2020 Volume 26 Issue 3 Pages 381-388
In order to reduce the environmental problems caused by vinegar residue emission, simultaneous saccharification and fermentation technology was used to produce alcohol and xylose from vinegar residue. Due to pretreatment of vinegar residue is vital for simultaneous saccharification and fermentation, uniform experimental designs were used to investigate each of the 5 factors involved in sodium hydroxide and xylanase pretreatment. The results showed that the optimal conditions for vinegar residue pretreatment were determined as solid-to-liquid ratio of 1:11(W/V), NaOH concentration of 2.2%, pretreatment temperature of 71 °C, pretreatment time of 80 min and xylanase usage of 0.3 mL/g. Under this condition, the total sugar yield of vinegar residue was 664 mg/g. Following simultaneous saccharification and fermentation, alcohol and xylose were produced, the yields of ethanol and xylose were 319 mg/g and 179 mg/g, respectively. Therefore, the feasibility of producing alcohol and xylose by enzymatic degradation of vinegar residue was achieved.
Vinegar is a condiment that has been enjoyed by the Chinese since ancient times (Li et al., 2014). Vinegar can increase appetite, relieve fatigue, protect the gastric mucosa, prevent osteoporosis, reduce blood pressure and blood lipids (Kishi et al., 1999; Johnston et al., 2005; Kondo et al., 2001). However, due to the characteristics of traditional solid-state fermentation process, a large amount of vinegar residue is produced. At present, China produces approximately 2.5 million tons of vinegar and 2 million tons of vinegar residue (Zhang et al., 2019). Due to its large quantity and lack of a reasonably effective reusing method, vinegar residue is often disposed irresponsibly. Moreover, this may affect the healthy development of the brewing industry. So the search of multichannel, high value-added reusable purposes of vinegar residue is a hot topic in the field. Nowadays, the main ways of reusing vinegar residue are to produce feed, as cultivation substrates and bioenergy, etc (Li et al., 2018; Zhang et al., 2018). The application of vinegar residue in feed production can improve the nutritive value and palatability of feed. However, as this process still produces by-products, the environmental pollution issue has not been completely solved (Song et al., 2012; Wang et al., 2010). As cultivation substrates, vinegar residue can reduce cultivation and production costs, which in turn increase economic efficiency. But the use of vinegar residue as cultivation substrates is mainly for inexpensive fungi or landscape plants (Wang et al., 2011; Li et al., 2015). As bioenergy, vinegar residue is mainly used for biogas production, pyrolysis and anaerobic fermentation (Smirnova, 2000; Ran et al., 2017; Liu et al., 2019). The research on vinegar residue as bioenergy is limited to the mechanistic and procedural aspects, whereas the applicability in actual energy production still requires further technology breakthrough.
Exploiting the high value use of fermentation secondary waste in the vinegar industry, transforming waste into valuable resources, as well as allowing the fermentation industry to achieve reasonable industrial ecology with the efficient use of secondary waste as resources, are of great significance to resource development and environmental protection. These are also important topics for the future of the brewing industry. This study primarily explored the stepwise degradation of vinegar residue into alcohol and xylose by complex enzyme and microorganism, and has provided a breakthrough for the effective recycling of vinegar residue. Due to vinegar residue forms a recalcitrant lignocellulose complex with lignin to limit the effective conversion to alcohol and xylose. Therefore, an effective pretreatment step is required to accelerate the hydrolysis process and enhance the digestibility of vinegar residue. For instance, vinegar residue has been pretreated by 3% NaOH and the methane yield increased by 53.99% (Shen et al., 2016). Thus, it is vital to discover the functions of NaOH pretreatment on vinegar residue utilization.
