Methods for pre-treatment of biological waste

Abstract

A method for pretreating biological waste is provided, the method comprising mixing the biological waste with an alkaline solution, an oxidant, and a synthesis catalyst to form pretreated biological waste.

Description

Technical Field

[0001] The invention relates to a method for pre-treating biological waste. Background Art

[0002] Cellulose is the main component of biowaste. However, cellulose is entangled in a cross-linked matrix of lignin and hemicellulose and cannot be easily decomposed. Therefore, various pretreatment steps are required to improve the decomposition of biowaste. Therefore, in biowaste management, pretreatment of biowaste is very important to enhance enzymatic degradability.

[0003] Current biowaste pretreatment methods include thermal treatment, acid treatment, alkaline treatment and oxidant-based treatment. However, thermal treatments such as steam explosion and oxidant-based treatments are energy-intensive because high temperatures are required. In addition, for acid treatments, high concentrations of acid are used, which are corrosive, not environmentally friendly, and produce inhibitory compounds that are harmful to subsequent treatments. On the other hand, alkaline treatment is a slow process that takes several days to weeks.

[0004] There is therefore a need for improved methods for pre-treating biological waste. Summary of the invention

[0005] The present invention seeks to address these problems and / or to provide improved methods for pre-treating biological waste.

[0006] According to a first aspect, the present invention provides a method of pretreating biological waste, the method comprising mixing the biological waste with an alkaline solution, an oxidant and a synthesis catalyst to form pretreated biological waste.

[0007] The biowaste material may be any suitable biowaste material. For example, the biowaste material may comprise lignocellulosic biomass.

[0008] The synthetic catalyst can be any suitable synthetic catalyst. For example, the synthetic catalyst can include a metal complex having a metal ion and surrounded by a synthetic ligand. In particular, the synthetic ligand can include at least one nitrogen. According to a particular aspect, the metal ion included in the synthetic catalyst can be a transition metal ion.

[0009] The mixing can be performed under appropriate conditions. For example, the mixing can be performed at a temperature of 20-100°C.

[0010] According to certain aspects, mixing can include adding biowaste to a solution comprising an alkaline solution, an oxidant, and a synthesis catalyst. A suitable amount of biowaste can be added to the solution. For example, adding can include adding 1-50 wt % of biowaste based on the total weight of the solution. Mixing can be performed at a temperature of 20-80° C. Mixing can be performed for ≤ 24 hours.

[0011] According to another particular aspect, the mixing may include:

[0012] - mixing the biowaste with an alkaline solution at a predetermined temperature for a predetermined period of time to form biowaste solids; and

[0013] - Adding biowaste solids to a solution containing an oxidant and a synthesis catalyst.

[0014] The predetermined time period may be any suitable time period. For example, the predetermined time period may be 1-72 hours. The predetermined temperature may be any suitable temperature. For example, the predetermined temperature may be ≤100° C. In particular, the predetermined temperature may be 20-100° C. Even more particularly, the predetermined temperature may be room temperature.

[0015] The solution may contain suitable amounts of the oxidant and the synthesis catalyst. For example, the solution may contain 0.1-10 vol% of the oxidant based on the total volume of the solution. For example, the solution may contain 0.1-1000 ppm of the synthesis catalyst.

[0016] The method may further include treating the pretreated biowaste. The treating may include any suitable type of treating. In particular, the treating may include fermentation, enzymatic saccharification, or a combination thereof of the pretreated biowaste.

[0017] According to certain aspects, treating the pretreated biogenic waste comprises fermenting the pretreated biogenic waste in the presence of bacteria. The fermentation can be carried out for a suitable period of time. In particular, the fermentation can be carried out for a period of 24-195 hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order that the invention may be fully understood and readily put into practice, exemplary embodiments shall now be described by way of non-limiting example only, with reference to the accompanying drawings. In the drawings:

[0019] Figure 1 A schematic workflow for conducting biowaste pretreatment and subsequent testing is shown;

[0020] Figure 2 Soluble lignin concentrations after pretreatment of biowaste using three different methods are shown; Figure 2 A shows incubation in 1% NaOH for 1 day before pretreatment (experimental group 1); Figure 2 B shows incubation in 1% NaOH for 2 days before pretreatment (experimental group 1); Figure 2 C shows the phenol concentration after pretreatment of biomass with alkaline peroxide (AP) method and catalytic alkaline peroxide (CAP) method;

[0021] Figure 3shows the scanning electron microscope (SEM) images of biomass after different pretreatment methods; Figure 3 A shows the control; Figure 3 B shows alkaline pretreatment (experimental group 1); Figure 3 C shows alkaline peroxide pretreatment (experimental group 1); Figure 3 D shows catalytic alkaline peroxide pretreatment (experimental group 1); Figure 3 E shows alkaline peroxide pretreatment (experimental group 2); Figure 3 F shows catalytic alkaline peroxide pretreatment (experimental group 2);

[0022] Figure 4 The changes after enzymatic saccharification of pretreated biomass are shown; Figure 4 A shows the reduction of biomass (experimental group 1); Figure 4 B shows glucose concentration (experimental group 1); Figure 4 C shows the reducing sugar concentration (experimental group 2);

[0023] Figure 5 Shows the changes in soluble lignin after fermentation using Clostridium sp. G117; Figure 5 A shows the gas chromatography results of the fermentation products of soluble lignin after 24 hours of fermentation; Figure 5 B shows the time course fermentation curves of the three main products, acetone, butanol and butyric acid; Figure 5 C shows the utilization of lignin and glucose by Clostridium G117;

[0024] Figure 6 The total methane produced after methanogenesis using three pretreatment methods for control, alkaline peroxide (AP), and catalytic alkaline peroxide (CAP) is shown;

[0025] Figure 7 Shown are the yields of products from fermentation of pretreated food waste using Clostridium acetobutylicum BOH3;

[0026] Figure 8 shows HPLC analysis of products from oxidation of vanillin using a control, H2O2 alone, and H2O2 with a catalyst; and

[0027] Fig. 9 NMR analysis of the products extracted from the aromatic liquid of the biomass is shown. DETAILED DESCRIPTION

[0028] As explained above, there is a need for improved methods for pre-treating biological waste.

