CN107523606B - Pre-evaluation method for lignocellulose enzymolysis sugar production capacity - Google Patents
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Abstract
The invention discloses a pre-evaluation method for the glucose production capability of lignocellulose enzymolysis. The enzymolysis sugar production capability of lignocellulose is closely related to the resource potential of lignocellulose, but the enzymolysis process is complex in operation, long in time consumption and high in enzyme price in the actual application and production process, and if the effect is not ideal, manpower and material resources are greatly wasted, and the process efficiency is influenced. The method respectively determines the contents of lignin and cellulose in the lignocellulose, comprehensively reflects the influence of the lignin and the cellulose on the sugar production capacity by enzymolysis by adopting the saccharification index, and constructs a binary relation model of the saccharification index and the reducing sugar yield, thereby effectively evaluating the sugar production capacity and the resource potential of the lignocellulose. The method is simple to operate, wide in adaptability, strong in reliability and high in practical application value.
Description
Technical Field
The invention belongs to the technical field of biomass new energy, and particularly relates to a pre-evaluation method for the enzymatic hydrolysis and sugar production capacity of lignocellulose.
Background
In recent years, biological energy sources represented by lignocellulose have been receiving much attention. As the most abundant renewable substance in nature, lignocellulose can be used as an ideal carbon source for bioethanol, biodiesel and various biomass materials. The resource utilization of lignocellulose is influenced by a highly complex structure, and the cellulose which is most easily hydrolyzed is protected by hemicellulose and lignin and is deeply buried in a highly heterogeneous and recalcitrant structure formed by the lignin and the hemicellulose. Therefore, lignocellulose is difficult to directly use for practical use. Generally, lignocellulose needs to be subjected to physical and chemical pretreatment, and cellulose is subjected to enzymolysis by using cellulase after separation and purification to generate reducing sugar as a raw material to enter a next-stage biotransformation program. The sugar production capacity by enzymolysis is one of the key evaluation parameters for the efficient resource utilization of lignocellulose. However, the enzymolysis process is complex in operation in actual production, the time consumption is long (1-3 days), the price of the cellulase is high, and once the enzymolysis effect is not ideal, a large amount of manpower and material resources are undoubtedly wasted, and the benefit is influenced. Therefore, if the capability of lignocellulose for producing sugar by enzymolysis can be pre-evaluated before enzymolysis, the situation can be effectively avoided, and the process efficiency is improved, but the pre-evaluation method is still lacked at present.
Due to the complex structure of lignocellulose, the enzymolysis process mechanism is complex, the factors influencing enzymolysis are more, and interaction influence exists, so that the main influencing factors are difficult to determine for subsequent evaluation. In the past, linear regression analysis was tried to be performed on the relationship between single variables such as cellulose content and lignin content and the enzymolysis sugar yield, but the results show that the enzymolysis process is a multi-factor and multi-dimensional complex system, and the change of the single variable is difficult to accurately describe the change of the enzymolysis sugar yield. Recently, we found that the lignin content and the cellulose content have strong correlation with the sugar yield of enzymolysis. Among them, a higher lignin content tends to result in a lower amount of sugars produced by enzymatic hydrolysis, and lignin is a complex, amorphous polymer formed by connecting phenylpropane as a unit through an ether bond and a carbon-carbon bond, and mainly provides mechanical support for plants while inhibiting microorganisms, viruses, water and the like from invading the plants. Due to its impedance, lignin hinders the invasion of enzymes into the biomass and inactivates the enzymes by non-productive adsorption, which reduces the efficiency of enzymatic hydrolysis. Therefore, in practical application, it is necessary to remove the lignin component in the lignocellulose by means of pretreatment or the like. Cellulose is used as a main carbon source of a subsequent process, and the higher the content of the cellulose, the more reducing sugar is generated by enzymolysis. Therefore, it is generally desirable to preserve and expose the cellulosic components as much as possible during the pretreatment of the lignocellulose to facilitate the contact and action of the cellulase enzymes with the cellulose. Based on the relationship among the lignin content, the cellulose content and the enzymolysis sugar yield, the invention carries out statistical analysis on the basis of simultaneously considering two main influence factors, constructs and obtains a comprehensive parameter, namely the saccharification index, and establishes a relationship model between the saccharification index and the lignocellulose enzymolysis sugar yield, so that the method can be used for accurately pre-evaluating the lignocellulose enzymolysis sugar yield, and the pre-evaluation method is intuitive and effective and can play an important role in practical application.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide a pre-evaluation method for the glucose-producing capability of lignocellulose enzymatic hydrolysis, which can simply and efficiently evaluate the glucose-producing capability and the resource potential of the lignocellulose enzymatic hydrolysis.