Materials Fresh vinegar residue (unprocessed wet residues after vinegar extraction, stored at 4 °C), cottonseed hulls and bran were both provided by Si Po Vinegar Co., Ltd (Yibin, China); Glucose kit, Huili Biotech Co., Ltd (Changchun, China); dextran amylase NS22035 (enzyme activity was 21 IU/mL, 1 IU of dextran amylase is defined as the enzymatic loading that releases 1 µmol glucose/min from starch at 37 °C and pH 6.8), xylanase NS22083 (enzyme activity was 17 IU/mL, 1 IU of xylanase is defined as the enzymatic loading that releases 1 µmol xylose/min from xylan at 50 °C and pH 4.8), cellulose NS22086 (enzyme activity was 25 IU/mL, 1 IU of cellulase is defined as the enzymatic loading that releases 1 mg glucose/min from Whatman 1 filter paper) and complex enzyme NS22002 (enzyme activity of the main β-glucosidase was 7 IU/mL, 1 IU of β-glucosidase is defined as the enzymatic loading that releases 1 µmol glucose/min from salicin at 37 °C and pH 5.0) were purchased from Novozymes Biotechnology Ltd. Among them, xylanase NS22083 was a kind of xylan degrading enzyme system, including β-1,4-endoxylanase, β-xylosi-dase, α-L-arabinofuranosidase, α-D-glucuronidase, acetyl xylanase and phenolic acid esterase; Saccharomyces cerevisiae 1300 was purchased from Sichuan Microbial Resources Infrastructure and Culture Collection Center (Chengdu, China); xylose, phloroglucinol, sodium hydroxide were of analytical grade.
Test flow Test flow shown was in Fig. 1.
The test flow of this research
Recovery of residual starch from fresh vinegar residue In a 500-mL glass flask, 100 g fresh vinegar residue (68% water content) was mixed with 100 mL of 0.05 mol/L citrate buffer solution (pH was 6.0), and the pH was adjusted to 5 with NaOH solution. Different amount of dextran amylase was added. The flasks were placed on HZO-X100 air bath shaker (Haocheng Experimental Instrument Manufacturing Co., Ltd, China) for 72 h at 50 °C and 150 r/min. After the reaction, vacuum filtration was used for solid-liquid separation. The solid portion was dried at 60 °C and used for follow-up tests. The recovered liquid was used as supplemental sugar solution to simultaneous saccharification and fermentation.
NaOH pretreatment on dried vinegar residue after recovery of residual starch Dried vinegar residue obtained after recovery of residual starch was crushed and mixed homogeneously with NaOH solution at different conditions: NaOH solution concentrations were between 0.5% and 4%, solid-to-liquid ratios were between 1:6 and 1:14 (W/V), reaction times were between 30 min and 90 min, and reaction temperatures were between 45 °C and 85 °C. After the pretreatment, solid-liquid separation was performed. The solid portion was washed with warm water until neutralized, and was dried at 60 °C to constant weight.
Xylanase pretreatment on dried vinegar residue In a 250-mL flask, 12 g dried vinegar residue, which obtained after NaOH pretreatment, 1.2∼3.6 mL xylanase (0.10, 0.15, 0.20, 0.25, 0.30 mL/g [enzyme/solid], respectively) and 120 mL of 0.05 mol/L citrate buffer solution was mixed. The pH of the solution was adjusted to 5, and the solution was placed on an air bath shaker for 96 h at 60 °C and 150 r/min. Solid-liquid separation was performed after the reaction. The solid portion was dried at 60 °C to constant weight, and the liquid portion was recycled.
Uniform experimental design With reference to the tests of the enzymatic hydrolysis of sugar from the residues of Baijiu production, 5 factors were found to affect saccharification and degradation: X1, X2, X3, X4, and X5. X1: solid-to-liquid ratio (W/V); X2: NaOH concentration (%); X3: pretreatment temperature (°C); X4: pretreatment time (min); X5: usage of xylanase (mL/g) (Liu et al., 2011). The total sugar yield was used as a reference index, and data processing system (DPS) was used to design the uniform design table U8 (85) (Cai et al., 2014). The levels of different factors were shown in Table 1, and the uniform design table U8 (85) was shown in Table 2.