[0029] Generally speaking, the present invention relates to a method for pre-treating biowaste to further decompose the components of the biowaste to increase its biodegradability. The method of the present invention is also an environmentally friendly method, which does not use extreme heat and temperature and also uses less chemicals. The method also does not affect the subsequent treatment of the pre-treated biowaste. In particular, the method of the present invention is energy-saving and environmentally friendly.

[0030] According to a first aspect, the present invention provides a method of pretreating biological waste, the method comprising mixing the biological waste with an alkaline solution, an oxidant and a synthesis catalyst to form pretreated biological waste.

[0031] Biowaste can be any suitable biowaste. For the purposes of the present invention, biowaste can be defined as biodegradable organic waste. Biowaste can include residual biomass, food waste, agricultural waste, waste sludge such as from wastewater treatment plants, etc. According to particular aspects, biowaste can include lignocellulosic biomass. In particular, biowaste can include cellulose, which is a cross-linked matrix of lignin and hemicellulose.

[0032] The synthetic catalyst can be any suitable synthetic catalyst. For example, the synthetic catalyst can include a metal complex having a metal ion and surrounded by a synthetic ligand. The metal ion can be any suitable metal ion. According to a particular aspect, the metal ion included in the synthetic catalyst can be a transition metal ion. In particular, the metal ion can be, but is not limited to, an ion of iron, copper, or an alloy thereof.

[0033] The synthetic ligand may include at least one nitrogen. For example, the synthetic ligand may include, but is not limited to, one or more of the following: diglycyl-glycerol (GGG), histidyl-glycyl-glycerol (HGG), glycyl-glycyl-histidine (GGH), phthalocyanine (Pc), 2,2'-bipyridine (bpy), ethylenediamine-N,N,N',N'-tetraacetic acid (EDTA), trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (Cy-DTA), diethylenetriamine-N,N,N',N",N"- pentaacetic acid (DTPA), N-(2-hydroxyethyl)ethylenediamine-N',N',N'-triacetic acid (EDTA-OH), triethylenetetramine-N,N,N',N",N'",N'"-hexaacetic acid (TTHA), O,O'-di(2-aminoethyl)ethylenediol-N,N,N',N'-tetraacetic acid (GEDTA), ethylenediamine-N,N'-dipropionic acid dihydrochloride (EDDP), tetraamido macrocyclic ligand (TAML), and 2,2'-bipyridine (BPY).

[0034] According to certain aspects, the synthesis catalyst may be, but is not limited to, Cu II -(bpy),CuII -GGG, Cu II -GGH, Cu II -DTPA, Cu II -BPY and Fe II -Pc. In particular, the synthesis catalyst may be iron-tetraamido macrocyclic ligand (Fe-TAML).

[0035] The oxidant can be any suitable oxidant. For example, the oxidant can be, but is not limited to, molecular oxygen, ozone, fluorine, chlorine, perchlorate, hypochlorite, permanganate compounds, hydrogen peroxide (H2O2), and peroxides such as benzyl peroxide. According to certain aspects, the oxidant can be H2O2.

[0036] The alkaline solution can be any suitable alkaline solution. For example, the pH of the alkaline solution can be 10-14. The alkaline solution can be, but is not limited to, sodium hydroxide, potassium hydroxide, magnesium hydroxide, sodium orthosilicate, sodium metasilicate, sodium carbonate, ammonia, ammonium hydroxide, calcium carbonate, or a combination thereof.

[0037] Mixing can be carried out under suitable conditions. According to certain aspects, mixing can be carried out at a temperature of 20-100° C. For example, mixing can be carried out at a temperature of 22-95° C., 25-90° C., 28-85° C., 30-80° C., 35-75° C., 40-70° C., 42-68° C., 45-65° C., 50-60° C., 55-58° C. In particular, the temperature can be 42-100° C.

[0038] The mixing can be performed by any suitable method. For example, the mixing of the alkaline solution, the oxidant and the synthesis catalyst can be performed simultaneously or sequentially.

[0039] According to a particular aspect, mixing can include mixing simultaneously. For example, mixing can include adding the biowaste to a pretreatment solution comprising an alkaline solution, an oxidant, and a synthesis catalyst. Therefore, the method can further include preparing a pretreatment solution before mixing. Preparing the pretreatment solution can include mixing an appropriate amount of alkaline solution, an oxidant, and a synthesis catalyst. Water can also be added to form the pretreatment solution. The alkaline solution, the oxidant, and the synthesis catalyst can be as described above.

[0040] In particular, the preparation may include mixing 0.1-10 vol% of the alkaline solution based on the total volume of the pretreatment solution to be prepared. For example, the preparation may include mixing 0.5-9.5 vol%, 1.0-9.0 vol%, 1.5-8.5 vol%, 2.0-8.0 vol%, 2.5-7.5 vol%, 3.0-7.0 vol%, 3.5-6.5 vol%, 4.0-6.0 vol%, 4.5-5.5 vol%, 5.0-5.2 vol% of the alkaline solution based on the total volume of the pretreatment solution to be prepared. Even more particularly, the preparation may include mixing 0.5-2.0 vol% of the alkaline solution based on the total volume of the pretreatment solution to be prepared.

[0041] Preparation may include mixing 0.1-10 vol% of the oxidant based on the total volume of the pretreatment solution to be prepared. For example, preparation may include mixing 0.5-9.5 vol%, 1.0-9.0 vol%, 1.5-8.5 vol%, 2.0-8.0 vol%, 2.5-7.5 vol%, 3.0-7.0 vol%, 3.5-6.5 vol%, 4.0-6.0 vol%, 4.5-5.5 vol%, 5.0-5.2 vol% of the oxidant based on the total volume of the pretreatment solution to be prepared. Even more particularly, preparation may include mixing 0.5-3.0 vol% of the oxidant based on the total volume of the pretreatment solution to be prepared.