The purpose of the invention is realized by the following method:
a pre-evaluation method for lignocellulose enzymolysis sugar production capacity estimates lignocellulose enzymolysis sugar production capacity by determining a saccharification index of lignocellulose, wherein the relation between the saccharification index and the lignocellulose enzymolysis sugar production capacity is that the saccharification index is less than 0.25, the estimated reducing sugar yield is less than 400mg/g, the saccharification index is less than 0.25, the estimated reducing sugar yield is 400-800 mg/g, the saccharification index is more than 0.65, the estimated reducing sugar yield is more than 800mg/g, and the saccharification index is 1.27, 1.27 × cellulose content and 2.65 × lignin content.
The saccharification index is determined by the lignin content and the cellulose content in lignocellulose, and the relationship among the saccharification index, the lignin content and the cellulose content is obtained by statistical data.
The yield of the enzymatic hydrolysis reducing sugar is determined by a saccharification index, and the relationship between the saccharification index and the enzymatic hydrolysis reducing sugar is obtained through statistical data, wherein the yield of the enzymatic hydrolysis reducing sugar is 150.606+999.817 × saccharification index.
The invention is realized by the following method:
1) the lignocellulosic lignin content and the cellulose content were determined. The lignin content is expressed by the Clarsen lignin (Klason lignin) content. The test mode is that the solid-to-liquid ratio of powdery and dry biomass is not lowAdding the obtained product into concentrated sulfuric acid with the mass fraction not lower than 70% (w/w) at a ratio of 1:15, stirring and reacting at 10-30 ℃ for at least 3 hours, adding deionized water with the volume of 30-45 times that of the concentrated sulfuric acid, boiling and refluxing for at least 3 hours, filtering residues, washing the residues to be neutral by using boiling water, accurately weighing the cleaned Clarsen lignin after vacuum drying, wherein the mass ratio of the Clarsen lignin to a raw biomass is the lignin content, the cellulose content is represented by α -cellulose content, and the test method is that sodium hypochlorite with the mass concentration not lower than 1.0% (w/v) is dissolved in dilute acetic acid with the volume fraction not lower than 8% (v/v), and the mass of powdered and dried biomass is m1Mixing the obtained product with a mixed solution according to a solid-to-liquid ratio of not less than 1:50, sealing and reacting at 70-80 ℃ for at least 1 hour, filtering residues, washing the residues with acetone and deionized water, and drying in vacuum for at least 24 hours to obtain holocellulose with the mass m2(ii) a Taking part of holocellulose, and recording the mass as m3Adding sodium hydroxide solution with the mass concentration not lower than 17.5% (w/v) according to the solid-to-liquid ratio not lower than 1:20, stirring and reacting for at least 40 minutes at room temperature, adding equal volume of deionized water, continuing to react for 3-10 minutes, filtering residues, adding dilute acetic acid with the volume fraction not lower than 8% (v/v) of which the solid-to-liquid ratio to the original holocellulose is not lower than 1:30, further filtering the residues after reaction, washing the residues to be neutral by using boiling water, and accurately weighing the weight m of the washed α -cellulose after vacuum drying4The cellulose content is represented by the formula: cellulose content ═ m4/m3)×(m2/m1) And (4) calculating.
