane units (coumaryl alcohol, coniferyl alcohol, sinapyl alcohol) linked by ether bonds (such as β-O-4 bonds) and carbon-carbon bonds. Among them, the ether bonds and other linkages have low stability and are prone to breakage under acidic / alkaline conditions, high temperature or mechanical shear (such as ultrasonic treatment) in the fermentation system, which leads to the depolymerization of lignin macromolecules and releases small molecule phenolic monomers or dimers such as vanillin, p-hydroxybenzaldehyde, and eugenol. Therefore, the high molecular weight lignin obtained after alkali dissolution, ultrasound, and dialysis is less likely to generate small molecule phenolic compounds in the fermentation system. This is mainly due to the improved structural stability and the precise removal of easily degradable components by the pretreatment. Alkali dissolution can break some weak ether bonds (such as β-O-4 bonds) in lignin, removing only easily degradable low molecular weight fragments. Ultrasound treatment mainly promotes the dispersion of high molecular weight lignin through mechanical vibration, rather than deep depolymerization, avoiding excessive breakage that generates small molecules. Dialysis further screens out the small molecule phenols (such as vanillin and p-hydroxybenzaldehyde) generated during the pretreatment. The high molecular weight lignin that is ultimately retained has a stable carbon-carbon bond backbone. These bonds have high bond energies, much higher than those of easily broken ether bonds, making them difficult to break spontaneously under mild fermentation conditions.
[0028] The above-mentioned soluble lignin is used as an enhancer in ethanol fermentation.
[0029] The above-mentioned soluble lignin is used to improve the stress resistance of yeast under fermentation environmental stress conditions.
[0030] Preferably, the fermentation environmental stress conditions include low pH stress conditions, high temperature stress conditions, and / or heavy metal stress conditions.
[0031] More preferably, the heavy metal stress conditions in the fermentation environment are those containing cadmium, copper, and / or chromium.
[0032] A method for improving the efficiency of ethanol fermentation involves adding the aforementioned soluble lignin to the ethanol fermentation system.
[0033] A method for improving the stress resistance of yeast under fermentation environmental stress conditions involves adding the aforementioned soluble lignin to the fermentation system.
[0034] Compared with the prior art, the present invention has the following beneficial effects:
[0035] This invention discloses the application of soluble lignin as an enhancer in ethanol fermentation and / or in improving the stress resistance of yeast under fermentation environmental stress. Adding the soluble lignin of this invention to the ethanol fermentation system can not only increase the concentration of the product ethanol, but also improve the stress resistance of yeast under stress environmental conditions and increase the survival rate of yeast.
[0036] In addition, the present invention has the following advantages:
[0037] Simplified process: The addition of a single soluble lignin can solve three major problems at the same time, eliminating the need for separate addition of pH adjusters, antioxidants and heavy metal removers, reducing equipment usage and operation steps;
[0038] Cost reduction: Lignin is derived from lignocellulose byproducts (such as pulping black liquor), and its raw material cost is cheaper than chemical buffers. It can be recycled and can still maintain a high synergistic effect after multiple recyclings.
[0039] Environmentally friendly: It avoids chemical reagent residues, and the lignin is eventually biodegradable, meeting the safety standards for ethanol production;
[0040] High versatility: It is suitable for fermentation systems of various raw materials such as corn stalks and sugarcane bagasse, and is stable and effective in a wide temperature range, adaptable to different regional industrial conditions. Attached Figure Description
[0041] Figure 1 The concentration of ethanol in the system after 24 h of fermentation under high temperature stress is given.
[0042] Figure 2 The effect of adding lignin on the concentration of heavy metal ions in the supernatant of the fermentation system.
[0043] Figure 3 The concentration of ethanol in the system after 24 h of fermentation under heavy metal stress.
[0044] Figure 4 The survival rate of yeast cells after 24 h of fermentation under heavy metal stress is given.