[0042] Preparation can include mixing 0.1-1000ppm of the synthetic catalyst based on the total volume of the pretreatment solution to be prepared. For example, the amount of the synthetic catalyst can be 0.5-900ppm, 1.0-750ppm, 5-500ppm, 10-400ppm, 15-350ppm, 20-300ppm, 50-150ppm, 75-100ppm. Even more particularly, preparation can include mixing 0.5-5.0ppm of the catalyst based on the total volume of the pretreatment solution to be prepared.

[0043] A suitable amount of biowaste can be added to the pretreatment solution. For example, the addition can include adding 1-50 wt% of biowaste based on the total volume of the pretreatment solution. In particular, the amount of biowaste added can be 5-45 wt%, 10-40 wt%, 15-35 wt%, 20-30 wt%, 25-28 wt%. Even more particularly, the amount of biowaste can be 5-20 wt% based on the total volume of the pretreatment solution to be prepared.

[0044] Mixing can be carried out at a suitable temperature. For example, the temperature can be 20-80°C. In particular, the temperature can be 25-75°C, 30-60°C, 40-58°C, 45-55°C, 48-50°C. Even more particularly, the temperature can be 40-50°C.

[0045] Mixing can be performed for a suitable period of time. For example, mixing can be performed for ≤ 24 hours. In particular, mixing can be performed for 5-22 hours, 10-20 hours, 12-18 hours, 15-16 hours. Even more particularly, mixing can be performed for 3-8 hours.

[0046] According to another specific aspect, mixing can include sequential mixing. For example, mixing can include a first mixing of the biowaste with a first solution comprising an alkaline solution at a first predetermined temperature for a first predetermined time period to form biowaste solids. Subsequently, mixing can include mixing the biowaste solids with a second solution comprising an oxidant and a synthesis catalyst at a second predetermined temperature for a second predetermined time period. The biowaste can be washed between the first mixing and the second mixing. For example, the biowaste can be mixed with water between the first mixing and the second mixing.

[0047] The first solution may have a suitable pH. For example, the pH of the first solution may be about 10-14. The first predetermined time period may be any suitable time. For example, the first predetermined time period may be 1-72 hours. In particular, the first predetermined time period may be 6-60 hours, 12-48 hours, 18-42 hours, 24-36 hours. Even more particularly, the first predetermined time period may be 12-24 hours.

[0048] The first predetermined temperature may be any suitable temperature. For example, the first predetermined temperature may be ≤100° C. In particular, the first predetermined temperature may be 20-100° C., 25-90° C., 30-75° C., 45-70° C., 50-60° C. Even more particularly, the first predetermined temperature may be about 20-25° C.

[0049] The second solution may include an appropriate amount of an oxidant and a synthesis catalyst. For example, the second solution may include an oxidant of 0.1-10 vol% based on the total volume of the second solution. In particular, the second solution may include an oxidant of 0.5-9.5 vol%, 1.0-9.0 vol%, 1.5-8.5 vol%, 2.0-8.0 vol%, 2.5-7.5 vol%, 3.0-7.0 vol%, 3.5-6.5 vol%, 4.0-6.0 vol%, 4.5-5.5 vol%, 5.0-5.2 vol% based on the total volume of the second solution. Even more particularly, the second solution may include an oxidant of 0.5-2.5 vol% based on the total volume of the second solution.

[0050] The second solution may include 0.1-1000ppm of a synthetic catalyst based on the total volume of the second solution. For example, the second solution may include 0.5-900ppm, 1.0-750ppm, 5-500ppm, 10-400ppm, 15-350ppm, 20-300ppm, 50-150ppm, 75-100ppm of a synthetic catalyst. Even more particularly, the second solution may include 0.5-3.0ppm of a catalyst based on the total volume of the second solution.

[0051] The second predetermined temperature may be any suitable temperature. For example, the second predetermined temperature may be ≤100°C. In particular, the second predetermined temperature may be 20-100°C, 25-90°C, 30-75°C, 45-70°C, 50-60°C. Even more particularly, the second predetermined temperature may be about 20-25°C.

[0052] The second predetermined time period can be any suitable time. For example, the second predetermined time period can be 1-72 hours. In particular, the second predetermined time period can be 6-60 hours, 12-48 hours, 18-42 hours, 24-36 hours. Even more particularly, the second predetermined time period can be 12-24 hours.

[0053] The method of the present invention enables an oxidant and a synthetic catalyst to decompose the resistant structure of biomass contained in the biowaste without forming inhibitory compounds that may be harmful to subsequent processing or treatment steps of the treated biowaste. In particular, the metal complex contained in the synthetic catalyst can combine with the oxidant and catalyze the oxidation of organic molecules such as lignin contained in the biowaste by decomposing cell walls and releasing lignin from the biowaste.

[0054] The method of the present invention may further include processing the pretreated biowaste. Further processing may enable the pretreated biowaste to be converted into value-added products such as sugars and biofuels. In particular, the lignin released from the biowaste after pretreatment of the biowaste may be converted into chemicals such as acetone, ethanol, butanol, etc. In addition, subjecting the pretreated biowaste to a fermentation process may enable the biowaste to be converted into biofuels to a greater extent, because the pretreated biowaste can be more easily degraded.

[0055] The processing may include any suitable type of processing. In particular, the processing may include fermentation, enzymatic saccharification, or a combination thereof of the pretreated biowaste.

[0056] According to a particular aspect, further processing can include making pretreated biowaste enzymolysis saccharification to form sugars such as but not limited to glucose, xylose, fructose, galactose, lactose, maltose and sucrose etc. Any suitable enzyme can be used to carry out enzymolysis saccharification, such as but not limited to endoglucanase, cellulase, hemicellulose or its mixture. Especially, enzyme can be cellulase. Sugar can be made to carry out further processing such as fermentation etc. to form biofuel.