2) Substituting the measured lignin content and cellulose content into formula 1:
saccharification Index (SI) ═ 1.27 × cellulose content-2.65 × lignin content (equation 1)
And calculating to obtain the lignocellulose saccharification index.
3) Evaluating the capability of the pretreated lignocellulose for producing sugar by hydrolysis according to the saccharification index:
the yield of reducing sugar is 150.606+999.817 × saccharification index SI
When the saccharification index is less than 0.25, the yield of the biomass enzymatic hydrolysis reducing sugar is estimated to be less than 400mg/g, and the enzymatic hydrolysis sugar production potential is low; when the saccharification index is 0.25-0.65, the yield of the biomass enzymatic hydrolysis reducing sugar is estimated to be 400-800 mg/g, and the enzymatic hydrolysis sugar production potential is moderate; when the saccharification index is greater than 0.65, the yield of the biomass enzymatic hydrolysis reducing sugar is estimated to be greater than 800mg/g, and the enzymatic hydrolysis sugar production potential is high.
The lignocellulose sample is derived from one or more of corn stalks, rice stalks and wheat stalks.
The basis for evaluating the sugar yield of biomass enzymolysis by adopting the saccharification index is as follows:
1) the feasibility of using lignin content and cellulose content to evaluate reducing sugar production was first determined. Taking 48 lignocellulose samples (comprising 16 corn stalk samples, 16 rice stalk samples and 16 wheat stalk samples), respectively determining the lignin content, the cellulose content and the enzymolysis reducing sugar yield of the samples, and averaging the results of repeating the experiments for three times, wherein the results are shown in table 1, and through SPSS (shortest path secure) correlation analysis, the correlation coefficient is as high as-0.899, and the correlation coefficient is consistent with the proved trend; the cellulose content and the reducing sugar yield have obvious positive correlation, the correlation coefficient reaches 0.802, and the result is inferred to be the combined action result of removing the lignin and the hemicellulose. Correlation analysis determines that the lignin content, the cellulose content and the reducing sugar yield are strongly related. Further multivariate linear regression analysis gave good results and the reducing sugar yield can be fitted with equation 2:
the yield of reducing sugar is 145.264-2649.154 × lignin content +1266.692 × cellulose content (formula 2)
The result shows better fitting performance, the Pearson's correlation is 0.954, and the fitting coefficient R is20.910; formula F value up to 227.51>The significance level critical value is 15.00, and the linear fitting is good; the linear fitting process variance inflation factor, VIF, is only 0.579<The collinearity diagnostic cutoff value was 10.000, indicating that no collinearity existed in the fit. All analyses demonstrated the feasibility of using lignin content and cellulose content to assess reducing sugar yield.
TABLE 1 Multi-sample parameter measurements and statistical analysis
2) The Saccharification Index is used for comprehensively representing two parameters of lignin content and cellulose content, a ternary system of the lignin content, the cellulose content and the reducing sugar yield can be reduced into a binary system of the Saccharification Index and the reducing sugar Index, and the direct guidance of the actual production is facilitated20.910, the feasibility of assessing the ability of lignocellulose to hydrolyze to produce sugar based on the saccharification index was demonstrated.
3) According to the analysis results, the saccharification index and the enzymolysis sugar yield of lignocellulose can be divided into 3 intervals, as shown in fig. 1, and the range of the saccharification index, the prediction result of the reducing sugar yield and the evaluation of the sugar production capacity in each interval are shown in table 2. In the actual process, the saccharification index data is compared with the conditions in the table, so that the enzymolysis sugar production capacity of the biomass can be qualitatively evaluated, and a reducing sugar yield prediction interval is obtained, thereby guiding the optimization of the actual production process.