| Serial number | Factors | ||||
|---|---|---|---|---|---|
| Solid-to-liquid ratio (W/V) | NaOH concentration (%) | Pretreatment temperature (°C) | Pretreatment time (min) | Usage of xylanase (mL/g) | |
| 1 | 1:09 | 0.5 | 60 | 60 | 0.05 |
| 2 | 1:10 | 1.0 | 65 | 65 | 0.10 |
| 3 | 1:11 | 1.5 | 70 | 70 | 0.15 |
| 4 | 1:12 | 2.0 | 75 | 75 | 0.20 |
| 5 | 1:13 | 2.5 | 80 | 80 | 0.25 |
| 6 | 1:14 | 3.0 | 85 | 85 | 0.30 |
| 7 | 1:15 | 3.5 | 90 | 90 | 0.35 |
| 8 | 1:16 | 4.0 | 95 | 95 | 0.40 |
| Experimental level | Factors | ||||
|---|---|---|---|---|---|
| X1 | X2 | X3 | X4 | X5 | |
| N1 | 1 | 5 | 4 | 6 | 8 |
| N2 | 4 | 1 | 3 | 7 | 2 |
| N3 | 3 | 6 | 5 | 2 | 4 |
| N4 | 6 | 7 | 7 | 8 | 5 |
| N5 | 2 | 3 | 8 | 4 | 3 |
| N6 | 7 | 8 | 2 | 3 | 1 |
| N7 | 5 | 2 | 6 | 1 | 7 |
| N8 | 8 | 4 | 1 | 5 | 6 |
Simultaneous saccharification and fermentation Cellulase and complex enzyme were added to xylanase degraded dried vinegar residue for simultaneous saccharification and fermentation. First, 96 mL sugar solution obtained from the recovery of residual starch step was mixed with 5 g dried vinegar residue, and 10 mL S. cerevisiae 1 300 seed solution to a 250-mL flask. Meanwhile, 1 mL cellulase (0.2 mL/g [enzyme/solid]) and 0.25 mL complex enzyme (0.05 mL/g [enzyme/solid]) were added to the flask. The flask was covered with plastic to capture the CO2 produced during fermentation, and was placed in the air bath shaker set at 120 r/min and 37 °C for 24 h. Second, 5 g dried vinegar residue, 1.0 mL cellulase, and 0.25 mL complex enzyme were incubated at 37 °C for 24 h on a shaker at 120 r/min. Third, 5 g dried vinegar residue, 1.0 mL cellulase, and 0.25 mL complex enzyme were incubated at 37 °C for 24 h on a shaker at 120 r/min. Fourth, the rest of dried vinegar residue, 0.3 mL cellulase, and 0.1 mL complex enzyme were incubated at 37 °C for 48 h on a shaker at 120 r/min.
Ethanol yield calculation During the test, the yields of total sugar, glucose, xylose, and alcohol were calculated as Equation 1 and Equation 2.
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Where SY is the yield (mg/g) of total sugar, glucose or xylose, WTSP is the weight (g) of total sugar, glucose or xylose after degradation, WPDVR is the weight (g) of the vinegar residue. EP is the yield (mg/g) of alcohol, WEPF is the weight (g) of alcohol after simultaneous saccharification and fermentation.
Mass balance calculation Mass balance of matter was calculated using Equation 3 and Equation 4 (Nwobi et al., 2015; Awedem et al., 2019).
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Analytical method The cellulose, hemicellulose and lignin of dried vinegar residue were measured by the improved National Renewable Energy Laboratory (NREL) method (Sluiter et al., 2012). Total sugar was measured by dinitrosalicylic acid (DNS) method, glucose was measured by glucose assay kit, xylose was measured by the phloroglucinol method, and alcohol was measured by TRACE GC DSQ II gas chromatography (Thermo, United States) using isopropanol as internal standard (Peng et al., 2013; Kiszonas et al., 2012; Chen et al., 2007). The main components in dried vinegar residue are shown in Table 3.
| Component (%) | Dried vinegar residue |
|---|---|
| Residual starch | 16.32 ± 0.22 |
| Cellulose | 26.11 ± 0.69 |
| Hemicellulose | 24.56 ± 0.87 |
| Lignin | 16.72 ± 0.21 |
| Ash | 9.81 ± 0.63 |
| Other | 6.50 ± 0.70 |
Statistical analysis Origin Pro 10.2 software was used for data mapping, statistical analysis of results were performed using SPSS 19.0 software. Significant differences between means were identified using Duncan's multiple range test (p < 0.05).
