3.1. Effects of pretreatment conditions on enzymatic hydrolysis of mono-saccharides release
Characteristics of Q. infectoria was listed in Table 1. Glucan and xylan contents were 16.2% TS and 12.8% TS, respectively, which are similar with the reports on compositions of other oak leaves (Baber et al. 2014). Lignin content of 29.6 % was significantly higher than glucan and xylan in the leaf biomass.
Dilute acid pretreatment was first investigated to conclude the preferred reaction conditions. 12.5% of dry biomass were used for the pretreatment. Enzymatic hydrolysis was then applied on the pretreated slurry to release mono-sugars, which was used to evaluate performance of the pretreatment. Multiple-way ANOVA analysis shows that acid concentration and temperature had significant (P<0.05) influences on glucose production. Xylose production was significantly (P<0.05) impacted by all three factors of acid concentration, temperature, and reaction time. Two-way and three-way interactions did not have significant (P>0.05) influences on glucose production. While, xylose production was significantly (P<0.05) influenced by the interaction of acid concentration and pretreatment time.
Glucose concentrations of Q. infectoria in the hydrolysates under different pretreatment conditions were shown in Figures 1a-c. Compared to lower acid concentrations (0.5 and 1%) and lower reaction temperature (105°C), higher glucose concentrations of 8.7, 8.4, and 8.6 g/L under 120°C and 2% acid were achieved for 0.5, 1 and 2 hours, respectively, with no significant (P>0.05) difference between each other. For the pretreatment at 105°C, there were also no significant (P>0.05) differences between different reaction times and acid concentrations. The glucose concentrations were around 7.5 g/L at 105°C, which were significantly (P<0.05) lower than them from the pretreatment at 120°C. The results clearly demonstrate that glucose release of Q. infectoria was mainly dependent on reaction temperature and acid concentration. 2% acid at 120 °C for 2 hours was the preferred pretreatment conditions for glucose release from Q. infectoria. In comparison with other major herbaceous energy crops of switchgrass and miscanthus (Ruan et al. 2013), the glucose conversion of the leaves was 43%, which is lower than 60 and 55% from switchgrass and miscanthus, respectively, under similar pretreatment and enzymatic hydrolysis conditions. The possible reason might be that high lignin content in the leaves (30%) absorbed cellulase and prevented some of them from hydrolyzing glucan in the pretreated leaves (Yang and Wyman 2006; Tengborg et al. 2001; Chen et al. 2012).
Xylose concentrations of Quercus infectoria under different pretreatment conditions are shown in Figures 1d-1f. Unlike glucose production, xylose concentration was significantly influenced by acid concentration, temperature and reaction time. Increasing acid concentration from 0.5 to 1% exhibits little impact on xylan conversion at 105 °C for all three reaction times, comparing to 30-60% increase of xylose concentration at 120°C at the corresponding reaction times. Xylose concentrations were further increased by 30-40% at 105°C and 20-30% at 120°C with the increase of acid concentration from 1% to 2%. Under 2% acid and 105°C, xylose concentrations reached 10.3, 13.3, and 14.1 g/L for 0.5, 1, and 2 hours of reaction time, respectively. Meanwhile, under 2% acid and 120°C, xylose concentrations reached 15.9, 15.4, and 17.0 g/L for 0.5, 1, and 2 hours of reaction time, respectively. The data show that xylose concentration was significantly (P<0.05) increased with extending the pretreatment time from 0.5 to 2 hours and increasing the reaction temperature from 105 to 120°C. The highest xylose concentration of 17.0 g/L was achieved under the conditions of 2 hours of reaction time, 2% of acid concentration, and 120°C of reaction temperature. Considering total mono-sugar conversion (both xylose and glucose), 2% acid at 120 °C for 2 hours was adopted to carry out pretreatment for enzymatic hydrolysis. Switchgrass and miscanthus were used again to compare xylose conversion. The xylose conversion of the leaves was 93%, which is higher than switchgrass (79%) and miscanthus (70%) (Ruan et al. 2013). Due to the fact that majority of xylose was released during the pretreatment step, lignin absorption of enzymes had less effect on xylose release than glucose release.