TABLE 2 evaluation indexes of sugar production ability of biomass, each level of index name and characteristics
The evaluation method provided by the invention has the advantages that:
1) the method is characterized in that a comprehensive index saccharification index constructed by the lignin content and the cellulose content is provided as an evaluation index for evaluating the enzymatic hydrolysis sugar production capacity of biomass by demonstrating the correlation between the lignin content and the cellulose content and the yield of reducing sugar and performing multiple linear regression analysis for the first time. The method can evaluate the sugar production capability of the lignocellulose by enzymolysis only by measuring the content of lignin and cellulose in the lignocellulose, and the required equipment is simple. By evaluating the enzymolysis potential of lignocellulose, the enzymolysis process is effectively guided, low-efficiency enzymolysis is avoided, and the efficiency is improved.
2) The method does not need to carry out a complex pretreatment process on lignocellulose, can be suitable for evaluating the primary biomass and various pretreated biomasses, and has the advantages of strong reliability, wide application and obvious practical significance.
Drawings
FIG. 1: the relation graph of biomass reducing sugar yield and saccharification index.
Detailed Description
The invention is described in further detail below with reference to the figures and the examples, but without limiting the invention.
The method for testing the content of the lignocellulose lignin in the following embodiment is that powdered and dried biomass is added into 72% (w/w) concentrated sulfuric acid according to the solid-liquid ratio of 1:15, the mixture is stirred and reacted for 4 hours at 20 ℃, deionized water with the volume being 35 times that of the concentrated sulfuric acid is added, the mixture is boiled and refluxed and reacted for 4 hours, residues are filtered and washed to be neutral by using boiling water, the cleaned Clarsen lignin is dried in vacuum, the weight of the Clarsen lignin is accurately weighed, and the mass ratio of the obtained product to the original biomass is the lignin content. The cellulose content was measured by dissolving 1.33% (w/v) sodium hypochlorite in 10% (v/v) dilute acetic acid, the mass of the powdered, dried biomass being m1Mixing the obtained product with the mixed solution according to the solid-liquid ratio of 1:60, sealing and reacting at 75 ℃ for 1 hour, filtering residues, washing with acetone and deionized water, and vacuum drying at 60 ℃ for 24 hours to obtain holocellulose with the mass m2(ii) a Taking part of holocellulose, and recording the mass as m3Adding 17.5% (w/v) sodium hydroxide solution according to the solid-to-liquid ratio of 1:25, stirring at 20 ℃ for reaction for 40 minutes, adding the same volume of deionized water for continuous reaction for 5 minutes, filtering the residue, and adding the filtered residue into the reactor10% diluted acetic acid with a solid-to-liquid ratio of cellulose of 1:40, further filtering the residue after reaction, washing the residue to neutrality by using boiling water, drying the washed α -cellulose in vacuum, and accurately weighing the weight m of the cellulose4The cellulose content is represented by the formula: cellulose content ═ m4/m3)×(m2/m1) And (4) calculating.
Example 1
Sample 1 (cornstalk) was measured to have a lignin content of 0.235 with a standard deviation of 0.003, a cellulose content of 0.500 with a standard deviation of 0.004, and a glycation index of 0.01225 was calculated.0.5 g of sample 1 was subjected to an enzymatic hydrolysis test (20 m L0.5 mM citric acid buffer (pH 4.8) and 30 μ l novicen (finland) CTec cellulase 2 was added and hydrolyzed at 50 ℃ for 72 hours at 120 rpm), followed by three replicates of DNS to determine the reducing sugar yield, which was 261.11mg/g and a standard deviation of 4.332. as can be seen from table 2, a glycation index of 0.01225<0.250 belongs to the low potential region, the reducing sugar yield was predicted to be <400mg/g, and the actual sugar yield was 261.11mg/g, which proved to be accurately predicted, and was low in potential sugar yield and was not suitable for practical use.