[0057] According to another specific aspect, further processing can include fermenting the pretreated biowaste in the presence of bacteria to form value-added products such as, but not limited to, acetone, ethanol, butanol, etc. The bacteria can be any suitable bacteria. For example, the bacteria can be, but not limited to, Saccharomyces, Streptococcus, Lactobacillus, Bacillus, Escherichia, Salmonella, Clostridium, or a combination thereof. In particular, the bacteria can be Clostridium. Even more particularly, the bacteria can be Clostridium BOH3.

[0058] Fermentation can be carried out in a suitable culture medium with pretreated biological waste as a culture medium substrate. In particular, the culture medium can contain a substrate loading of 30-200 / L of pretreated biological waste.

[0059] Fermentation can be carried out for a suitable period of time. In particular, fermentation can be carried out for a period of 24-195 hours. In particular, fermentation can be carried out for a period of 36-192 hours, 48-180 hours, 60-156 hours, 72-144 hours, 96-120 hours.

[0060] Having now generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the invention.

[0061] Example

[0062] Example 1 - Pretreatment of biomass

[0063] pH Optimization

[0064] The effect of pH on reducing sugar concentration was investigated. The biomass was pretreated with catalytic alkaline peroxide (CAP) solutions (1% H2O2 and 1ppm Fe-TAML catalyst) with different pH. After pretreatment, the pretreated samples were subjected to enzymatic hydrolysis for 24 hours to determine how much reducing sugars could be obtained from the biomass. When the CAP method was implemented at pH 3, the reducing sugar concentration was only 15.9±0.56g / L, indicating that weakly acidic conditions are not suitable for CAP. Similarly, when the CAP method was performed under neutral conditions of pH 7, the reducing sugar concentration was 16.7g / L. When the CAP method was performed under alkaline conditions of pH 10, the reducing sugar concentration increased to 19g / L.

[0065] However, further increase in pH to 11.5 does not lead to an increase in reducing sugar concentration, which remains at 18.4 g / L. Finally, at pH 13, the highest reducing sugar concentration is around 24.5 g / L. However, high pH requires a large amount of sodium hydroxide, which is environmentally unfriendly. Since pH 10 is the optimal pH for Fe-TAML and less sodium hydroxide is required, pH 10 is used in the following examples to determine the reaction conditions that can result in reducing sugar concentrations above 24.5 g / L at pH 10.

[0066] Materials and methods

[0067] Experimental Group 1:

[0068] Dry biomass (corn stover) samples containing about 18 wt% lignin were used in this study. The samples were ground and sieved with uniform particle size. 2 g of biomass was immersed in 20 mL of 1 vol% sodium hydroxide (NaOH) solution for one or two days (Stage 1).

[0069] Then, the solution was centrifuged at 7,000 rpm for 4 minutes and the residual solid was recovered. The recovered solid was mixed with 20 mL of hydrogen peroxide (H2O2) solution (5000 ppm) to form a mixture. In the case of catalytic alkaline peroxide pretreatment, iron-tetraamido macrocyclic ligand (Fe-TAML) was added to the mixture at a final concentration of 0.5 ppm. The pH of the mixture solution was adjusted to 10 using 1 M NaOH solution. Subsequently, the mixture was placed in an incubator at 50 ° C for 24 hours (stage 2).

[0070] Experimental Group 2:

[0071] Corn stalks were used as model biomass in Experimental Group 2. The average size of corn stalks was about 3 mm. They were stored in a drying oven before use. Different alkaline H2O2 (0.5-2 vol%) liquids were prepared as follows: 20 vol% H2O2 solution was added to deionized water, and the pH was adjusted to 10 with 1 M NaOH solution. Then, 2 g of biomass was added to 20 mL of liquid, and the final concentration was 100 g / L. Next, a catalyst (5 ppm) was added. The liquid was placed in an incubator and shaken at 50 ° C for different pretreatment times at a constant speed of 155 rpm. After pretreatment, the liquid was filtered, and all remaining solids were collected and dried at 55 ° C for 10 hours.

[0072] After pretreatment, the samples were tested for soluble lignin and the remaining solid residues were further degraded using Cellic CTec 2 enzyme. A schematic flow chart of the process is shown in Figure 1 shown.

[0073] Soluble lignin test

[0074] The Folin-Ciocardio (FC) reagent was diluted at a ratio of 1:9. After the pretreatment step, 0.3 mL of the diluted FC reagent was added to 0.3 mL of the sample. After 5 minutes, 0.6 mL of a 15 vol% sodium carbonate solution was added to the sample to develop color. After 1 hour, the absorbance of the sample was measured using a UV-Vis spectrophotometer to determine the lignin concentration.

[0075] Enzymatic saccharification / hydrolysis of residual solids

[0076] The residual solid after pretreatment was mixed with 20 mL of CellicCtec 2 solution (100 U) in 12% (w / v) sodium acetate buffer (pH = 4.8). The sample was placed in an incubator (T = 50 ° C). After 24 hours, the remaining solid was separated from the supernatant and then dried. The amount of reducing sugars in the supernatant was determined using the dinitrosalicylic acid (DNS) method.

[0077] DNS reagent was prepared by mixing 100 mL of distilled water with 2 g of 3,5-dinitrosalicylic acid. Then, 60 g of Rochelle's salt (sodium potassium tartrate) was added to the solution until it was completely dissolved. NaOH solution (3.2 g in 40 mL of water) was added to the reagent and mixed evenly.

[0078] To determine the reducing sugar concentration, 2 mL of DNS reagent was added to the 1 mL sample solution in a test tube. The mixture was placed in boiling water for 5 minutes. The mixture was cooled to room temperature and 7 mL of distilled water was added to the test tube. The absorbance of the sample at 540 nm was measured using a UV-Vis spectrophotometer to determine the reducing sugar concentration. Finally, the concentration of reducing sugar was determined from the calibration curve.

[0079] Fermentation of lignin and reducing sugars

[0080] The microorganism used in this study was Clostridium G117. Commercially available reinforced Clostridium medium (RCM) was used to culture strain G117.

