KR101544188B1 - Recovery and reuse of organic acids as catalysts used in biomass pretreatment process - Google Patents

Abstract

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an organic acid catalyst recovery and reuse technology used in biomass pretreatment. More specifically, the present invention relates to a biomass pretreatment using an organic acid and recovering the used organic acid by electrodialysis and using the recovered organic acid for biomass pretreatment Technology.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention [0001] The present invention relates to an organic acid catalyst used in a biomass pretreatment,

The present invention relates to an organic acid catalyst recovery and reuse technique, and more particularly to an organic acid catalyst recovery and reuse technique used in biomass pretreatment.

With the depletion of fossil fuels due to the progress of industrialization in the world, deepening instability of the energy market caused by high oil prices, and increasing environmental pollution, the need for renewable and alternative energy is increasing. As part of the development of alternative energy, bio-ethanol production technology from biomass is attracting attention. Biomass refers to a carbon assimilation process for fixing carbon dioxide using sunlight, that is, a saccharide biosynthesized through a photosynthesis process, and a whole organism including the same. The production and export of bioethanol is led by the United States and Brazil, using corn-derived starch as a raw material and fermenting sugarcane-derived sugar to produce ethanol. However, since corn and sugarcane are major food resources, they are used to produce bioethanol, which is a major cause of rising grain prices. Therefore, bio-ethanol production technology derived from lignocellulosic biomass can be proposed as an alternative to overcome the drawbacks of grain-derived bioethanol.

Lignocellulosic biomass, also called lignocellulosic biomass, commonly includes waste wood, rice straw, and plant scum (grass, weeds, waste vegetables, etc.) and is not competing with food resources because it is organic waste. It is easy to secure the raw material in the apparatus and can be regenerated. Lignocellulose is a complex of lignin, a refractory aromatic polymer, and carbohydrates, cellulose and hemicellulose (Perlack et al., Industrial Bioprocessing, 27 (5): 2, 2005). In order to produce ethanol using lignocellulose as a raw material, pretreatment of lignocellulose to convert the polysaccharide constituting lignocellulose to a fermentable sugar (GLUCOSE, xylose, etc.) capable of ethanol fermentation pretreatment process. In other words, the purpose of pretreatment is to increase the efficiency of the hydrolysis and fermentation process of cellulose and polysaccharide in the subsequent saccharification process. The pretreatment process removes lignin and hemicellulose partially or loosely bonds with cellulose, effectively extracts cellulose, and partially degrades cellulose to make cellulase more accessible. Effective pretreatment has the following criteria. That is, maximizing the action of enzymes, minimizing the formation of fermentation inhibitors of decomposition products, minimizing energy input, and being cost-effective. In addition to these criteria, several other factors need to be considered. Recovery of high value-added co-products (eg lignin, protein), pretreatment catalysts, catalyst recycling and waste disposal. For pre-treatment of lignocellulosic biomass, mechanical methods, chemical methods, biological methods, and hybrid methods between them are applied.

So far, diluted sulfuric acid pretreatment has been mainly used as a pretreatment method. However, strong acids such as sulfuric acid have various problems such as corrosion of the pretreatment device, difficulty in picking up the acid after treatment, and production of many fermented seafood in the pretreatment process. Recently, organic acids such as oxalic acid have been introduced as acid catalysts to replace sulfuric acid. Oxalic acid in the organic acid catalyst has two carboxyl groups, and these functional groups have a characteristic of selectively decomposing hemicellulose during the pretreatment. Therefore, the pretreatment effect is superior to that of sulfuric acid, and fermentation of low-seafood products can be minimized. In addition, the used organic acid catalyst has an advantage of being easily recovered by using a method such as electrodialysis.

Regarding the recovery of organic acid by electrodialysis, Korean Patent No. 0288001 recycles the microorganism to the fermentation tank using a microfiltration membrane of a fermentation liquid of organic acid, recovering the organic acid through the electrodialysis process of the filtrate from which the microorganism has been removed, The present invention relates to a method for recovering an organic acid used as a reusing medium after recycling to a fermentation process after adding a minimum amount of nutrients to a fermentation waste liquid remaining as a waste after a dialysis process. The prior art documents are similar to those of the present invention in that the organic acid is recovered by being input to the electrodialysis apparatus, but it is a partial technique of the present invention in which the objects and effects of the present invention are different. It will not be a problem.

Korean Patent Publication No. 2010-0072212 also relates to a method for producing a fermentation product from lignocellulosic biomass, a reactor for carrying out the method, and the use of a reactor for producing a fermentation product. The prior art is directed to a method for preparing an organic acid from the lignocellulosic biomass as a fermentation product by pretreating a lignocellulosic biomass with an alkaline solution, while the present invention relates to a method for preparing a bioethanol Oxalic acid, an organic acid, can be reused by electrodialysis.