Example 2
The lignin content of the sample 2 (cornstalk) is 0.125, and the standard deviation is 0.005; the cellulose content was 0.485 and the standard deviation was 0.004, the glycation index was calculated to be 0.2847, the reducing sugar assay procedure was the same as in example 1, and three replicates showed a reducing sugar yield of 430.76mg/g and a standard deviation of 8.182. As can be seen from Table 2, the saccharification index of 0.650>0.2847>0.250 belongs to a middle potential region, the predicted value of the reducing sugar yield is 400-800 mg/g, the actual sugar yield is 430.76mg/g, the prediction is accurate, and the potential sugar yield of the sample is moderate.
Example 3
The content of lignin measured by the sample 3 (corn stalk) is 0.015, and the standard deviation is 0.001; the cellulose content was 0.728, the standard deviation was 0.008, the glycation index was 0.88481, the reducing sugar determination procedure was the same as in example 1, and after three replicates, the reducing sugar yield was 924.56mg/g, and the standard deviation was 7.541. As can be seen from Table 2, the saccharification index 0.88481 is more than 0.650, the yield of reducing sugar is more than 800mg/g, the actual yield of sugar is 924.56mg/g, the prediction is accurate, and the potential yield of sugar of the sample is high.
Example 4
The lignin content of the sample 4 (rice straw) is 0.232, and the standard deviation is 0.005; the cellulose content was 0.418 and the standard deviation was 0.006, the glycation index was calculated to be-0.08394, the reducing sugar assay procedure was the same as in example 1, and three replicates were tested to determine a reducing sugar yield of 243.26mg/g and a standard deviation of 4.324. As can be seen from Table 2, the saccharification index-0.08394 <0.250 belongs to a low potential region, the predicted value of the reducing sugar yield is <400mg/g, the actual sugar yield is 243.26mg/g, the prediction is proved to be accurate, and the sample has low potential sugar yield and is not suitable for practical application.
Example 5
The lignin content of the sample 5 (rice straw) is 0.095 by measurement, and the standard deviation is 0.001; the cellulose content was 0.585 with a standard deviation of 0.004, the glycation index was calculated to be 0.4912, the reducing sugar assay procedure was the same as in example 1, and three replicates showed a reducing sugar yield of 530.26mg/g with a standard deviation of 6.982. As can be seen from Table 2, the saccharification index of 0.650>0.4912>0.250 belongs to a middle potential region, the predicted value of the reducing sugar yield is 400-800 mg/g, the actual sugar yield is 530.26mg/g, the prediction is accurate, and the potential sugar yield of the sample is moderate.
Example 6
The lignin content of the sample 6 (rice straw) is 0.032, and the standard deviation is 0.001; the cellulose content was 0.638 and the standard deviation was 0.007, the glycation index was calculated to be 0.72546, the reducing sugar assay procedure was the same as in example 1, and after three replicates, the reducing sugar yield was 828.56mg/g and the standard deviation was 9.867. As can be seen from Table 2, the saccharification index 0.72546 is more than 0.650, the yield of reducing sugar is more than 800mg/g, the actual yield of sugar is 828.56mg/g, the prediction is accurate, and the potential yield of sugar of the sample is high.
Example 7
The lignin content of the sample 7 (wheat straw) is measured to be 0.198, and the standard deviation is 0.009; the cellulose content was 0.458, the standard deviation was 0.006, the glycation index was calculated to be 0.05696, the reducing sugar assay procedure was the same as in example 1, and three replicates showed a reducing sugar yield of 285.26mg/g and a standard deviation of 4.324. As can be seen from Table 2, the saccharification index 0.05696<0.250 belongs to a low potential region, the predicted value of the reducing sugar yield is <400mg/g, the actual sugar yield is 285.26mg/g, the prediction is proved to be accurate, and the sample has low potential sugar yield and is not suitable for practical application.