[0081] 20 mL of RCM was dispensed into 120 mL serum bottles while purging the bottles with nitrogen to remove oxygen. The bottles were sealed with rubber septa and aluminum caps to provide anaerobic conditions. Subsequently, the bottles containing RCM were autoclaved at 121° C. for 20 minutes. At the same time, the hydrolyzate was also sealed with rubber septa and aluminum caps and autoclaved. The hydrolyzate was added to the RCM alone and then inoculated immediately.

[0082] After inoculation, the bottles were incubated at 37°C with constant shaking (140 rpm). After 24 hours of fermentation, 1 mL of the culture medium was collected from the bottles and centrifuged at 9,000 rpm for 10 minutes. The clear supernatant obtained was used for determination of the fermentation product.

[0083] Analysis of fermentation products

[0084] The supernatant was analyzed using a gas chromatograph (GC; Model 7890A; Agilent Technologies, USA) equipped with a flame ionization detector (FID). Fermentation products containing acetone, butanol, butyric acid, etc. were detected based on their retention times.

[0085] result

[0086] Three different pretreatment methods, alkali (NaOH), alkaline peroxide (NaOH+H2O2), and catalytic alkaline peroxide (NaOH+H2O2+catalyst), were compared in two different experimental groups using the experimental conditions listed in Table 1.

[0087]

[0088] Table 1: Pretreatment conditions of different pretreatment groups

[0089] Soluble lignin test

[0090] After pretreatment, the soluble lignin concentration in the solution was measured. Figure 2 A shows that when biomass is only processed with NaOH and incubation time is 1 day, the lignin concentration in solution is only 43.6g / L, shows that NaOH can remove some lignin from biomass.By contrast, when NaOH and H2O2 (5000ppm) both are used, soluble lignin concentration further increases to 50.4g / L.When three kinds of components (NaOH, H2O2 and catalyst) are all added to experimental group 1, soluble lignin concentration increases to 51.6g / L.The result shows, use catalytic alkaline peroxide (CAP) method to release more lignin from biomass.Removing lignin from biomass helps to destroy cellulose-hemicellulose and produce more glucose for fermentation.

[0091] When the incubation time in NaOH was 2 days, the lignin concentration (e.g. Figure 2 B) is almost the same as that of 1 day incubation time. However, when CAP pretreatment was used, the lignin concentration was only 47.9 g / L, which was lower than that of 1 day incubation time. Some lignin was oxidized by H2O2 to form reducing sugars.

[0092] like Figure 2C It can be seen that when the biomass was soaked in NaOH, the phenol concentration after the AP method was higher than that after the CAP method. This may be because the phenolic compounds were oxidized in the presence of Fe-TAML. Because lignin is composed of phenolic compounds, the presence of Fe-TAML can accelerate the oxidation and dissolution of lignin. As a result, more lignin was removed using the CAP method. After the oxidation of the biomass, the soluble phenolic compounds were further oxidized by H2O2 in the presence of Fe-TAML, resulting in the lower phenol concentration shown.

[0093] When combined, the results showed that the use of H2O2 and Fe-TAMLs resulted in an overall increase in soluble lignin concentration.

[0094] Appearance

[0095] The appearance of the samples of Experimental Group 1 after pretreatment was observed. In the control and alkali pretreatment samples, the biomass remained basically intact. No significant changes were observed. In contrast, after alkaline peroxide and catalytic alkaline peroxide pretreatment, H2O2 made the samples white. In addition, the biomass became fragile and easily broken. In particular, when the CAP method was used, the biomass samples became white and powdery, which well indicated that the pretreatment was very effective.

[0096] Scanning electron microscopy (SEM)

[0097] Scanning electron microscopy (SEM) was used to study the microscopic images of the pretreated biomass to better understand the conversion of biomass at the microscopic level. Figure 3 A As can be seen, there is no surface damage in the control sample. The surface is hard and tightly packed with almost no pores.

[0098] Figure 3 B shows that the surface of the sample pretreated with alkali in experimental group 1 was damaged, with some cracks and holes on the surface. The results show that NaOH can hydrolyze biomass to a certain extent, but the biomass remains intact overall. Figure 3 In C, some cylindrical fibers can be observed on the surface of the alkaline peroxide pretreated samples in Experimental Group 1. This result indicates that the AP method enhances the delignification process and leads to the detachment of holocellulose from the matrix. Figure 3 D shows that the CAP method in experimental group 1 resulted in a very rough surface, and some parts of the surface were carved, and some cylindrical fibers and deep valleys also appeared on the surface.

[0099] Figure 3 E shows that there are many cylindrical fibers on the surface after AP pretreatment in experimental group 2. This further suggests that alkaline peroxide enhances the pretreatment process, leading to delignification. Figure 3F shows that after CAP pretreatment in experimental group 2, the sample surface showed a distorted internal structure, with mainly cylindrical fibers on the surface. Based on the clear and more fragmented fiber structure observed after CAP pretreatment, it is clear that the CAP method improved the enzymatic hydrolysis efficiency and could obtain a higher reducing sugar concentration.

[0100] X-ray diffractometer (XRD)

[0101] X-ray diffractometer (XRD) was used to characterize the pretreated biomass samples of experimental group 2. The crystallinity index of the control, AP pretreated and CAP pretreated samples was 58.36%, 60.17% and 67.02%, respectively. An increase in the crystallinity index was observed after pretreatment, which may be due to the hydrolysis of glycosidic bonds in the accessible region of cellulose resulting in more exposure of crystalline cellulose regions to the enzyme.

[0102] Enzymatic saccharification / hydrolysis of residual solids

[0103] After pretreatment, the pretreated solid biomass was subjected to enzymatic saccharification to determine how much biomass could be hydrolyzed into reducing sugars. After 24 hours of incubation with Cellic CTec 2, the residual biomass that could not be hydrolyzed in the process was separated from the liquid. After drying, the residual biomass was weighed and the results were as follows: Figure 4 As shown in A.