Japanese Patent Application Laid-Open No. 2010-081917 relates to the recycling of materials used for the recovery of lignin at the same time as efficiently recovering the lignin contained in the total amount of the organisms at low cost. The prior art document is a method for producing a fat or alcohol from a saccharide component obtained by saccharifying cellulose or hemicellulose in a fermentation process for producing a desired substance by a microorganism using a saccharide obtained according to a saccharification process, (Oxalic acid) used in the pretreatment process during the ethanol production process is reused by the electrodialysis device.

Korean Patent Publication No. 2006-0047945 discloses a method for recovering a high purity oxalic acid aqueous solution by separating a dissolved metal component from an oxalic acid etching waste solution treated with an indium containing etched material, Oxalic acid aqueous solution. The prior art is a method of extracting a high purity oxalic acid aqueous solution by extracting all or a part of oxalic acid and separating the dissolved metal components by contacting with an anion exchange resin, whereas the present invention is a method of recovering an organic acid used for a pretreatment And the organic acid is recovered and reused by a process using a dialysis apparatus.

However, the possibility of commercialization of organic acid as a solvent for extracting or dissolving cellulose from woody biomass including the above-mentioned prior art has been studied, but organic acid is very expensive and has a limited industrial application. In the case of oxalic acid organic acid catalysts, the cost is higher than sulfuric acid on the basis of weight, so economical aspects should be considered. Therefore, it is necessary to recover the organic acid by the recovery process such as electrodialysis and to use the recovered catalyst again for pretreatment. The electrodialysis process is an electrochemical process that concentrates ionic materials by using the selective permeability of ions in the ion exchange membrane. The cation exchange membrane and the anion exchange membrane are alternately arranged. The dilution liquid in which the initial input solution is diluted and the ionic substance are concentrated Concentrate. Ionic organic acid catalysts such as oxalic acid are concentrated and recovered under an electric field in an electrochemical process.

The present invention has been made to solve the above-mentioned problems, and it is an object of the present invention to provide a pretreatment process capable of recovering and reusing oxalic acid as an organic acid catalyst, and a method for producing bioethanol containing the same. However, these problems are exemplary and do not limit the scope of the present invention.

According to one aspect of the present invention, there is provided a biomass pretreatment method comprising: a pretreatment step of treating an organic acid to a pulverized biomass; A separation step of dividing the pretreated biomass into a solid fraction and a liquid fraction; And an electrodialysis step of electrodialysis of the liquid fraction and separating the liquid fraction into a recovered organic acid and a hydrolysis fraction. The biomass pretreatment method enables recovery and reuse of organic acid.

In the biomass pretreatment method, an additional pretreatment step of treating the recovered organic acid to the pulverized biomass; Further separating the further pretreated biomass into a solid fraction and a liquid fraction; And an additional electrodialysis step of electrodialysis of the liquid fraction and separating it into a re-circulating organic acid and a hydrolysis fraction, is further provided, wherein a biomass pretreatment method capable of recovering and reusing an organic acid is further provided .

In the pretreatment method for biomass that enables the recovery and reuse of the organic acid, the organic acid addition recovery process may be repeated one to ten times.

In the biomass pretreatment method, the organic acid may be a biomass pretreatment method capable of recovering and reusing an organic acid treated with 5 to 10 parts by weight based on 100 parts by weight of the biomass dry weight.

In the biomass pretreatment method, the pretreatment step may be a biomass pretreatment method which enables the recovery and reuse of the organic acid having a pH of 1 to 4.

In the biomass pretreatment method, the electrodialysis step may be a biomass pretreatment method capable of recovering and reusing organic acids carried out at a voltage of 3 V to 12 V.

In the biomass pretreatment method, the organic acid may be an organic acid such as oxalic acid, acetic acid , malic acid, citric acid, butyric acid, palmitic acid, tartaric acid or glutaric acid.

In the biomass pretreatment method, the biomass may be a lignocellulosic biomass composed of cellulose, hemicellulose, lignin and the like.

In the biomass pretreatment method, the organic acid or the recovered organic acid may be treated in an amount of 5 to 10 parts by weight based on 100 parts by weight of the dried biomass.

In the biomass pretreatment method, the pH of the pretreatment step or the additional pretreatment step may be 1 to 4.

 In the biomass pretreatment method, the electrodialysis step or the additional electrodialysis step may be performed at a voltage of 3 V to 12 V.

According to one aspect of the present invention, there is provided a biomass comprising: a pretreatment step of treating an organic acid to a pulverized biomass; A separation step of dividing the pretreated biomass into a solid fraction and a liquid fraction; An electrodialysis step of electrodialysis of the liquid fraction to separate it into recovered organic acid and hydrolysis fraction; A saccharification step of producing a fermentable sugar by enzymatic hydrolysis of the solid fraction; And a fermentation step of culturing the hydrolyzed fraction, the fermentable sugar or the mixture of the hydrolyzed fraction and the fermentable sugar in an anaerobic condition by inoculating the fermentation broth with ethanol.