[0045] Figure 5 This represents the ethanol concentration in the system during the recycling of lignin 1 to 10 times. Detailed Implementation
[0046] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.
[0047] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.
[0048] Example 1: Preparation of soluble lignin
[0049] This embodiment provides a method for preparing soluble lignin, the specific steps of which are as follows:
[0050] 16 g of enzymatically hydrolyzed lignin was added to 184 mL of ultrapure water, the pH was adjusted to 12 with NaOH, and the mixture was sonicated (300 W, 40 kHz) for 1 h. After filtration, the mixture was transferred to a 500 Da dialysis bag and dialyzed in ultrapure water at room temperature for 48 h (with water changed every 6 h). The lignin powder was obtained by rotary evaporation (50 °C).
[0051] Comparative Example 1
[0052] This comparative example follows the procedure of Example 1, except that vacuum drying is performed directly after ultrasonication. The specific steps are as follows:
[0053] 16 g of enzymatically hydrolyzed lignin was added to 184 mL of ultrapure water, the pH was adjusted to 12 with NaOH, and the mixture was ultrasonically treated (300 W, 40 kHz) for 1 h. After filtration, the mixture was vacuum dried to obtain lignin powder.
[0054] Comparative Example 2
[0055] This comparative example was conducted in accordance with Example 1, except that acid precipitation and vacuum drying were performed after ultrasonication. The specific steps are as follows:
[0056] 16 g of enzymatically hydrolyzed lignin was added to 184 mL of ultrapure water, and the pH was adjusted to 12 with NaOH. The mixture was then sonicated (300 W, 40 kHz) for 1 h. The pH of the treated lignin solution was then adjusted to 3 with 0.2 M HCl solution. The resulting precipitate was filtered under vacuum and repeatedly washed with distilled water, followed by vacuum drying to obtain lignin powder.
[0057] Example 2: Effects of different preparation methods on the solubility and molecular weight of lignin
[0058] I. Experimental Methods
[0059] The lignin powders obtained in Example 1, Comparative Example 1, and Comparative Example 2 were added to water to a final concentration of 2 g / L. After thorough stirring, the mixture was filtered through a 0.45 μm filter membrane, and the filtration results were observed.
[0060] Weigh 10 mg of lignin powder obtained in Example 1, Comparative Example 1, and Comparative Example 2, and add 10 mL of solvent (DMSO solution containing 0.1% LiBr, w / v). After thorough stirring, filter the solution through a 0.45 μm organic phase filter membrane to remove undissolved particles or impurities. Collect the filtrate as the sample to be tested. Perform gel permeation chromatography. The data workstation converts the retention time of the sample into the corresponding molecular weight according to the calibration curve, calculates the area of each chromatographic peak as the weight, and finally determines the weight-average molecular weight and number-average molecular weight of each lignin.
[0061] II. Experimental Results
[0062] According to the results in Table 1, in terms of solubility, the lignin prepared using the methods of Example 1 and Comparative Example 1 are all soluble lignin, while the lignin prepared using the method of Comparative Example 2 is insoluble.
[0063] Enzymatic hydrolysis of lignin is a lignin preparation isolated from plant raw materials through cellulase hydrolysis. Enzymatic hydrolysis of lignin removes small-molecule phenolic inhibitors while retaining the active groups such as phenolic hydroxyl and carboxyl groups of large-molecule lignin, thus reducing its molecular weight. According to the results in Table 1, the molecular weights of the lignin prepared in Comparative Examples 1 and 2 are both lower than those of the lignin prepared in Example 1.
[0064] Table 1. Solubility and molecular weight of lignin
[0065]
[0066] Example 3: Effects of lignin prepared by different methods on ethanol fermentation
[0067] I. Experimental Methods
[0068] Prepare the basal culture medium: 100 g / L glucose (derived from straw hydrolysis), 1 g / L NH4Cl, 1 g / L KH2PO4, 0.3 g / L MgSO4·7H2O, pH 5.5.