[0104] Depend on Figure 4 AIt can be observed that after adding H2O2, the reduction of biomass increased from 71.5% to 92%. The results show that H2O2 is able to oxidize the lignin of biomass and release more holocellulose for enzymatic hydrolysis. Moreover, when using a catalyst, more than 98% of the solid biomass is degraded and becomes soluble in water. After the CAP pretreatment method, only 2% of the biomass remains as solids. The high degradation efficiency (>92%) of AP- or CAP-pretreated biomass also suggests that the Cellic CTec2 enzyme is not inhibited by residual H2O2 and catalyst.

[0105] Reducing sugar concentration

[0106] For the liquid portion after enzymatic hydrolysis, a DNS test is performed to determine the amount of reducing sugars in the liquid, such as Figure 4 B and 4C. The results were used to evaluate the effectiveness of the three pretreatment methods.

[0107] The reducing sugar concentrations in the liquid fraction after enzymatic hydrolysis showed a similar trend. In experimental group 1, the reducing sugar concentration was higher than 50.9 g / L in the presence of 5000 ppm H2O2 during pretreatment, but in the absence of H2O2, the reducing sugar concentration was only 37.8 g / L. The highest reducing sugar concentration produced by the biomass pretreated by the CAP method was 58.7 g / L ( Figure 4 B).

[0108] In experimental group 2, the reducing sugar concentration obtained by the AP method was 23.71 g / L, of which the glucose concentration was 23.46 ± 0.03 g / L and the xylose concentration was 0.250 ± 0.002 g / L, which was 2 times higher than that of the control. The results showed the synergistic effect of NaOH and H2O2 in the pretreatment process. Figure 4 C also showed that if the CAP method was used, the reducing sugar concentration further increased to 27.91 g / L, with glucose concentration of 27.58 ± 0.15 g / L and xylose concentration of 0.326 ± 0.002 g / L ( Figure 4 C). The results show that the addition of Fe-TAML is beneficial to the pretreatment process.

[0109] The higher reducing sugar concentration is due to two factors. First, lignin dissolution causes the biomass to become more porous and susceptible to enzymatic hydrolysis. Second, some phenolic compounds such as vanillin are strong enzyme inhibitors, which are oxidized by H2O2 and Fe-TAML in the CAP process. In other words, the CAP process is also a detoxification method for removing strong inhibitors during pretreatment.

[0110] Generally, a reducing sugar concentration of about 50-60 g / L is ideal for acetone-butanol-ethanol (ABE) fermentation. A rough mass balance between the solid and liquid fractions indicated that an additional 7-8 g / L of the CAP process pretreated biomass was converted to reducing sugars after enzymatic hydrolysis.

[0111] Fermentation of lignin and reducing sugars

[0112] After the pretreatment method, soluble lignin and residual sugars were released. To use it as a useful substrate, fermentation was performed with the pretreated sample from experimental group 1. Soluble lignin was used as a carbon source for fermentation. About 14 g / L lignin was added to the RCM medium.

[0113] Initially, dark fermentation broth could be observed after the inoculation process. After 24 hours of fermentation, the fermentation broth became lighter in color and became cloudy. Bubbles were formed, which indicated that Clostridium strain G117 was able to grow on soluble lignin and residual glucose as substrates. Figure 5 A shows the products formed during the fermentation process. From the retention times, acetone, butanol and butyric acid are formed. Figure 5 B, The concentrations of acetone, butanol, and butyric acid increased as the fermentation progressed, but stopped increasing after 100 h of fermentation. Figure 5 C shows that soluble lignin and glucose were rapidly consumed by Clostridium G117 during the fermentation process.

[0114] It is believed that inhibitors such as phenols, furfural and hydroxymethyl furfural are generated from lignin after acid or base pretreatment. These inhibitors can inhibit microbial growth and impair the yield of fermentation products. However, there is no evidence that inhibitory compounds are present in the soluble lignin obtained from the CAP pretreatment process.

[0115] Optimization of pretreatment methods

[0116] The effects of H2O2 concentration, catalyst concentration and time on reducing sugar concentration were analyzed and optimized using the conditions of Experimental Set 2 using Response Surface Methodology (RSM). The experimental results were visualized in a three-dimensional response surface plot and contour plots were also generated to study the effects of any two processing variables once the other variables were kept constant at the central level. Each response surface plot had clear peaks within the design boundaries, suggesting that the maximum concentration of reducing sugars could be obtained within that boundary.

[0117] The effect of H2O2 concentration and time on reducing sugar concentration at a fixed catalyst concentration of 2.75 ppm was studied. As H2O2 concentration increased, the concentration of reducing sugar increased with the length of pretreatment time. However, there was a maximum reducing sugar concentration at 38.66 g / L. This suggests that excessive use of H2O2 leads to the production of enzyme inhibitors and reduces reducing sugar concentrations.

[0118] Next, the effects of catalyst concentration and time on reducing sugar concentration at a fixed H2O2 concentration of 12,500 ppm were investigated. Higher reducing sugar concentrations were obtained with higher catalyst concentrations and longer pretreatment times. However, when the reducing sugar concentration reached a maximum value of 38.66 g / L, further increases in catalyst concentration or pretreatment time only resulted in a decrease in reducing sugar concentration. This may be due to the catalase activity of the catalyst and the decomposition of H2O2 when the catalyst concentration was too high. Excessive catalyst may inhibit the enzymatic activity and lead to a decrease in reducing sugar concentration.

[0119] The effects of H2O2 concentration and catalyst concentration on reducing sugar concentration were studied when the pretreatment time was fixed at 4.5 hours. The increase in reducing sugar concentration was more sensitive to H2O2 concentration than to catalyst concentration. However, the optimal H2O2 concentration was about 12,500 ppm. Beyond this point, the reducing sugar concentration remained constant. This is because the catalyst was not fast enough to catalyze all the H2O2 molecules within the pretreatment time, thus resulting in an excess of H2O2 in the system.