The method for producing bioethanol may further include: an additional pretreatment step of treating the recovered organic acid to the pulverized biomass after the electrodialysis step; Further separating the further pretreated biomass into a solid fraction and a liquid fraction; And an additional electrodialysis step of electrodialysis of the liquid fraction and separating it into reac- tive water organic acid and hydrolysis fraction.

According to one embodiment of the present invention as described above, the organic acid catalyst is recovered economically by the electrodialysis method, the bioethanol fermentation efficiency of the separated hydrolyzate after recovery is increased, and the biomass pretreatment organic acid catalyst It is possible to implement a pretreatment process method that enables the recovery and reuse of phosphorous oxalic acid. Of course, the scope of the present invention is not limited by these effects.

FIG. 1 is a block diagram showing a pre-treatment process for recovering and reusing oxalic acid using electrodialysis (ED) according to an embodiment of the present invention.
Figure 2 is a graph showing the effect of electrical potential on conductivity changes in an electrodialysis process of a synthesis solution according to one embodiment of the present invention.
3 is a graph showing the permeation rate of oxalic acid in the electrodialysis process (potential 10 V, pH 2.2) of the synthesis solution according to one embodiment of the present invention.
4 is a graph showing the results of the influence of the pH of the solution on the transport of oxalic acid in the electrodialysis process of the synthesis solution according to an embodiment of the present invention.
FIG. 5 is a graph showing the permeation rate of oxalic acid in the electrodialysis process under the condition of 10 V of the hydrolyzate obtained by pretreating the mushroom waste medium with oxalic acid according to an embodiment of the present invention. FIG.
Figure 6 using the hydrolyzate did not use the mushroom waste after the oxalic acid pre-treatment medium hydrolyzate and electrodialysis with electrodialysis process in the first step, the process according to one embodiment of the present invention by yeast cells (Pichia stipitis) This graph shows consumption of fermentable sugars and production of ethanol.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples. It should be understood, however, that the invention is not limited to the disclosed embodiments and examples, but may be embodied in many different forms and should not be construed as limited to the exemplary embodiments set forth herein. It is provided to fully inform the scope of the invention to the person.

Example 1 Recovery and Reuse in Organic Acid Catalyst Pretreatment Process

In the present invention, the oxalic acid initially used was continuously recovered by electrodialysis and reused while performing the biomass pretreatment process three times in succession. The present invention will be described with reference to Fig. The hydrolyzate (H1) generated after using oxalic acid in the first pretreatment step was obtained. At this time, oxalic acid was recovered and an electrodialysis process was used to treat the hydrolyzate. (EDO1) in which oxalic acid is concentrated in the electrodialysis process and a diluent (EDH1) in which the ionic substance is diluted. For the second pretreatment, the concentration of oxalic acid fraction recovered by electrodialysis was adjusted to maintain the same concentration (0.064 g / g) as the first pretreatment (115).

In the second pretreatment and the third pretreatment, the biomass pretreatment was carried out using oxalic acid recovered from the previous stage, respectively, and the hydrolyzate from which the oxalic acid was removed was collected separately or added to the fermentation process for bioethanol production. In the embodiment of the present invention, the results of three times of reuse are shown. However, in the pretreatment method for biomass which enables the recovery and reuse of the organic acid, the organic acid addition recovery process may be repeated one to ten times.

Example 2 Biomass and Oxalic Acid Pretreatment

Production of Zinnia mushrooms (cauliflower mushroom. Sparassis crispa) and lignocellulosic remaining mushroom waste medium was used as biomass (South Jeolla Province Forest Resources Institute, Republic of Korea), the components are 80% (v / v) Douglas Fir sawdust (Douglas fir sawdust ), 10% (v / v) wheat powder, 10% (v / v) corn powder, 16 Brix oligosaccharides and water. The biomass was pulverized to a size of 40-60 mesh at a J-NCM Wiley factory (Jisco, Korea) and stored at 4 ° C under a moisture content of 10% or less.

Pretreatment was carried out at 130 ° C for 25 minutes by treating 0.064 g of oxalic acid per gram of biomass dry weight, after which the temperature was rapidly increased to 170 ° C. Specifically, a 500 ml cylindrical stainless steel reaction vessel placed in a tumbling digester was charged with biomass and oxalic acid solution, and each vessel was charged with 50 g of biomass on a dry weight basis and a total solids and liquid ratio of 1 : 4 (w / w). Heat was then applied to raise the reaction temperature and the biomass was rotated to maintain contact with the liquid during the pretreatment. After the pretreatment was completed, the liquid fraction (hydrolyzate) was separated from the pretreated biomass by vacuum filtration. The hydrolyzate was separated from the pretreated biomass by vacuum filtration and stored at 4 ° C.