[0069] 2 g / L of the lignin prepared in Example 1, Comparative Example 1, and Comparative Example 2 were added to the basal culture medium, respectively, and a control group (without lignin) was set up. Then, *Saccharomyces cerevisiae* (1×10⁻⁶) was inoculated. 8 Fermentation was carried out at 34℃ and 150 rpm for 24 h (CFU / mL).
[0070] After fermentation, the sample liquid was centrifuged at 10,000 rpm for 3 min, and the supernatant was collected. The ethanol concentration was determined by liquid chromatography. 0.5 mL of the sample liquid and 0.5 mL of 0.1% basic methylene blue staining solution (v / v) were mixed in a centrifuge tube and incubated at 34°C for 5 min. A small volume of the mixture was then transferred to a cell counting chamber for microscopic observation. The viability of the yeast cells was determined based on the color difference between live and dead cells, and the survival rate was calculated.
[0071] II. Experimental Results
[0072] According to the results in Table 2, the lignin prepared in Example 1 can improve the efficiency of ethanol fermentation, increase the concentration of ethanol in the product, and improve the survival rate of yeast.
[0073] Based on the molecular weight results of Example 2, it can be seen that although the lignin prepared in Comparative Examples 1 and 2 has a smaller molecular weight, it inhibits ethanol fermentation. After adding the lignin prepared in Comparative Examples 1 and 2 to the ethanol fermentation system, the ethanol concentration decreased by 9.1%–29.8% compared to the control group, and the yeast survival rate also decreased by 10.3%–42.0% compared to the control group. These results indicate that the lignin prepared in Example 1 has a synergistic effect in the ethanol fermentation system, can resist low pH stress during fermentation, and can reduce the toxic effects of lignin on yeast cell membranes, thereby improving yeast survival rate.
[0074] Table 2. Effects of lignin obtained by different preparation methods on ethanol fermentation
[0075]
[0076] Example 4: Effects of soluble lignin on ethanol fermentation under high temperature stress
[0077] I. Experimental Methods
[0078] Prepare the basal culture medium: 100 g / L glucose (derived from straw hydrolysis), 1 g / L NH4Cl, 1 g / L KH2PO4, 0.3 g / L MgSO4·7H2O, and add acetic acid and sodium acetate to the basal culture medium to make the system a 50 mmol / L pH 6.0 buffer solution.
[0079] Add 2 g / L of the lignin prepared in Example 1 to the basal culture medium, and set up a control group (without lignin), then inoculate with Saccharomyces cerevisiae (1×10⁻⁶). 8 Fermentation was carried out at 45℃ and 150 rpm for 24 h (CFU / mL).
[0080] After fermentation, the ethanol concentration in the fermentation system was measured according to Example 3.
[0081] II. Experimental Results
[0082] The results are as follows Figure 1 As shown, after fermentation at 45℃ for 24 h, the ethanol concentration of the product in the group with added lignin was 30.8 g / L, which was 22% higher than that of the control group (25.2 g / L). This indicates that the lignin prepared in Example 1 can resist the inhibitory effect of high temperature stress on ethanol fermentation.
[0083] Example 5: Effects of soluble lignin on ethanol fermentation under heavy metal stress.
[0084] I. Experimental Methods
[0085] Prepare the basal culture medium: 100 g / L glucose (derived from straw hydrolysis), 1 g / L NH4Cl, 1 g / L KH2PO4, 0.3 g / L MgSO4·7H2O, pH 5.5. Introduce Cu into the basal culture medium. 2+ Pb 2+ and Cr 6+ The final concentration was 9.8 mg / L.
[0086] Add 2 g / L of the lignin prepared in Example 1 to the basal culture medium, and set up a control group (without lignin), then inoculate with Saccharomyces cerevisiae (1×10⁻⁶). 8 Fermentation was carried out at 34℃ and 150 rpm for 24 h (CFU / mL).
