Method for quickly judging removal end point of inhibitor in lignocellulose hydrolysate on line

Abstract

The invention belongs to the field of biological energy sources and biological refinery, and discloses a method for quickly judging a removal end point of an inhibitor in lignocellulose hydrolysate on line, which comprises the following steps: pretreating a lignocellulose biomass raw material, and preparing hydrolysate containing fermentable monosaccharide and the inhibitor by enzymatic saccharification; preparing a biological detoxification microbial strain seed solution; a microbial degradation and removal process of the inhibitor; and detecting the pH value of the hydrolysate on line in the inhibitor degradation process, and ending ventilation. The method provided by the invention is simple, feasible, rapid and accurate, and the inhibitor removal end point is judged by monitoring the pH change of the lignocellulose hydrolysate on line. The method has the advantage that the removal end point of the inhibitor in the lignocellulose hydrolysate can be accurately judged by detecting the time point when the pH value of the hydrolysate reaches the peak value on line. The method is rapid, simple and convenient, and has wide industrial production application potential.

Description

Technical Field

[0001] The invention belongs to the field of bioenergy and biorefining, and particularly relates to a method for online rapid determination of an inhibitor removal endpoint in a lignocellulose hydrolyzate. Background Art

[0002] After pretreatment, enzymatic saccharification, lignocellulose raw materials can obtain a hydrolyzate containing fermentable sugars and inhibitors. In order to prevent the subsequent biotransformation from threatening the vitality of fermenting microorganisms, it is necessary to remove the inhibitors in the hydrolyzate. The biodegradation method has less environmental impact and lower cost, and is a favorable way to properly remove these fermentation inhibitors. However, in the process of inhibitor removal, as the inhibitors are degraded, the growth of the detoxified strain gradually becomes vigorous. If the detoxification process is not stopped in time, it is very easy to cause a large amount of fermentable sugars to be lost, reducing the subsequent biotransformation yield. For example, Zhang Jianan et al. (CN105420291A) of Tsinghua University found that when using a sticky red yeast to remove inhibitors from corn cob hydrolyzate: when the 5-hydroxymethylfurfural and furfural in the hydrolyzate were completely removed and 2.1g / L acetic acid was retained, only 6% of glucose and 8% of xylose were lost; however, further cultivation lost 100% of glucose and 73% of xylose. Therefore, it is necessary to quickly and accurately determine the endpoint of the entire inhibitor removal process to prevent the loss of fermentable sugars.

[0003] Some patents point out that it is possible to determine whether the removal of inhibitors is complete by offline detection of whether the main inhibitors remain. For example, Bao Jie et al. (CN105734087A) from East China University of Science and Technology disclosed a method for rapid biodegradation of inhibitors from lignocellulose. During the inhibitor biodegradation process, they sampled the pretreated materials containing inhibitors and tested the inhibitor content, and used the complete degradation of one or several inhibitors with the highest toxicity and content in the lignocellulose pretreated raw materials (i.e., the presence of these inhibitors could not be detected) as a sign of judging the completion of inhibitor biodegradation.

[0004] However, the above method of judging the inhibitor removal endpoint by offline detection of the residual concentration of the inhibitor is quite time-consuming and laborious, requiring not only frequent sampling, sample preparation and sample testing, but also the sample testing process inevitably requires the use of some expensive analytical equipment to accurately determine the residual concentration of the inhibitor, such as high-performance liquid chromatography or gas chromatography-mass spectrometry, etc. In addition, when the inhibitor is removed to a low concentration level, it is necessary to wait for the latest inhibitor test results to appear before judging whether the inhibitor removal process should be stopped. Therefore, the untimely nature of this judgment method is likely to lead to the occurrence of excessive consumption of fermentable sugars. Summary of the invention

[0005] In view of the shortcomings of the current methods for judging the end of inhibitor removal, the present invention provides a method for quickly judging the end point of inhibitor removal in a lignocellulose hydrolysate by online pH observation. That is, during the inhibitor removal period, the pH change of the lignocellulose hydrolysate is observed online. When the pH rises to the highest point and begins to decline, it is considered that the inhibitor removal is completed. At this time, the hydrolysate from which the inhibitor removal is completed can be used for the subsequent bioconversion process. The method for judging the end point of inhibitor removal is simple and feasible. It does not require the use of any high-precision and expensive analytical equipment. The end point of inhibitor removal in the lignocellulose hydrolysate can be quickly and accurately judged only by online monitoring of the pH change of the hydrolysate. Therefore, the problem of excessive consumption of fermentable sugars caused by untimely judgment of the removal end point is successfully avoided, and it has the potential for industrial production application.