[0120] Using RSM, it was found that the optimal H2O2 concentration, catalyst concentration, and time for reducing sugar concentration were 8850 ppm, 0.91 ppm, and 4.44 hours, respectively. Applying the newly optimized conditions generated by Design Expert, the maximum reducing sugar obtained was 36.2 g / L, which was 36.6% higher than the reducing sugar concentration obtained at pH 13. Two replicates were performed to verify the optimal conditions, and the results were consistent with the predicted values. The average value of reducing sugar concentration was 37.31 ± 0.08 g / L, of which the glucose concentration was 36.54 ± 0.08 g / L and the xylose concentration was 0.769 ± 0.002 g / L, which was 3.08% higher than the optimized sugar concentration. The low xylose concentration may be due to the ineffective hydrolysis of hemicellulose by the enzyme used. Finally, the prediction accuracy was 96.92%, which is acceptable.

[0121] Example 2 - Pretreatment of sludge

[0122] Materials and methods

[0123] Sludge samples were collected from a municipal wastewater treatment plant in Singapore. Sludge degradation was performed on the samples before and after anaerobic degradation (using bacteria to break down organic matter in the sludge) using three treatments including control, alkaline peroxide, and catalytic alkaline peroxide.

[0124] For alkaline peroxide treatment, 20 mL of sludge sample was mixed with 2 mL of 20% H2O2 solution, and the pH of the mixture was adjusted to 10 using 1.0 M NaOH solution. For catalytic alkaline peroxide, Fe-TAML was also added to the mixture at a final concentration of 1 ppm. The mixture was placed in a 50 ° C water bath for treatment. After 1 hour, the pretreated sludge underwent methane generation. Biogas from methane generation was analyzed using a gas chromatograph (GC; Model 7890A; Agilent Technologies, USA) equipped with a flame ionization detector (FID). Biogas (methane) was detected.

[0125] result

[0126] These treatments are important for sludge degradation because the dissolution of carbon in the sludge into dissolved organic matter will further enhance methane production. For the sludge sampled before the anaerobic digester, the appearance of the sludge was slightly lighter after treatment. This indicates that some sludge was degraded during the process, however, the change was not significant because the sludge sampled before the anaerobic digester was very concentrated and viscous.

[0127] The appearance of the sludge sampled after the anaerobic digester became lighter after the alkaline peroxide and catalytic alkaline peroxide pretreatment methods. This may be because the sludge becomes less concentrated after passing through the anaerobic digester. Some sludge solids may have been degraded by anaerobic microorganisms to produce methane during the process. Moreover, there are obvious changes after pretreatment. For both alkaline peroxide and catalytic alkaline peroxide treatments, a clear supernatant layer appeared after pretreatment. The liquid layer can be easily separated from the solid sediment. Moreover, the best sludge degradation efficiency was observed for the catalytic alkaline peroxide treatment because the liquid volume was greater than the liquid volume after the alkaline peroxide treatment. This is because a catalyst that helps the hydrogen peroxide oxidation process is added.

[0128] Methane production

[0129] Methane generation was performed to determine how much methane was produced after pretreatment. Figure 6 It can be seen that after adding H2O2, the total amount of methane produced increased from 7.46mmol to 12.5mmol. The results show that H2O2 can help the digestion of waste sludge. The maximum methane production produced by the sludge pretreated with the CAP method was 19.3mmol. This shows that the CAP method improves digestibility and does not inhibit the methane-producing organisms in the production of methane gas.

[0130] Example 3 - Fermentation of food waste

[0131] Materials and methods

[0132] The fermentation medium in the bioreactor consisted of (per liter): 0.5 g KH2PO4; 0.5 g K2HPO4; 0.2 g MgSO4; 2.2 g CH3COONH4; 0.05 g MnSO4; 0.01 g FeSO4·7H2O; 1 g NaCl; 3 g yeast extract, and 1 mL of trace element solution having the following concentrations (per liter): 0.006 mg H3BO3, 0.024 mg NiCl2·6H2O; 0.1 mg ZnCl2; 1.9 mg CoCl2·6H2O; 0.036 mg Na2MoO4·2H2O; and 0.05 mg CuCl2·2H2O.

[0133] For microbial seed culture, the medium was first boiled with 0.25 mL of resazurin solution (0.1%) and cooled to room temperature under nitrogen flow. Anaerobic medium (pH 6.5) was prepared, and 0.0242 g / L L-cysteine ​​and 0.048 g / L Na2S·6H2O were added, respectively. 5 mL of glucose sterile stock solution (300 g / L) and 1 mL of yeast extract sterile stock (150 g / L) were added to the medium. Subsequently, active cells (4%, vol / vol) were inoculated, incubated at 37°C for 24-30 hours, and stirred at 150 rpm on a rotary shaker.

[0134] Three stages of carbon source supplementation were performed: an initial dose of 80 g / L food waste, a second dose of 80 g / L food waste at 28-36 hours, and a third dose of 60 g / L food waste at 48-60 hours (220 g / L food waste equals approximately 100 g / L starch).

[0135] 1g / L CaCO3(Ca 2+ As a metal cofactor for α-amylase in Clostridium strain BOH3) was supplemented into food waste-based fermentation medium to enhance α-amylase activity for starch hydrolysis and butanol production.

[0136] Tryptophan-induced redox regulation of ABE fermentation by strain BOH3: 1 g / L L-tryptophan (precursor in de novo synthesis of NADH and NADPH) was added to the defined medium to enhance reducing power, thereby altering the flow via triggering the availability of NADH and NADPH. A synthetic catalyst was also added at a concentration of 1-10 ppm.

[0137] A two-stage pH switching strategy was used: the pH was first controlled at 6.0 during the first 6 hours (excluding the lag phase), and then allowed to drop to 5.0 as the culture proceeded. Subsequently, the pH was automatically maintained at 5.0-5.3 for continued reaction.