Example 3 Effect of Potential in Electrodialysis Process for Recovery of Oxalic Acid in Synthetic Solution

In the present invention, an experiment was conducted to recover and reuse oxalic acid in the lignocellulosic biomass pretreatment process using electrodialysis. Cell cell structure was assembled into a CJ-S3 electrodialysis stack (Creation Techno, Korea) having a total effective membrane area of 550 cm ² , NEOSEPTA CMX (Astom Corp., Japan) as a cation exchange membrane, anion exchange membrane NEOSEPTA AMX (Astom Corp., Japan) was used.

The effect of solution potential on oxalic acid transport in the electrodialysis process was investigated in a previous study using a synthetic solution consisting of 50 g / L glucose and 15 g / L oxalic acid. 500 ml of sterile distilled water was used as the initial concentrate of the electrodialysis unit and 500 ml of 3% Na ₂ SO ₄ solution was used as an electrode rinse solution in all experimental procedures. The diluent, concentrate and electrode wash solution circulated at a flow rate of 1.56 L / min. The organic fouling was controlled by filtering the hydrolyzate with a 0.45 mu m filter before the first supply. A distilled water wash procedure was performed between runs performed with a base solution (1.0 wt.% NaOH). After the washing, 500 ml of 0.1 M NaCl dilution and 500 ml of 0.05 M NaCl concentrate were used for electrodialysis. The potential was kept constant. The characteristics of the ion exchange membranes used in this Example are shown in Table 1.

Characteristics of ion exchange membranes
AMX ^a

CMX ^b
Electrical Resistance (Ohm cm ² )

2.2

3.2

Water content (g H ₂ O / g dried membrane)

0.32

0.24

Exchange capacity (meq / g dried membrane)

2.3

1.6

Number of counter ions

0.96

0.95

^a commercial cation exchange membrane
^b Commercial anion exchange membrane

Figure 2 shows the change in electrical conductivity with time in diluted solutions according to different potentials. At a potential above 7 V, the electrical conductivity decreased to zero within 50 minutes. Oxalic acid was removed within 30 minutes in the electrodialysis process of the example and was then kept constant. 3 is a graph showing the results of electrochemical transport of oxalic acid at 10 V potential. Table 2 shows the effect of dislocations in the electrodialysis process. The power consumption value increased as the applied potential increased. Oxalic acid was almost completely removed at a potential of 7 V or higher.

Effect of electric potential on electrodialysis of synthetic solutions
pH _ave
(pH change)

Current efficiency of oxalic acid (%)

Transport of oxalic acid in the concentrate (g / h)

Final concentration in concentrate

Glucose (g / L)

Oxalic acid (g / L)

3V

1.6 (1.31.8)

52.3

1.4

2.4

10.5

5V

2.0 (1.62.5)

44

2.8

1.7

11.8

7V

2.1 (1.52.6)

49

5.7

1.3

11.9

10V

2.2 (1.62.7)

48.3

7.7

1.1

12

12V

2.0 (1.42.7)

51

11.6

1.6

12.2

Example 4 Effect of pH on electrodialysis for recovery of oxalic acid in synthetic solution

Under the same conditions as in Example 2 of the present invention, the influence of the pH of the solution in the electrodialysis step was examined at a constant potential of 10 V. The effect of pH of the solution on oxalic acid transport in the electrodialysis process was investigated in a previous study using a synthetic solution consisting of 50 g / L glucose and 15 g / L oxalic acid. In an embodiment of the present invention, pH _ave is the arithmetic mean of the values of the various pH values during the process, and pH _ave 2.2 except for the pH of the dilute solution was adjusted using 5.0 N NaOH. 4 shows the electric transport of oxalic acid depending on the pH value of the different solutions at a constant potential of 10 V. The recovery efficiency of oxalic acid reached 100% in 60 minutes in all processes depending on the pH value of different solutions. As shown in Fig. 4, the removal rate of oxalic acid in the solution of pH _ave 2.2 was much higher than that of the solution having the other pH _ave . In the electrodialysis process shown in Table 3, the conductivity and oxalic acid transport ratio of the solution having pH _ave higher than 4.2 were low due to the presence of oxalic acid in the form of bivalent ions. In addition, the conductivity was low and the high power consumption was high in pH _ave 1.5 solution. This was a result of the neutral form of oxalic acid (Weng et al., Bioresour. Technol., 101: 48894894, 2010; Wang et al., J. Membr. Sci., 385-386, 226-233 , 2011). Since oxalic acid is present in monovalent form at pH _ave 2.2, the current efficiency is much higher, and the highest oxalic acid conductivity and lowest power consumption are observed. The current efficiency and transport ratio of oxalic acid increased as the potential increased.