[0006] In order to achieve the purpose of quickly and accurately judging whether the inhibitor in the lignocellulose hydrolysate has been completely removed, the present invention is achieved through the following technical solutions:

[0007] A method for quickly determining the end point of inhibitor removal in lignocellulose hydrolysate online, comprising the following specific steps:

[0008] (1) Pretreatment and enzymatic saccharification of lignocellulosic biomass raw materials to prepare a hydrolyzate containing fermentable monosaccharides and inhibitors;

[0009] (2) Preparation of seed solution of biologically detoxified microbial strains;

[0010] (3) inoculating the hydrolyzate obtained in step (1) with the seed solution of the detoxified microbial strain obtained in step (2), and introducing air to maintain a certain dissolved oxygen level, thereby initiating a microbial degradation and removal process of the inhibitor;

[0011] (4) Online detection of the pH of the hydrolyzate during the inhibitor degradation process and observation of its changes. When the pH rises to a peak value and a downward inflection point appears, it is considered the end point of the inhibitor removal operation;

[0012] (5) After the ventilation is terminated, the corresponding target product fermentation strain is immediately inoculated to start the subsequent fermentation process to produce bio-liquid fuels or bio-based chemicals such as ethanol, chiral lactic acid, microbial lipids, sugar acids, etc.

[0013] In the present invention, the pretreatment method of the lignocellulosic biomass raw material in step (1) includes but is not limited to acid pretreatment, alkali pretreatment, steam expansion pretreatment, ammonia fiber expansion pretreatment, etc.;

[0014] In the present invention, the lignocellulose hydrolysate in step (1) has an initial pH of 4.0 to 5.5 and a solid content of 10% (w / w) to 35% (w / w), and is prepared by cellulase hydrolysis, wherein the enzyme dosage is 0.5 to 10 mg enzyme protein / g dry material.

[0015] In the present invention, the biological detoxification strain used in step (2) is Paecilomyces variotii FN89 (CGMCC17665). The strain seed liquid is obtained by expanding the spores of Paecilomyces variotii FN89 in a certain amount of synthetic culture medium.

[0016] In the present invention, the inhibitor removal process described in step (3) does not require adjusting the pH of the system.

[0017] In the present invention, the pH of the system described in step (4) will change regularly, that is, the initial system pH (4.0-5.5) gradually begins to rise after the inhibitor biodegradation strain is inoculated, reaches the highest point (5.5-7.5), and then begins to decline. The time point when the pH reaches the highest point is the end point of inhibitor removal.

[0018] In the present invention, the inhibitor removal endpoint is reached when the system pH in step (4) reaches a peak value, furfural and 5-hydroxymethylfurfural are not detected in the hydrolyzate, the acetic acid concentration is lower than 2 g / L, and the fermentable sugar consumption from the inoculation of the biological detoxification strain to the inhibitor removal endpoint is 1% to 10%.

[0019] Further, step (3) and step (4) are specifically as follows: the seed solution of step (2) is inoculated into the hydrolyzate obtained in step (1) at an inoculation amount of 10-20% (v / w). o C, 750rpm, 0.5-1.5vvm sterile air culture for 16-24h, and no pH adjustment or control was made during the whole process. After 12.5-20.5 hours, the pH of the straw hydrolyzate increased from the initial 4.0-5.5 to the highest point (5.5-7.5) and began to decrease.

[0020] In the present invention, the subsequent bio-based chemical production process described in step (5) includes but is not limited to all bioconversion processes using lignocellulosic biomass as raw materials, such as ethanol, chiral lactic acid, microbial lipids, and sugar acids.

[0021] The main innovation of the present invention is to determine the inhibitor removal endpoint (for example: the detoxification endpoint of Paecilomyces variotii FN89) in the lignocellulose hydrolysate by pH change.

[0022] In the process of lignocellulose biorefining, the pretreated lignocellulose raw materials are enzymatically saccharified to obtain a hydrolyzate rich in fermentable monosaccharides such as glucose, xylose, arabinose, mannose and galactose. The hydrolyzate contains a large number of inhibitors such as furfural, 5-hydroxymethylfurfural, acetic acid, etc., which seriously affect the subsequent fermentation process. The inhibitors are fully biodegraded by specific biological detoxification microorganisms, which is conducive to the subsequent fermentation strains to efficiently ferment and produce bio-based chemicals such as ethanol, chiral lactic acid, sugar acid, etc. The biological detoxification microorganisms will not consume fermentable monosaccharides before the inhibitors are completely degraded, but will begin to consume fermentable monosaccharides after the inhibitors are completely degraded. Therefore, accurately judging the end point of inhibitor removal can avoid the problem of excessive consumption of fermentable sugars by biological detoxification microorganisms.