[0138] result

[0139] The fed-batch fermentation process involving Clostridium acetobutylicum BOH3 can directly ferment a wide range of pre-treated food wastes with high yields. Figure 7It can be seen that by applying a two-stage pH conversion strategy, cofactor availability, redox regulation, and a three-stage feeding strategy, the process produced 16.5 g / L butanol and 24.1 g / L total solvent (ABE), corresponding to a butanol yield of about 16.5% and a butanol production of 0.229 g / (L·h). The experimental results showed that the yield and productivity of the obtained strain were better than those of known strains. Therefore, the wild-type acetobutylic acid Fusobacterium strain BOH3 is able to ferment a wide range of low-cost and readily available carbon sources with high yield and yield, and it is easy to culture and develop for commercial purposes.

[0140] Example 4 - Detoxification of a Potential Inhibitor (Vanillin)

[0141] Materials and methods

[0142] Detoxification was performed using three treatments, including control, H2O2 only, and catalytic alkaline peroxide containing H2O2 and Fe-TAML catalyst. These treatments are important because these potential inhibitors may be present after the pretreatment process, which will inhibit enzymatic activity.

[0143] 10 mg / mL vanillin was dissolved in ethanol / deionized water (ratio = 1:9). The pH of the solution was adjusted to 10 with 1 M NaOH solution to obtain an inhibitor solution. For the method using only H2O2, 100 mg / mL hydrogen peroxide liquid was prepared by adding 20% ​​hydrogen peroxide solution to deionized water and added to the inhibitor solution. For the catalytic alkaline peroxide method, 0.01 mg / mL Fe-TAML catalyst was also added to the inhibitor solution.

[0144] After 1 hour, the oxidation products were further analyzed by high performance liquid chromatography (HPLC) (Agilent, USA) equipped with a photodiode array detector (PDA, 280 nm).

[0145] result

[0146] According to HPLC analysis ( Figure 8 ), no change was observed in the control group. With H2O2 alone, vanillin had been oxidized to 4.096 mg / mL vanillic acid. For the catalytic alkaline peroxide method, no peak was observed, indicating successful detoxification using the model inhibitor.

[0147] Further enzymatic hydrolysis was performed to determine the amount of sugar that could be produced after the detoxification process. As can be seen in Table 2, after the addition of H2O2, the total amount of sugar produced did not change much from 5.91 g / L to 5.36 g / L. The results showed that after oxidation using only H2O2, there was still vanillic acid that inhibited the enzymatic activity. The maximum sugar yield produced by the detoxification method using CAP was 22.06 g / L. This shows that the CAP method was able to remove potential inhibitors and did not inhibit the enzymes in sugar production.

[0148] experiment Comparison <![CDATA[H2O2]]> <![CDATA[H2O2 + Catalyst (CAP)]]> Time (hours) 1 1 1 <![CDATA[H2O 2( mg / mL)]]> - 100 100 Catalyst (mg / mL) - - 0.01 Vanillin (mg / mL) 9.134 0.0141 Not detected Vanillin reduction (%) 0 97.68 100 Vanillic acid (mg / mL) - 4.096 Not detected Sedimentation (yes / no) no no yes Sugar concentration (g / L) 5.91 5.36 22.06

[0149] Table 2: Oxidation of vanillin at 10 mg / mL

[0150] Example 5 - Production of polyphenolic acids from aromatic liquids

[0151] Soluble lignin was precipitated using 3M sulfuric acid. The acidified lignin (precipitate) was then filtered and the aromatic liquid was solvent extracted using ethyl acetate. After solvent extraction, the polyphenolic acids were further purified using column chromatography. Thus, crystallization was used to obtain pure polyphenolic acids in solid form.

[0152] Different polyphenolic acids were obtained. For example, pure p-coumaric acid ( Fig. 9 ). This indicates that all by-products of the treatment process can be used to produce valuable polyphenolic acids. In addition, they are easy to isolate, produce and can be further developed for commercial purposes.

[0153] While the foregoing description has described exemplary embodiments, those skilled in the relevant art will appreciate that many variations can be made without departing from the invention.

Claims

1. A method for pretreating biological waste, the method comprising mixing the biological waste with a solution comprising an alkaline solution, an oxidant and a synthesis catalyst to form a pretreated biological waste, wherein the synthesis catalyst comprises Fe-TAML iron-tetraamido macrocyclic ligand, wherein the oxidant comprises hydrogen peroxide, wherein the solution comprises 0.1-10 vol% of the oxidant based on the total volume of the solution, wherein the synthesis catalyst has a concentration of 0.5-10 ppm and wherein the mixing is carried out at a pH of 10-11.

5.

2. The method of claim 1, wherein the biowaste comprises lignocellulosic biomass.

3. The method according to claim 1, wherein the solution comprises 0.1-3.0 vol% of an oxidizing agent based on the total volume of the solution.

4. The method according to claim 1, wherein the mixing is performed at a temperature of 20-100°C.

5. The method of claim 1, wherein the mixing comprises adding the biological waste to a solution comprising the alkaline solution, the oxidant, and the synthesis catalyst.

6. The method of claim 5, wherein the adding comprises adding 1-50 wt% of biological waste based on the total weight of the solution.

7. The method according to claim 5, wherein the mixing is performed at a temperature of 20-80°C.

8. The method of claim 5, wherein the mixing is performed for ≤ 24 hours.

9. The method of claim 1, wherein the mixing comprises: - mixing the biowaste with the alkaline solution at a predetermined temperature for a predetermined period of time to form biowaste solids; and - Adding biowaste solids to a solution comprising said oxidant and said synthesis catalyst.

10. The method of claim 9, wherein the predetermined period of time is 1-72 hours. The method according to claim 9 , wherein the predetermined temperature is ≤100° C.

12. The method according to claim 11, wherein the predetermined temperature is 20-100°C. The method according to claim 11 , wherein the predetermined temperature is room temperature.

14. The method of claim 1, wherein the method further comprises treating the pretreated biological waste.

15. The method of claim 14, wherein the processing comprises fermentation, enzymatic saccharification, or a combination thereof of the pretreated biowaste.

16. The method of claim 15, wherein the fermenting comprises fermenting the pretreated biowaste in the presence of bacteria.

17. The method of claim 15, wherein the fermentation is carried out for a period of 24-195 hours.

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