Analysis of residual starch recovered from fresh vinegar residue The effects of dextran amylase on the yield of residual starch recovered from fresh vinegar residue were studied. The total sugar concentration was measured after the experiment, the solid and liquid were recovered. As shown in Fig. 2, when the dextran amylase were at 0.16 mL/100 g, 0.18 mL/100 g and 0.20 mL/100 g, the total sugar concentrations were 19.5±1.1 g/L, 20.0±1.5 g/L and 20.9±1.1 g/L, respectively. The yield of total sugar increased along with the amount of amylase used. The increase in yield of total sugar was less obvious when more than 0.16 mL/100 g of enzymes was used (p < 0.05). Considering the cost of enzymes, 0.16 mL/100 g of enzymes should be used for recovering residual starch from fresh vinegar residue. Upon calculation, the amount of recovered liquid was 96 mL, the yield of total sugar was 19.5 g/L. The percentage of residual starch recovered from fresh vinegar residue was 32.3%, the amount of recovered solid was 27.8 g, the recovery rate of solid was 86.9% and the recovery rate of liquid was 57.1%.
Effects of different usage of dextran amylase on residual starch recovered from fresh vinegar residue (mean ± SD, n = 3)
Effects of NaOH concentration on enzymatic degradation of dried vinegar residue The effects of different NaOH concentrations on enzymatic degradation of dried vinegar residue obtained after residual starch recovered were studied under the following conditions: a solid-to-liquid ratio of 1:10 (W/V), pretreatment temperature of 65 °C, pretreatment time of 60 min and xylanase usage of 0.30 mL/g. The results were shown in Fig. 3. As NaOH concentrations increased, the recovery rate of solid decreased, indicating that higher concentrations of NaOH had higher capabilities to dissolve the lignin in the raw materials. In addition, the increase in NaOH concentrations also led to losses of other materials, such as cellulose and hemicellulose, in the vinegar residue. Due to the effect of sodium hydroxide and xylanase, hemicellulose in vinegar residue can be degraded to glucose, xylose, arabinose, mannose and other reducing sugars (Tian et al., 2001; Gao et al., 2016). Therefore, the concentrations of total sugar, glucose, and xylose in the enzymatic hydrolysate gradually increased. After pretreatment, S. cerevisiae 1300 can ferment glucose and mannose to produce alcohol (Liu et al., 2011). When the NaOH concentrations were 2%, 3% and 4%, the recovery rate of solid were 64.7%, 60.1% and 54.2%, respectively. Considering factors such as the recovery rate of solid and sugar concentration, 2% NaOH was chosen for the pretreatment of dried vinegar residue obtained after residual starch recovered. In such conditions, the total sugar, glucose, and xylose concentrations in the enzymatic hydrolysates reached 56.3±1.5 g/L, 28.4±1.1 g/L and 15.1±2.1 g/L, respectively, with the corresponding sugar yields reaching 387±10 mg/g, 195±8 mg/g and 104±14 mg/g, respectively.
Effects of different NaOH concentrations on enzymatic degradation of dried vinegar residue obtained after residual starch recovered (mean ± SD, n = 3)
Effects of solid-to-liquid ratio on enzymatic degradation of dried vinegar residue The effects of solid-to-liquid ratio for enzymatic hydrolysis were evaluated with 2% NaOH, pretreatment temperature of 65 °C, pretreatment time of 60 min and xylanase usage of 0.30 mL/g. As the solid-to-liquid ratio decreased, the recovery rate of solid also gradually decreased. This might be due to a higher concentration of NaOH were lead to increase the contact surface of the raw materials. The higher solid-to-liquid ratio also resulted in gradual increase in the concentrations of total sugar, glucose, and xylose in the enzymatic hydrolysate. As shown in Fig. 4, considering the cost factor, the optimal effect was achieved when the solid-to-liquid ratio was 1:12(W/V). The recovery rate of solid was 65.1%, and the concentrations of total sugar, glucose, and xylose in the enzymatic hydrolysate reached 63.1±1.8 g/L, 38.7±1.8 g/L and 13.7±1.8 g/L, respectively, with the corresponding sugar yields reaching 456±12 mg/g, 280±13 mg/g and 99±13 mg/g, respectively.