[0023] Compared with the existing method for determining the endpoint of inhibitor removal of lignocellulose pretreated materials or their hydrolyzates, the present invention has the following beneficial effects:

[0024] (1) During the inhibitor removal period, there is no need to frequently take samples, prepare samples, and test samples to monitor the degradation degree of inhibitors offline, which greatly reduces the workload and time cost;

[0025] (2) During the inhibitor removal process, there is no need to use expensive analytical equipment such as HPLC and GC-MS to accurately detect the residual concentration of inhibitors in the system;

[0026] (3) During the removal of inhibitors, the change in the pH of the system can be used to accurately determine whether the removal of inhibitors has been completed. This is very simple and quick.

[0027] (4) During the inhibitor removal period, the removal endpoint can be quickly and accurately determined by online monitoring of the pH change of the system, which can greatly avoid excessive consumption of fermentable sugars by inhibitor biodegrading microorganisms.

[0028] The biological detoxification strain used is Paecilomyces variotii FN89, which is classified and named Paecilomyces variotii FN89, deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration, with a deposit number of CGMCC17665 and a deposit date of May 8, 2019. Specific implementation plan

[0029] The following examples are provided for a better understanding of the present invention and are not intended to limit the present invention. The experimental methods in the following implementation methods are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0030] The present invention is further described in detail below in conjunction with examples, but the implementation mode of the present invention is not limited to the scope represented by the examples.

[0031] Embodiment 1:

[0032] In this embodiment, a seed solution of Paecilomyces variotii FN89 with good activity was prepared at an inoculation amount of 20% (v / v), and the inhibitors were removed from a straw hydrolyzate (solid content 30% (w / w)) containing inhibitors. The changes in the system pH and the inhibitor concentration were used to determine whether the inhibitors in the hydrolyzate had been completely removed.

[0033] (1) Preparation of wheat straw hydrolysate

[0034] The pH of wheat straw pretreated with sulfuric acid and steam was adjusted to about 5.5, and the solid content was 30% (w / w) and enzymatic saccharification was performed for 12 hours, and the amount of cellulase Cellic CTec2 (purchased from Novozymes (China) Biotechnology Co., Ltd.) added was 4 mg enzyme protein / g dry material.

[0035] (2) Preparation of biological detoxification microbial strain seed solution

[0036] The spores of Paecilomyces variotii FN89 were inoculated into the synthetic medium at a 10% (v / v) inoculum volume. o C. Aerobic culture at 300 rpm for 20 hours to obtain the seed solution required for inhibitor removal. The synthetic culture medium used was 20 g / L glucose, 1 g / L ammonium sulfate, 2 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate, 1 g / L yeast extract, and 0.5 g / L calcium chloride.

[0037] (3) Rapid removal of inhibitors from straw hydrolysate and determination of the removal endpoint

[0038] The seed solution from step (2) was inoculated into the hydrolyzate at a 20% (v / w) inoculation rate. o C, 750rpm, 1.5vvm sterile air culture for 16h, the whole process was detected by pH meter (D0912-0016, Suzhou Hanxing Analytical Sensor Co., Ltd.) online pH hydrolyzate, and the process did not make any adjustment and control of pH. After 12.5 hours, the pH of the straw hydrolyzate rose from the initial 4.64 to the highest point (6.82) and began to decline. HPLC detection showed that the concentrations of acetic acid, furfural, and 5-hydroxymethylfurfural were 0.19g / L, 0.00g / L, and 0.00g / L, respectively, and the concentrations of glucose and xylose were 82.93g / L and 35.32g / L, respectively (the material will continue to enzymolysis during the inhibitor removal process, resulting in an increase in sugar concentration). In addition, if the inhibitor removal process is not terminated for 12.5h of detoxification, obvious fermentable sugar consumption (especially glucose) can be observed. The results are shown in Table 1.

[0039] Table 1. Changes in pH and concentrations of major inhibitors in straw hydrolysate during inhibitor removal

[0040]

[0041]

[0042] Embodiment 2:

[0043] In this embodiment, a seed solution of Paecilomyces variotii FN89 with good activity was prepared at an inoculation amount of 10% (v / v), and the inhibitors were removed from a straw hydrolyzate (solid content 30% (w / w)) containing inhibitors. The changes in the system pH and the inhibitor concentration were used to determine whether the inhibitor removal in the hydrolyzate was complete.

[0044] (1) Preparation of wheat straw hydrolysate

[0045] Proceed according to step (1) of Example 1.