Example 8
The lignin content of a sample 8 (wheat straw) is measured to be 0.137, and the standard deviation is 0.006; the cellulose content was 0.545 with a standard deviation of 0.004, the glycation index was calculated to be 0.3291, the reducing sugar assay procedure was the same as in example 1, and three replicates showed a reducing sugar yield of 498.66mg/g with a standard deviation of 8.282. As can be seen from Table 2, the saccharification index of 0.650>0.3291>0.250 belongs to a middle potential region, the predicted value of the reducing sugar yield is 400-800 mg/g, the actual sugar yield is 498.66mg/g, the prediction is accurate, and the potential sugar yield of the sample is moderate.
Example 9
The lignin content of the sample 9 (wheat straw) is measured to be 0.298, and the standard deviation is 0.009; the cellulose content was 0.388 and the standard deviation was 0.006, the glycation index was calculated to be-0.29694, the reducing sugar assay procedure was the same as in example 1, and three replicates showed a reducing sugar yield of 182.26mg/g and a standard deviation of 4.324. As can be seen from Table 2, the saccharification index-0.29694 <0.250 belongs to a low potential region, the predicted value of the reducing sugar yield is <400mg/g, the actual sugar yield is 182.26mg/g, the prediction is proved to be accurate, and the sample has low potential sugar yield and is not suitable for practical application.
Claims (2)
1. A pre-evaluation method for lignocellulose enzymolysis sugar production capacity is characterized in that lignocellulose enzymolysis sugar production capacity is estimated by determining a saccharification index of lignocellulose, wherein the relation between the saccharification index and the lignocellulose enzymolysis sugar production capacity is that the saccharification index is less than 0.25, the estimated reducing sugar yield is less than 400mg/g, the saccharification index is more than 0.25 and less than 0.65, the estimated reducing sugar yield is 400-800 mg/g, the saccharification index is more than 0.65, the estimated reducing sugar yield is more than 800mg/g, and the saccharification index is 1.27 × cellulose content-2.65 × lignin content;
the lignocellulose sample is derived from one or more of corn stalk, rice stalk and wheat stalk;
the cellulose content is represented by α -cellulose content;
dissolving sodium hypochlorite with the mass concentration not lower than 1.0% (w/v) in dilute acetic acid with the volume fraction not lower than 8% (v/v), mixing powdered and dried biomass with the mass m1 and a mixed solution according to the solid-to-liquid ratio not lower than 1:50, sealing and reacting at 70-80 ℃ for at least 1 hour, filtering the residue, cleaning the residue with acetone and deionized water, drying in vacuum for at least 24 hours to obtain holocellulose with the mass m2, taking part of holocellulose with the mass m3, adding sodium hydroxide solution with the mass concentration not lower than 17.5% (w/v) according to the solid-to-liquid ratio not lower than 1:20, stirring and reacting for at least 40 minutes under the same volume at room temperature, adding deionized water to continue reacting for 3-10 minutes, filtering the residue, adding dilute acetic acid with the volume fraction not lower than 1:30 to obtain the cellulose content 35 4, further filtering the residue, and washing the cellulose content 355634 m/368626 to obtain the cellulose content 355636 m;
the lignin content is represented by clarsen lignin content;
the lignin content test mode is that powdery and dried biomass is added into concentrated sulfuric acid with the mass fraction not lower than 70% (w/w) according to the solid-to-liquid ratio not lower than 1:15, the mixture is stirred and reacted for at least 3 hours at 10-30 ℃, then deionized water with the volume 30-45 times that of the concentrated sulfuric acid is added for boiling reflux reaction for at least 3 hours, residues are washed to be neutral by using boiling water after being filtered, the cleaned Clarsen lignin is dried in vacuum, the weight of the cleaned Clarsen lignin is accurately weighed, and the mass ratio of the cleaned Clarsen lignin to the original biomass is.
2. The method of claim 1, wherein the enzymatic reducing sugar yield is 150.606+999.817 × saccharification index.
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| CN105424539A (en) * | 2015-11-06 | 2016-03-23 | 中国科学院天津工业生物技术研究所 | Neural network-based method for predicting sugar yield produced through corn straw hydrolysis |
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