Effect of pH and electrodialysis on solution of synthetic solution

pH _ave
(pH change) ^a


Current efficiency of oxalic acid (%)


Transport of oxalic acid in the concentrate (g / h)

Final concentration in concentrate

Glucose (g / L)

Oxalic acid (g / L)

1.5 (1.31.8)

19.8

6.7

1.6

10

2.2 (1.62.7)

48.3

7.7

1.1

12

4.2 (4.73.8)

17.2

4.7

1.6

10

5.2 (5.94.5)

17.1

4.4

1.6

10

6.0 (6.75.3)

20.4

5.9

1.6

12.7

It kept constant at ^a potential V 10

Example 5: Electrodialysis process for oxalic acid recovery from oxalic acid pretreated hydrolyzate

In the electrodialysis process for the recovery of oxalic acid, the hydrolyzate produced in the pretreatment process of the mushroom waste medium was used as the initial diluent. As shown in FIG. 5, the concentration of oxalic acid in the hydrolyzate during electrodialysis performed at 10 V potential resulted in the removal of oxalic acid from the diluent within about 30 minutes. Table 4 summarizes the transportation process of oxalic acid and saccharide in the electrodialysis process for the hydrolyzate. Electrodialysis results for oxalic acid-treated hydrolyzate of biomass were as follows. Oxalic acid was recovered at 100% efficiency in the concentrate and the losses of glucose and xylose during electrodialysis were 1.8% and 15.0%, respectively. Oxalic acid-treated hydrolyzate of biomass In the electrodialysis, diluted solution (treatment solution) was hydrolyzed and concentrated solution was distilled water.

Transport of oxalic acid and sugar from electrodialysis to hydrolyzate
Electrodialysis (10 V)

Oxalic acid

Glucose

Xylose

Transport speed to concentrate (g / h)

4.97

0.23

0.11

Removal rate in dilution (g / h)

3.48

0.38

1.11

Removal efficiency (%)

100

1.8

15

Final concentration in concentrate (g / L)

9.84

1.3

0.89

Example 6: Cleaning effect of oxalic acid pretreatment hydrolyzate electrodialysis process

In Example 5 of the present invention, the process performance of the hydrolyzate and the synthesis solution was compared in terms of oxalic acid transport rate and current efficiency. The transport rate of oxalic acid in the hydrolyzate was 3.48 g / h, which corresponds to half of the synthesis solution (7.7 g / h, Table 3). The current efficiency of oxalic acid was also 40.6%, which was somewhat lower than that of 48.3% of the synthesis solution. In the present embodiment, degradation of electrodialysis performance is a phenomenon of contaminant contamination due to deposition of organic matter on the surface of an anion exchange membrane. Membrane contamination of organic matter can be classified as reversible or irreversible depending on the forces of interaction between the foulants and the surface of the ion membrane. Reversible contamination was removed by washing, while irreversible contamination was not removed (Table 3). Chemical washing was carried out between electrodialysis, and desalting was carried out using NaCl solution to investigate the effect of contaminants during electrodialysis. Electrodialysis performance, such as transport rate, current efficiency and power consumption, did not decrease during the NaCl desalination process, which was not even reduced after electrodialysis experiments on hydrolyzate. Even though the electrodialysis performance was slightly reduced by the adsorption of the organic material during the electrodialysis experiment, no significant effect on the electrodialysis performance occurred because the adsorbed organic material was removed by the chemical washing solution due to reversible contamination.

Example 7: Electrodialysis process application Fermentable sugars of hydrolyzate of oxalic acid pretreatment

Table 5 shows the compositional analysis of the hydrolysis at each step. As the preprocessing step using oxalic acid recovered by electrodialysis was repeated, the concentrations of fermentable sugars and fermentation inhibiting substances contained in the hydrolysates (H1, H2, H3, Fig. 1) were slightly increased. This is due to unidentified toxic substances present in the hydrolyzate, and similar results have been reported previously (Cheng et al ., Biochem. Eng. J., 38: 105109, 2008). In the hydrolyzate of each step, the sugar was mostly glucose, whereas xylose was relatively less released from hemicellulose. Unlike mushroom waste media, S. crispa is a brown rot fungi that secretes a variety of hemicellulases during culture. Therefore, most of the waste media from cultivated mushrooms contain cellulose and lignin. (Sato et al., Bioresour Technol., 101: 6006-6011, 2010; Asada et al., Bioresource Technol., 102: 10052-10056, 2011). In addition, the mushroom waste medium contained a large amount of glucan such as mycelium of S. crispa and oligosaccharide, so that high concentration of glucose was detected in the hydrolyzate of each step.

After the hydrolyzate was electrodialyzed to collect oxalic acid, the initial hydrolyzate was separated into oxalic acid fractions (EDH, EDO, Figure 1) and concentrated hydrolyzate. Most of the oxalic acid was collected in the oxalic acid fractions and could be reused in the next pretreatment process by adding oxalic acid to provide the same conditions as the first pretreatment.