Effects of different solid-to-liquid ratio on enzymatic degradation of dried vinegar residue obtained after residual starch recovered (mean ± SD, n = 3)
Effects of pretreatment time on enzymatic degradation of dried vinegar residue The effects of pretreatment time for enzymatic hydrolysis were evaluated with 2% NaOH, solid-to-liquid ratio of 1:12, temperature of 65 °C and xylanase usage of 0.30 mL/g (Fig. 5). The prolongation of pretreatment time resulted in a gradual decrease of the recovery rate of solid. This might be due to the better dissolutions of the raw materials with longer pretreatment time. As shown in Fig. 5, the overall effect was better at a pretreatment time of 75 min. The recovery rate of solid was 67.3%, and the concentrations of total sugar, glucose, and xylose in the enzymatic hydrolysate reached 84.6±1.2 g/L, 48.2±0.7 g/L and 16.6±0.8 g/L, respectively, with the corresponding sugar yields at 618±9 mg/g, 352±5 mg/g and 121±6 mg/g.
Effects of different pretreatment time on enzymatic degradation of dried vinegar residue obtained after residual starch recovered (mean ± SD, n = 3)
Effects of pretreatment temperature on enzymatic degradation of dried vinegar residue The effects of pretreatment temperature for enzymatic hydrolysis were evaluated with 2% NaOH, solid-to-liquid ratio of 1:12, pretreatment time of 75 min and xylanase usage of 0.30 mL/g (Fig. 6). The increase in NaOH pretreatment temperature resulted in a gradual decrease of the recovery rate of solid, indicating that a higher pretreatment temperature led to a better dissolution of lignin in the raw materials. The concentrations of total sugar, glucose, and xylose first increased, followed by a decrease. The overall effect was better seen at a pretreatment temperature of 75 °C: the recovery rate of solid was 65.4%, and the concentrations of total sugar, glucose and xylose in the enzymatic hydrolysate reached 85.7±1.5 g/L, 49.8±1.1 g/L and 14.7±2.1 g/L, respectively, with the corresponding sugar yields at 628±11 mg/g, 365±8 mg/g and 108±15 mg/g, respectively.
Effects of different pretreatment temperature on enzymatic degradation of dried vinegar residue obtained after residual starch recovered (mean ± SD, n = 3)
Effects of xylanase usage on enzymatic degradation of dried vinegar residue The effects of xylanase usage for enzymatic hydrolysis were evaluated with 2% NaOH, solid-to-liquid ratio of 1:12, pretreatment time of 75 min and pretreatment temperature of 75 °C. As shown in Fig. 7, with the increase of xylanase usage, the recovery rate of solid decreased gradually, which indicated that xylanase had better hydrolysis effect on vinegar residue. However, when the xylanase usage increased from 0.20 mL/g to 0.30 mL/g, the effect of hydrolysis was not obvious. The concentration of total sugar, glucose and xylose in the enzymatic hydrolysate increased by 1.05%, 1.02% and 14.29%, respectively. This was proved that although xylanase NS22083 was a kind of xylan degrading enzyme system, the efficiency of xylose production was the highest. When the usage of xylanase was used at 0.20 mL/g, the concentrations of total sugar, glucose and xylose were 85.7±1.5 g/L, 49.8±1.1 g/L and 14.7±2.1 g/L, respectively, and the corresponding sugar 628±11 mg/g, 365±8 mg/g and 108±15 mg/g, respectively.
Effects of xylanase usage on enzymatic degradation of dried vinegar residue obtained after residual starch recovered (mean ± SD, n = 3)
DPS analysis of NaOH pretreatment of dried vinegar residue Quadratic regression analyses were performed to determine the significance of the effects of each factor on the total sugar yield after enzymatic degradation of vinegar residue using results from uniform design (Table 4) and DPS (data processing system). The equation for the quadratic regression was Y=611.5−14.9X1+3868.5X4+26.4X3×X3−150.6X4×X4−456.7X5×X5+158.9X2×X5.