Figure 1. Effects of pretreatment conditions on mono-sugar release from Quercus infectoria
In order to increase the amount of mono-sugars for following fermentation, the biomass amount was further increased to 15% of TS for the pretreatment, which was the highest TS content with appropriate rheological property for the pretreatment and hydrolysis. Starting from 2.9 g/L in the pretreated slurry of Q. infectoria, glucose concentration quickly increased to 9 g/L in the first 6 hours and then leveled off after reaching 10.5 g/L at 12 hours (Figure 2). Compared to glucose production, xylose started from much higher concentrations of 16.8 g/L that was the result of acid pretreatment (Figure 2). The xylose concentration was increased to 22.0 g/L in 12 hours and then leveled off following the same trend of glucose production. Since acetate was mainly generated during the acid pretreatment, the acetate concentration remained relatively constant during the course of the enzymatic hydrolysis, which the acetate concentration was 1.3 g/L at the beginning and slightly increased to 1.9 g/L at the end of the hydrolysis.
Figure 2. Enzymatic hydrolysis of the acid treated Q. infectoria
3.2. Ethanol fermentation on the hydrolysate using marxianus
Ethanol production from the hydrolysate was subsequently carried out using K. marxianus fermentation. The fermentation data were shown in Figure 3. It has been reported that K. marxianus is able to utilize both glucose and xylose in the hydrolysates with a preference of glucose (Signori et al. 2014). The changes of glucose, xylose, acetate, and ethanol during K. marxianus fermentation were presented in Figure 3. K. marxianus exhibits relatively fast rates of sugar consumption and ethanol production. There was no lag phase for K. marxianus growth on the hydrolysate. Glucose concentration dropped to 1.8 g/L at 12 hours from 10.9 g/L at the beginning of the fermentation. The corresponding glucose consumption rate was 0.76 g/L/h. There was no further glucose consumption after 12 hours fermentation. Xylose consumption was accelerated after 6 hours fermentation when glucose concentration dropped below 4.0 g/L. A total of 6.3 g/L xylose was consumed during the 36 hours fermentation with a xylose consumption rate of 0.18 g/L/h. The data show that K. marxianus fermentation accumulated ethanol once glucose maintained at higher concentrations in the fermentation broth. Ethanol concentration reached 7.5 g/L within the first 12 hours, giving an ethanol production rate of 0.63 g/L/h. After glucose consumption leveled off at 12 hours of the fermentation, both ethanol production and xylose consumption were stopped. This result suggests that K. marxianus might require the existence of glucose to metabolize xylose to produce ethanol, which is consistent with the observations from the literature reports (Signori et al. 2014; Du et al. 2019). Meanwhile, the data also demonstrate that a good amount of 9 g/L of xylose was consumed from 6 to 12 hours once glucose reached a low level of 3.3 g/L at 6 hours, which provides a potential strategy to apply K. marxianus to utilize xylose rich biomass for high-efficiency ethanol production. A small amount of glucose could be added into the fermentation broth to maintain the minimum amount of glucose for K. marxianus metabolism of xylose consumption and ethanol production. The fermentation results concluded that K. marxianus cultivation on the hydrolysate had a short fermentation time of 12 hours to produce 7.5 g/L with an ethanol production rate of 0.63 g/L/h.
Figure 3. Ethanol production from the hydrolysate using K. marxianus
3.3. Techno-economic analysis (TEA)
According to above experimental results, mono-sugar and ethanol production from Q. infectoria leaves are summarized in Table 2, and will be used for TEA. The selected pretreatment conditions are 120°C, 2% (w/w) H2SO4, and 2 hours followed by a 24-hour enzymatic hydrolysis. At 15% dry matter of Q. infectoria in the processing solution, the pretreatment and enzymatic hydrolysis had a glucose conversion of 42.8% and a xylose conversion of 99.8% with production of 0.076 kg glucose and 0.145 kg xylose per kg dry leaf biomass. The ethanol fermentation of the concentrated hydrolysate using K. marxianus for the TEA were set at 48 hours and 30°C of anaerobic cultivation. The extended fermentation time is due to the fact that a concentration step of the hydrolysate was introduced in the TEA analysis. The fermentation produced 0.050 kg ethanol per kg dry leaf biomass, respectively. The corresponding ethanol yield from mono-sugars consumed was 27.0%, and the corresponding ethanol yield from the dry leaf biomass was 5.0%.