Fermentable sugars of hydrolysates produced by pretreatment of oxalic acid in mushroom waste media
ingredient
(Unit: g / L)
H1 ^a

EDH1 ^b

EDO1 ^c

H2

EDH2

EDO2

H3

EDH3

EDO3

Glucose

44.62
(1.31) ^d

42.03
(1.23)

1.46
(0.29)

46.33
(1.26)

40.85
(1.21)

1.51
(0.19)

54.01
(1.18)

50.31
(1.30)

1.75
(0.41)

Xylose

17.56
(1.14)

15.88
(1.09)

0.99
(0.15)

22.89
(1.10)

20.19
(1.20)

1.15
(0.09)

25.61
(1.24)

23.18
(1.08)

0.88
(0.66)

^a Hydrolyzate from first stage oxalic acid pretreatment

^b Hydrolysis fraction obtained by electrodialysis of the first step hydrolyzate

^c The oxalic acid fraction obtained by electrodialysis of the first step hydrolyzate

^d Brackets indicate standard deviation

Example 8: Electrodialysis process application Fermentation inhibitor of oxalic acid pretreated hydrolyzate

Treatment of acids with lignocellulosic biomass results in the production of furfural aldehydes (furfural and 5-hydroxymethyl furfural (HMF)), weak acids (acetic acid, formic acid, levulinic acid) and total phenolic compounds (TPCs) (Almeida et al., Appl. Microbiol. Biotechnol., 82: 625-638, 2009; Mosier et al., Biotechnol. Bioeng., 79: 610-618, 2002). The present inventors analyzed concentrations of formic acid, acetic acid, HMF and furfural, which are fermentation inhibitors in the multistage pretreatment process. Table 6 shows the concentrations of fermentation inhibiting substances in each step. The concentration of formic acid was 1.32-1.51 g / L, the concentration of acetic acid was 1.54-2.05 g / L, and the concentration of HMF was 1.13-2.13 g / L. Removal efficiencies of formic acid and acetic acid, which were fermentation inhibitors formed in oxalic acid pretreatment biomass, were 24-68% and 40-63%, respectively. Furthermore, neutral type HMF and furfural were significantly removed by the electrodialysis (Walton et al, Bioresour.Technol, 101 : 19351940, 2010; Weng et al, Bioresour Technol, 101:..... 48894894, 2010) . The maximum removal efficiency of TPCs from the hydrolyzate is 51%. Removal of the non-ionized hydrophobic inhibitors (HMF, furfural, TPCs) in this embodiment is preferred because the ion exchange membranes are hydrophilic (Lee et al., J. Membr. Sci., 203: 115126, 2002) It is presumed to be related to the rebound reaction of the material and the membrane surface.

Concentrations of fermentable sugars and fermentation inhibitors in the hydrolyzed fractions (EDH1, EDH2, FIG. 1) were somewhat reduced by electrodialysis due to contamination of the inhibitor and diffusion of sugars on the surface of the ion membrane during electrodialysis. In the oxalate fractions (EDO1, EDO2, Fig. 2), some of the sugar and fermentation inhibitors were collected by electrodialysis, similar to those reported previously (Weng et al ., Bioresour. Technol., 101: 48894894, 2010). Considering the above results, it is possible to provide an appropriate condition for ethanol fermentation because the hydrolysis fraction (EDH) by electrodialysis contains a high concentration of fermentable sugar and a low concentration of fermentation inhibitor. Formic acid and acetic acid were removed by electrodialysis using an ion exchange membrane because a large amount of anion was absorbed at pH, whereas low ion removal efficiency was achieved by eliminating ionic HMF, furfural, aldehyde and phenol compounds only by diffusion It looked.

Fermentation inhibitor content of hydrolyzate produced by pretreatment of oxalic acid in mushroom waste medium
ingredient
(Unit: g / L)
H1 ^a

EDH1 ^b

EDO1 ^c

H2

EDH2

EDO2

H3

EDH3

EDO3

Acetic acid

1.54
(0.20) ^d

0.64
(0.10)

0.84
(0.11)

1.93
(0.23)

1.15
(0.18)

1.15
(0.10)

2.05
(0.20)

0.76
(0.58)

0.55
(0.12)

Levulinic acid
2.54
(0.53)

2.35
(0.10)

NA

2.53
(0.25)

2.31
(0.20)

NA

2.32
(0.21)

2.03
(0.11)

NA

HMF

1.13
(0.47)

0.78
(0.11)

NA

1.34
(0.13)

1.12
(0.10)

0.25
(0.05)

2.13
(0.20)

1.62
(0.78)

0.30
(0.10)

Furfural

0.58
(0.21)

NA

NA

0.80
(0.12)

0.63
(0.13)

0.23
(0.05)

0.86
(0.17)

0.47
(0.09)

0.20
(0.07)

TPC

5.07
(0.79)

2.5
(0.42)

NA

3.39
(0.63)

2.0
(0.40)

NA

4.0
(0.48)

2.12
(0.21)

NA

^a Hydrolyzate from first stage oxalic acid pretreatment

^b Hydrolysis fraction obtained by electrodialysis of the first step hydrolyzate

^c The oxalic acid fraction obtained by electrodialysis of the first step hydrolyzate