[0046] (2) Preparation of seed solution

[0047] Proceed according to step (1) of Example 1.

[0048] (3) Rapid removal of inhibitors from straw hydrolysate and determination of the end of removal

[0049] The seed solution was inoculated into the hydrolyzate at a 10% (v / w) inoculation rate. o C, 750rpm, 0.5vvm sterile air culture for 24h, the whole process does not make any adjustment and control to pH. After 20.5 hours, the pH of the straw hydrolyzate rose from the initial 4.51 to the highest point (6.72) and began to decline. HPLC detection shows that the concentrations of acetic acid, furfural, and 5-hydroxymethylfurfural are 1.69g / L, 0.00g / L, and 0.00g / L, respectively, and the concentrations of glucose and xylose are 102.19g / L and 42.80g / L, respectively (the material will continue to be enzymatically hydrolyzed during the inhibitor removal process, resulting in an increase in sugar concentration). In addition, if the inhibitor removal process is not terminated for 20.5h of detoxification, obvious fermentable sugar consumption can be observed. The results are shown in Table 2.

[0050] Table 2. Changes in pH and concentrations of major inhibitors in straw hydrolysate during inhibitor removal

[0051]

[0052]

[0053] Embodiment 3:

[0054] In this embodiment, an inactivated Paecilomyces variotii FN89 seed solution was prepared at a 10% (v / v) inoculation amount to remove inhibitors from straw hydrolyzate (solid content 30% (w / w)) containing inhibitors, and changes in the system pH and inhibitor concentration were used to determine whether the removal of inhibitors in the hydrolyzate was complete.

[0055] (1) Preparation of wheat straw hydrolysate

[0056] Proceed according to step (1) of Example 1.

[0057] (2) Preparation of seed solution

[0058] The procedure was carried out in accordance with step (2) of Example 1, except that the seed solution after aerobic culture for 20 h was subjected to 121 o C. After 20 minutes of high-temperature sterilization, the final product is an inactivated seed liquid with no inhibitor removal ability.

[0059] (3) Rapid removal of inhibitors from straw hydrolysate and determination of the end of removal

[0060] The inactivated seed solution that had been sterilized by high temperature was inoculated into the hydrolyzate at an inoculation rate of 10% (v / w). o C, 750rpm, 0.5vvm sterile air culture for 24h, the whole process does not make any adjustment and control to pH. After 20.5 hours, the pH of wheat straw hydrolyzate hardly changes. HPLC detection shows that the concentrations of acetic acid, furfural and 5-hydroxymethylfurfural are 7.44g / L, 0.12g / L and 1.13g / L respectively (acetic acid and furfural are reduced because of their volatility). The results are shown in Table 3.

[0061] Table 3. Changes in pH and concentrations of major inhibitors in straw hydrolysate during inhibitor removal

[0062]

[0063]

[0064] Embodiment 4:

[0065] In this embodiment, a seed solution of Paecilomyces variotii FN89 with good activity was prepared at an inoculation rate of 10% (v / v) to remove inhibitors from a straw hydrolyzate (solid content 30% (w / w)) containing inhibitors, and the changes in the system pH and inhibitor concentration were used to determine whether the removal of inhibitors in the hydrolyzate was complete, and L-lactic acid fermentation was performed on the hydrolyzate when the removal was complete.

[0066] (1) Preparation of wheat straw hydrolysate

[0067] Proceed according to step (1) of Example 1.

[0068] (2) Preparation of seed solution

[0069] Proceed according to step (2) of Example 1.

[0070] (3) Rapid removal of inhibitors from straw hydrolysate and determination of the end of removal

[0071] The seed solution was inoculated into the hydrolyzate at a 10% (v / w) inoculation rate. o C, 750rpm, 1.0vvm sterile air culture for 18h, and no pH adjustment or control was made during the whole process. After 18 hours, the pH of the hydrolyzate rose from the initial 4.61 to the highest point (6.85) and began to decline. HPLC detection showed that the concentrations of acetic acid, furfural, and 5-hydroxymethylfurfural were 0.55g / L, 0.00g / L, and 0.00g / L, respectively. The results are shown in Table 4.1.

[0072] (4) L-lactic acid fermentation

[0073] The hydrolyzate from which the inhibitors were removed in step (3) of this example was used for L-lactic acid fermentation. The fermentation strain was Pediococcus acidilacticiZY271 (strain collection number: CGMCC#13611). The L-lactic acid yield after 96 hours of fermentation reached 112.84 g / L. The results are shown in Table 4.2.