Table 2. Sugar and ethanol production from Q. infectoria using K. marxianus
According to the leaves production in Zawita sub-district, this study assumed that 22% (4,000,000,000 kg) of the annual leaves production was used as the feedstock for an ethanol biorefinery. TEA was conducted based on the ethanol production 200,000,000 kg per year. The mass and energy balance calculation is detailed in the supplemental material.
3.3.1. Mass and energy balance
Mass balance data show that the amounts of fuel (diesel) for collecting and transporting the leaves to the biorefinery are 0.09 and 0.02 kg diesel/kg ethanol produced, respectively (Figure 3). Since the leaves available in Zawita sub-district is within 50 km radius, reference numbers of 5.43 and 1.45 L diesel/metric ton dry biomass for biomass collection and transportation, respectively, in the similar radius were used for the calculation (Morey et al. 2010). The corresponding energy consumption for the leaves collection and transportation is 6.22 MJ/kg ethanol produced (Table 3).
Once the leaves biomass arrives at the biorefinery, 20 kg of dry biomass is needed to be pretreated and hydrolyzed to release mono-sugars (glucose and xylose) for production of 1 kg of ethanol. During the pretreatment and hydrolysis and condition operation, 2.67 kg of sulfuric acid, 127 kg of water (64 kg of fresh water and 63 kg of condensation water from the hydrolysate concentration), 2 kg of calcium hydroxide, and 1.64 kg of enzymes are used to convert 20 kg of dry biomass into 102 kg of hydrolysate with glucose and xylose concentrations of 17 and 22 g/kg hydrolysate, 27 kg of wet hydrolysis residue with a moisture content of 36% (w/w), and 3.7 kg of CaSO4 (Figure 3). Energy consumptions for acid pretreatment, enzymatic hydrolysis, detoxification (using Ca(OH)2), and press filtration (liquid/solid separation) are 43.1, 0.9, 0, and 3.1 MJ/kg ethanol produced, which were calculated based on the approach described in a previous study (Zanotti et al. 2016). Total energy consumption for the pretreatment and hydrolysis step is 47.1 MJ/kg ethanol produced (Table 3), which is one of the largest energy demanding operations in the biorefinery.
Sugar concentrations of hydrolysate from the pretreatment and hydrolysis step are relatively low for ethanol fermentation, which leads to a large amount of water demand and a very low ethanol content from the fermentation step. A hydrolysate concentration step is needed to increase sugar concentration and recirculate water. A single-effect mechanical vapor recompression (MVR) evaporator is selected to carry out the concentration due to the better steam economy of MVR (Minton 1986). The evaporator produces 40 kg of concentrated hydrolysate with glucose and xylose concentrations of 44 and 56 g/kg hydrolysate, respectively, from 103 kg of dilute hydrolysate (Figure 3). Meanwhile, sixty-three kg of condensation water is recirculated back to the pretreatment and hydrolysis operation. The energy balance shows that the evaporator demands 13.8 MJ/kg ethanol produced to concentrate the hydrolysate and recovers 13.2 MJ/kg ethanol produced during the condensation. The recovered energy is used to raise and maintain the solution temperature of the pretreatment and hydrolysis and condition operation (Table 3).
During a 48-hour ethanol fermentation, thirty-seven kg of fermentation broth with an ethanol content of 2.7% (w/w) and 3 kg of wet yeast with a dry matter of 33% (w/w) are generated from 40 kg of concentrated hydrolysate for production of 1 kg ethanol (Figure 3). One tenths kg/kg ethanol produced of corn steep liquor (nitrogen source), 0.013 kg/kg ethanol produced of diammonium phosphate, and 0.01 kg/kg ethanol produced of air are used for seed culture and ethanol fermentation. Energy consumptions for yeast seed culture and ethanol fermentation are 2.94 and 0.91 MJ/kg ethanol produced, which were calculated based on a reference (Zanotti et al. 2016). Total energy consumption for the ethanol fermentation is 3.85 MJ/kg ethanol produced (Table 3).