^d Brackets indicate standard deviation

Example 9: Ethanol production of hydrolyzate

Pichia stipitis yeast strains were used for fermentation of hydrolysates after pretreatment with oxalic acid in the waste mushroom medium. Yeast cells were cultured in a shaking incubator for 24 hours in an Eklenmeyer flask containing 400 ml of YPD (10 g / L yeast extract, 10 g / L peptone and 20 g / L glucose). After cell culture, yeast cells were collected and washed in sterile distilled water. The hydrolyzate was diluted 1: 1 with distilled water, and the hydrolyzate hydrolyzed and the initial hydrolyzate were adjusted to pH 6.0 using calcium hydroxide, and then filtered through a sterilized 0.45 탆 filter to remove calcium hydroxide . Washed cells corresponding to a dry cell weight of 2.0 g / L were added to the hydrolyzate at 5 g / L yeast, 5 g / L urea, 0.5 g / L MgSO ₄ 7H ₂ O and 1 g / L KH ₂ PO ₄ Respectively. The fermentation process was carried out on a stirrer at 150 rpm at 30 ° C for 96 hours. All analyzes were performed in triplicate.

Table 7 shows the fermentation performance in terms of ethanol production and productivity, and the yield of ethanol after fermentation for 72 hours. The fermentation results of the hydrolysates (EDH1) and (EDH2) in Table 7 were compared with those of the untreated hydrolyzate (H1). The effect of removal of inhibitor was investigated by electrodialysis in the fermentation of bioethanol. Ethanol production increased to 19 g / L in electrodialyzed hydrolyzate, while hydrolyzate without electrodialysis was approximately 6 g / L. The productivity and yield of ethanol increased up to the third stage because the fermentation inhibitor was removed through the electrodialysis process (Cheng et al., Biochem. Eng. J., 38: 105109, 2008).

Ethanol yield was very low considering the ethanol yield in the fermentation process without the electrodialysis process. In the initial hydrolyzate, fermentable sugars were not completely consumed by yeast ( P. stipitis ) during the fermentation process. The ethanol yield of the first step hydrolyzate (H1) was 0.10 g / g, the ethanol yield of the second hydrolyzate (H2) was 0.12 g / g, and the third hydrolyzate H3) was 0.12 g / g. However, the ethanol yield of the hydrolysates (EDH1, EDH2, EDH3) at all stages of electrodialysis was about three times (0.30 g / g). Furthermore, electrodialysis increased ethanol production as well as ethanol production by removing fermentation inhibitors. Ethanol production of the first hydrolyzate (H1, H2, H3) is low, while the production of ethanol in the electrodialyzed hydrolyzate (EDH1, EDH2, EDH3) is low due to inhibition of fermentation by electrodialysis It was remarkably increased due to the removal of the material.

Figure 6 shows the consumption of fermentable sugars and the production of ethanol over time. The ethanol concentration obtained after fermentation for 72 hours at the initial hydrolyzate (H1) of 61.89 g / L in the initial fermentable sugars including glucose and xylose was only 6 g / L, and the maximum ethanol concentration obtained after 96 hours fermentation Was 19.66 g / L. The initial low ethanol production rate is due to the effect of the fermentation inhibitor on the hydrolyzate, which reduces yeast growth and respiration (Larsson et al., Enzyme Microbial. Technol., 24 (3-4): 151-159, 1999; Palmqvist et al., Bioresour. Technol., 74 (1): 25-33, 2000). On the other hand, the concentration of the fermentable sugar of the electrodialyzed hydrolyzate (EDH1) dropped sharply during 96 hours of ethanol production, and was almost undetectable after 72 hours. The highest ethanol production after 72 hours was 19.38 g / L. The hydrolyzate hydrolyzate treated with the electrodialysis as the ethanol production corresponding to the fermentation of the hydrolyzate (H1) for 96 hours has the effect of shortening the fermentation time . This value corresponds to an ethanol yield per 0.33 g / g and an ethanol productivity of 0.27 g / L / h. These results indicate that oxalic acid is recovered through electrodialysis during the hydrolysis process, the fermentation inhibitor is reduced, and the efficacy is improved.

Ethanol Fermentation Performance of Electrodialyzed Hydrolyzate
Electrodialysis untreated hydrolyzate
(H1) ^a

Electrodialysis hydrolyzate

(EDHl) ^b

(EDH2) ^c

Ethanol production (g / L)

5.92 (0.32) ^d

19.38 (0.68)

17.07 (0.52)

Ethanol productivity (g / L / h)

0.08 (0.1)

0.27 (0.01)

0.23 (0.01)

Ethanol yield (per g / g)

0.10 (0.01)

0.33 (0.01)

0.29 (0.01)

^a hydrolyzate from oxalic acid pretreatment in the first step

^b hydrolyzate was subjected to electrodialysis to obtain a hydrolyzed fraction

^c Hydrolysis fraction obtained by electrodialysis of the hydrolyzate obtained from the second pretreatment together with the recovered oxalic acid