| Experimental level | Factors | Total sugar yield | ||||
|---|---|---|---|---|---|---|
| X1 | X2 | X3 | X4 | X5 | Y (mg/g) | |
| N1 | 1 | 5 | 4 | 6 | 8 | 482 ± 9 |
| N2 | 4 | 1 | 3 | 7 | 2 | 438 ± 7 |
| N3 | 3 | 6 | 5 | 2 | 4 | 640 ± 11 |
| N4 | 6 | 7 | 7 | 8 | 5 | 475 ± 10 |
| N5 | 2 | 3 | 8 | 4 | 3 | 635 ± 12 |
| N6 | 7 | 8 | 2 | 3 | 1 | 205 ± 6 |
| N7 | 5 | 2 | 6 | 1 | 7 | 522 ± 9 |
The complex correlation coefficients R was 0.9986 and F was 58.1258, with a significance value P of 0.0078 and a standard deviation of 0.0095. The larger the values of the complex correlation coefficients R and F, the smaller the standard deviations, indicating a better fit between the data and the equation. The interactions between factor 2 and factor 5, i.e., interactions between NaOH concentrations and pretreatment time, could be seen from the regression equation. With higher NaOH concentrations, pretreatment time should be reduced. The most direct effect on total sugar yield was factor 5 amount of xylanase: higher amount of enzymes led to higher sugar yield. Table 5 showed the best combination of factors for the enzymatic degradation of vinegar residue based on the regression equation and DPS analyses. Under the condition of a solid-to-liquid ratio of 1:11, NaOH concentration of 2.2%, pretreatment temperature of 71 °C, pretreatment time of 80 min, and xylanase usage of 0.3 mL/g, enzyme degraded dried vinegar residue resulted in total sugar yield of 664 mg/g, glucose yield of 385 mg/g, xylose yield of 114 mg/g, respectively, and the amount of recovered solid was 16.4 g, the recovery rate of solid was 59.0%. In addition, 83 mL xylose solution was obtained and the recovery rate of liquid was 69.2%.
| Y (mg/g) | X1 (W/V) | X2 (%) | X3 (°C) | X4 (min) | X5 (mL/g) |
|---|---|---|---|---|---|
| 664 ± 10 | 1:10.977 | 2.241 | 70.881 | 80.213 | 0.287 |
Analysis of enzymatic degradation and simultaneous saccharification and fermentation of vinegar residue The cellulase was added to the dried vinegar residue that was pretreated with NaOH solution and degraded by xylanase for simultaneous saccharification and fermentation. Following the fermentation, the alcohol concentration of 46.8 g/L was detected in the fermentation broth using GC, which was equivalent to alcohol yield to 319 mg/g. After the fermentation, the dry weight of 6.9 g (the recovery rate of solid was 42.1%), the volume of the fermentation broth was 92 mL (the recovery rate of liquid was 83.6%), and the xylose was 9.5 g/L, the yield of xylose was 65 mg/g. This portion of sugar solution can be mixed with xylose-rich solution obtained from the xylanase degraded vinegar residue, so that the yield of xylose can reach 179 mg/g, which can be used for other purposes, such as xylitol production (Zhang et al., 2012; Zhang et al., 2013; Rao et al., 2006).
Residual starch in fresh vinegar residue was recovered by dextran amylase and resulted in a glucose solution. The glucose solution was consumed by the S. cerevisiae 1300 during the early stage of simultaneous saccharification and fermentation. The total sugar concentration obtained from the recovered starch was 19.5 g/L, and 32.3% of residual starch from the fresh vinegar residue was recovered.
Uniform experimental designs were used to investigate each of the 5 factors involved in the NaOH and xylanase pretreatment of dried vinegar residue, including solid-to-liquid ratio, NaOH solubility, pretreatment temperature, pretreatment time, and the usage of xylanase. Using DPS analyses, the optimal pretreatment conditions for dried vinegar residue were determined, which were 1:11 solid-to-liquid ratio, NaOH concentration of 2.2%, pretreatment temperature of 71 °C, pretreatment time of 80 min, and xylanase usage of 0.3 mL/g. These optimal conditions allowed more effective enzymatic degradation of the dried vinegar residue and resulted in total sugar yield of 664 mg/g. Following simultaneous saccharification and fermentation, alcohol and xylose were produced, the yields of ethanol and xylose were 319 mg/g and 179 mg/g, respectively. Therefore, the feasibility of producing alcohol and xylose by enzymatic degradation of vinegar residue was achieved.
Acknowledgments This work was financially supported by Key Laboratory of Wuliangye-flavor Liquor Solid-state Fermentation, China National Light Industry (No. 2018JJ020); Solid-state Fermentation Resource Utilization Key Laboratory of Sichuan Province of China (No. 2019GTJ012, 2018GTJ014); Scientific research project of Yibin Vocational and Technical College of China (No. ybzysc18-19); Research Initiation Project of Yibin University of China (No. 2016QD08).