[0074] Table 4.1 Inhibitor removal results and pH changes before L-lactic acid fermentation

[0075]

[0076] Table 4.2 L-lactic acid fermentation results

[0077]

[0078]

[0079] The above examples prove that the method for judging the end of inhibitor removal in lignocellulose hydrolyzate can not only judge the inhibitor removal end point simply, quickly and accurately, but also eliminates the need for frequent sampling, sample preparation and sample testing to monitor the degradation degree of inhibitors offline, thereby greatly reducing the workload and time cost. In addition, the use of this method only requires the use of a pH meter, without the need for expensive analytical equipment such as HPLC and GC-MS. This technology effectively improves the efficiency of judging the end point of inhibitor removal in the inhibitor removal stage, greatly avoids the problem of fermentable sugar consumption caused by untimely and inaccurate judgment of the inhibitor removal end point, and is beneficial to the subsequent bioconversion process.

[0080] The foregoing descriptions of specific exemplary embodiments of the present invention are for purposes of illustration and illustration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is apparent that many changes and variations are possible in light of the above teachings. The exemplary embodiments are selected and described for the purpose of explaining the specific principles of the present invention and its practical application, thereby enabling those skilled in the art to implement and utilize various exemplary embodiments of the present invention and various selections and variations. The scope of the present invention is intended to be defined by the claims and their equivalents.

Claims

1. A method for quickly determining the end point of inhibitor removal in a lignocellulose hydrolysate online, characterized in that: The specific steps are: (1) Pretreatment and enzymatic saccharification of lignocellulosic biomass raw materials to prepare a hydrolyzate containing fermentable monosaccharides and inhibitors; (2) Preparation of seed solution of biologically detoxified microbial strains; (3) inoculating the hydrolyzate obtained in step (1) with the seed solution of the detoxified microbial strain obtained in step (2), and introducing air to maintain a certain dissolved oxygen level, thereby initiating a microbial degradation and removal process of the inhibitor; (4) Online detection of the pH of the hydrolyzate during the inhibitor degradation process and observation of its changes. When the pH rises to a peak value and a downward inflection point appears, it is considered the end point of the inhibitor removal operation; (5) After the ventilation is terminated, the corresponding target product fermentation strain is immediately inoculated to start the subsequent fermentation process to produce bio-liquid fuels or bio-based chemicals such as ethanol, chiral lactic acid, microbial lipids, sugar acids, etc.

2. The method for online rapid determination of the end point of inhibitor removal in lignocellulose hydrolysate according to claim 1, characterized in that: In the step (1), the pretreatment methods of the lignocellulosic biomass raw materials used include but are not limited to acid pretreatment, alkali pretreatment, steam explosion pretreatment, ammonia fiber explosion pretreatment, etc.

3. The method for online determination of the end point of inhibitor removal in lignocellulose hydrolyzate according to claim 1, characterized in that: In the step (1), the initial pH of the prepared lignocellulose hydrolysate is 4.0-5.5, the solid content is 10% (w / w)-35% (w / w), and it is prepared by cellulase hydrolysis, wherein the enzyme dosage is 0.5-10 mg enzyme protein / g dry material.

4. The method for online rapid determination of the end point of inhibitor removal in lignocellulose hydrolyzate according to claim 1, characterized in that: In the step (2), the biological detoxification strains used are Paecilomyces variotii FN89, CGMCC17665.

5. The method for online rapid determination of the end point of inhibitor removal in lignocellulose hydrolyzate according to claim 1, characterized in that: In step (3), the entire inhibitor removal process does not require adjustment of the system pH.

6. The method for online rapid determination of the end point of inhibitor removal in lignocellulose hydrolyzate according to claim 1, characterized in that: In step (4), the system pH will change regularly, that is, the initial system pH of 4.0-5.5 gradually begins to rise after the inoculation of the inhibitor biodegradation strain, reaches the highest point of pH 5.5-7.5 and then begins to decline. The time point when the pH reaches the highest point is the end point of inhibitor removal.

7. The method for online rapid determination of the end point of inhibitor removal in lignocellulose hydrolysate according to claim 1, characterized in that: At the inhibitor removal endpoint where the pH reaches the highest point, furfural and 5-hydroxymethylfurfural are undetectable in the hydrolyzate, the acetic acid concentration is lower than 2 g / L, and the fermentable sugar consumption from the inoculation of the biological detoxification strain to the inhibitor removal endpoint is 1% to 10%.

8. The method for online rapid determination of the end point of inhibitor removal in lignocellulose hydrolyzate according to claim 1, characterized in that: In step (5), the subsequent bio-based chemical production process includes but is not limited to all bioconversion processes using lignocellulosic biomass as raw materials, such as ethanol, chiral lactic acid, microbial oils, sugar acids, etc.

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