^d Brackets indicate standard deviation

Example 10: Saccharification of pretreated biomass

The saccharification process (enzyme hydrolysis) was performed in the pretreatment biomass obtained by oxalic acid pretreatment using oxalic acid recovered through electrodialysis. 5 g of the pretreated biomass obtained in each pretreatment step was transferred into a 125 ml Eklenmeyer flask containing 50 ml of 50 mM NaCl (pH 4.8). For the hydrolysis, cellulase (Accellerase 1000, 500 CMC U / g) and β-glucosidase (80 pNPG U / g) purchased from Danisco, USA were added. Enzymatic hydrolysis was carried out on a stirrer for 96 hours at 150 rpm and 50 ° C, and all analyzes were repeated three times. Enzymatic conversion of cellulose to glucose was calculated based on the glucose concentration measured in the enzyme hydrolysis. As shown in Table 8, the conversion of glucan to glucose was in the range of 76.03% to 77.63%. These results indicate that the hydrolysis rate for each pretreated biomass did not change significantly when oxalic acid recovered by electrodialysis was used in the pretreatment process. The enzyme conversion rate for xylans was relatively low when compared to the conversion of cellulose. This may be due to the enzymatic conversion of insufficient zylanase and the resulting low xylan.

Pretreatment evaluation of oxalic acid pretreated with oxalic acid recovered from enzyme hydrolysis
Biomass

Glucose
(g / L) ^a

Conversion of Glucan to Glucose (%)

Xylose (g / L) ^a

Convert from Xylon to Xylose (%)

Biomass obtained from oxalic acid pretreatment (S1) ^c

43.80 (1.21) ^b

76.03

3.20 (0.31)

48.51

Biomass obtained from oxalic acid pretreatment (S2) ^c

43.32 (1.30)

76.77

2.96 (0.33)

51.26

Biomass from oxalic acid pretreatment (S3) ^c

42.72 (1.18)

77.63

2.92 (0.33)

50.77

^a Fermentable sugars from enzyme hydrolysis after 96 hours

^b Brackets indicate standard deviation

^c See Fig.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. I will understand. Accordingly, the true scope of the present invention should be determined by the technical idea of the appended claims.

Claims (13)

In the biomass pretreatment method,
A pretreatment step of treating the pulverized biomass with an organic acid;
A separation step of dividing the pretreated biomass into a solid fraction and a liquid fraction; And
Wherein the liquid fraction is subjected to electrodialysis under conditions of pH 2.1 to 4 and a voltage of 7 to 12 V to convert the organic fraction to a recovered organic acid and a hydrolysis fraction, thereby allowing recovery and reuse of the organic acid. The method according to claim 1,
An additional pretreatment step of treating the recovered organic acid to the pulverized biomass;
Further separating the further pretreated biomass into a solid fraction and a liquid fraction; And
Further comprising an additional electrodialysis step of separating the liquid fraction into a rehydratable organic acid and a hydrolytic fraction by electrodialysis under conditions of pH 2.1-4 and a voltage of 7-12 V, And biomass pretreatment methods that enable reuse. 3. The method of claim 2,
The method of claim 1, wherein the organic acid is recovered and reused from 1 to 10 times. The method according to claim 1,
Wherein the organic acid is treated in an amount of 5 to 10 parts by weight based on 100 parts by weight of the biomass dry weight, whereby the organic acid can be recovered and reused. delete delete 3. The method according to claim 1 or 2,
Wherein the organic acid is capable of recovery and reuse of organic acids such as oxalic acid, acetic acid, malic acid, citric acid, butyric acid, palmitic acid, tartaric acid or glutaric acid. The method according to claim 1 or 2, wherein
Wherein the biomass is a lignocellulosic biomass, which enables the recovery and reuse of the organic acid. 3. The method of claim 2,
Wherein the organic acid or the recovered organic acid is treated in an amount of 5 to 10 parts by weight based on 100 parts by weight of the biomass dry weight, whereby the organic acid can be recovered and reused. delete delete A pretreatment step of treating the pulverized biomass with an organic acid;
A separation step of dividing the pretreated biomass into a solid fraction and a liquid fraction;
An electrodialysis step of subjecting the liquid fraction to electrodialysis under conditions of pH 2.1 to 4 and a voltage of 7 to 12 V to separate the recovered organic acid and the hydrolyzed fraction;
A saccharification step of producing a fermentable sugar by enzymatic hydrolysis of the solid fraction; And a fermentation step of culturing the hydrolyzed fraction, the fermentable sugar or the mixture of the hydrolyzed fraction and the fermentable sugar in an anaerobic condition by inoculating the fermentation broth with ethanol. The method of claim 12, wherein
After the electrodialysis step,
An additional pretreatment step of treating the recovered organic acid to the pulverized biomass;
Further separating the further pretreated biomass into a solid fraction and a liquid fraction; And
Further comprising an additional electrodialysis step of separating the liquid fraction into a rehydratable organic acid and a hydrolyzed fraction by electrodialysis under conditions of pH 2.1 to 4 and a voltage of 7 to 12 V, Way.

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