[0087] After fermentation, the ethanol concentration and yeast survival rate in the fermentation system were measured according to Example 3, and the heavy metal ions (Cu) in the fermentation system were also measured. 2+ Pb 2+ and Cr 6+ To determine the ion concentration and calculate the heavy metal removal rate, the following steps were performed: The sampled liquid was centrifuged at 10,000 rpm for 3 minutes, and the supernatant was collected. The ion concentration in the supernatant was measured using inductively coupled plasma atomic emission spectrometry (ICP). The ion concentration before fermentation was recorded as A0, and the ion concentration after fermentation was recorded as A1. The removal rate was then calculated. The calculation formula is:
[0088]
[0089] II. Experimental Results
[0090] After 24 hours of fermentation, the concentration of heavy metal ions in the supernatant decreased to approximately 0.17 mg / L, with an average removal rate of 98.3%.Figure 2 The average ethanol concentration of the product was 2.5 times higher than that of the control group (without lignin). Figure 3 The average survival rate of yeast increased from 30.1% to 71.6%. Figure 4 The above results indicate that the lignin prepared in Example 1 can resist the inhibitory effect of heavy metal stress on ethanol fermentation and its toxic effect on yeast, thereby improving the ethanol fermentation efficiency under heavy metal stress conditions.
[0091] Example 6: Recycling of lignin in ethanol fermentation
[0092] I. Experimental Methods
[0093] Prepare the basal culture medium: 100 g / L glucose (derived from straw hydrolysis), 1 g / L NH4Cl, 1 g / L KH2PO4, 0.3 g / L MgSO4·7H2O, pH 5.5.
[0094] Add 2 g / L of the lignin prepared in Example 1 and Comparative Examples 1-2 to the basal culture medium, respectively, and set up a control group (without lignin). Then, inoculate with Saccharomyces cerevisiae (1×10⁻⁶). 8 Fermentation was carried out at 34℃ and 150 rpm for 24 hours (CFU / mL).
[0095] After fermentation, the precipitated lignin was collected by centrifugation, and the pH was adjusted to 5.5 with NaOH to redissolve the lignin. The collected lignin was then used to repeat the above fermentation process, and reused 10 times.
[0096] The ethanol concentration in the fermentation system was measured according to Example 3.
[0097] II. Experimental Results
[0098] The results are as follows Figure 5 As shown, after the 10th fermentation, the ethanol concentration of the product was 84.3% of that of the first fermentation. This demonstrates that the lignin prepared in Example 1 can be recycled and retains its synergistic effect on ethanol fermentation even after repeated use.
[0099] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.
Claims
1. A soluble lignin, characterized in that, The soluble lignin is prepared by alkali dissolution and sonication of a lignin solution, separation of the liquid after sonication, dialyzing of the liquid, and removal of the solvent after dialysis. The alkaline dissolution involves adjusting the pH of the lignin solution to 10-13. The ultrasound has a power of 200–400 W, a frequency of 30–50 kHz, and a duration of 0.5–2 h. The molecular weight cutoff of the dialysis bag used for dialysis is 500 Da; The lignin is enzymatically hydrolyzed lignin.
2. The use of the soluble lignin according to claim 1 as an synergist in ethanol fermentation.
3. The application of the soluble lignin according to claim 1 in improving the stress resistance of yeast under fermentation environmental stress conditions.
4. The application according to claim 3, characterized in that, The fermentation environment stress conditions include low pH stress conditions, high temperature stress conditions, and / or heavy metal stress conditions.
5. A method for improving the efficiency of ethanol fermentation, characterized in that, The soluble lignin described in claim 1 is added to the ethanol fermentation system.
6. A method for improving the stress resistance of yeast under fermentation environmental stress conditions, characterized in that, Add the soluble lignin as described in claim 1 to the fermentation system.