A conventional distillation tower is then used to extract ethanol from the fermentation broth. One kg bioethanol stream with an ethanol content of 95% (v/v) is obtained from the distillation (Figure 3). The distillation also generates 36 kg of wastewater, which is treated by a wastewater treatment operation before discharging. Based on ethanol content in the fermentation broth (2.7%w/w or 3.42%v/v), an energy demand of 18.5 MJ/kg ethanol produced for the distillation was calculated based on the numbers from a reference (Katzen et al. 1999) (Table 3).
Since the leaf has a relatively high lignin content (Table 1), recovering the lignin-rich hydrolysis residue and using it as the fuel to power the ethanol biorefinery are critical to sustain ethanol production from the leaves. Twenty-seven kg of wet residue with a dry matter of 36% (w/w) obtained from a press filter is dried using a triple-pass rotary dryer to produce 10 kg of dry residue with a lignin content of 60% (w/w) (Figure 3). The energy demand of the drying is 45.9 MJ/kg ethanol produced, which is the second largest energy demand in the biorefinery (Table 3). However, the energy content of dry lignin-rich residue is 131 MJ/kg ethanol produced, which is much higher than the energy demand of the drying. Therefore, the lignin-rich residue is used by a boiler and a turbo-generator to power the biorefinery.
Meanwhile, yeast biomass from the fermentation process rich in protein can be another value-added product of animal feed. Another drying process using the triple-pass rotary dryer consumes 5.3 MJ of thermal energy to produce 1 kg of dry yeast biomass (Figure 3 and Table 3).
Due to a large amount of wastewater (7,190,104,864 kg/year) generated from the biorefinery, it must be treated before discharging. An activated sludge wastewater treatment operation is implemented in the biorefinery to handle the wastewater. Ninety percent (6,471,094,377 kg/year) of the wastewater is reclaimed. Based on an average energy demand of 1,14 kJ/kg wastewater for conventional activated sludge wastewater treatment process reported by the Water Environment Federation (WEF), energy consumption of wastewater treatment at the biorefinery is 0.041 MJ/kg ethanol produced or 8,203,910 MJ/year for 200,000,000 kg/year of ethanol production.
Figure 4. Mass balance of 1 kg bioethanol production from the leaves
Table 3. Energy balance of ethanol production from the leaves a
The mass and energy balance data demonstrate that due to the fact that O. infectoria leaf has a high lignin content, the lignin-rich hydrolysis residue contains the energy that is able to power the entire biorefinery with a surplus net energy of 3.8 MJ/kg ethanol produced or 753,256,533 MJ/year for the biorefinery. However, the water demand (64 kg/kg ethanol produced) of the biorefinery is much higher than other reports ranging from 5 to 12 kg/kg ethanol produced (Humbird et al. 2011; Mu et al. 2010). In order to address the high water demand issue, efficiencies of both mono-sugar production and ethanol fermentation from the leaves need to be further improved.
3.3.2. Economic analysis
Economic feasibility is another important factor that determines commercial applicability of the ethanol biorefinery in the region. CapEx, OpEx, and revenues are the parameters to assess economic performance of the biorefinery. As presented in Table 4, the CapEx to establish the studied biorefinery in Zawita sub-district is $1,227,906,682. Due to the large amount of leaves required by the biorefinery and high lignin content in the leaves, the pretreatment/hydrolysis/condition of feedstock handling and the boiler and turbo-generator of hydrolysate residue utilization are the most expensive units ($104,731,510 and $413,881,582, respectively) in the biorefinery. The OpEx is $170,129,613/year, including feedstock collection and transportation, water, chemicals, enzymes, maintenance, and labor costs. The revenue streams of the biorefinery are ethanol, dry yeast, and energy saving from hydrolysis residue utilization. Ethanol as a biofuel ($1.11/kg), dry yeast as animal feed additive ($0.5/kg), and energy saving ($0.1/kwh) lead to a total revenue of $293,822,418/year, which is 1.73 times higher than the OpEx. Correspondingly, a net positive revenue of $123,692,804/year is realized from the biorefinery operation.
The cash flow analysis predicts that payback period of the biorefinery is 10 years (Figure 5). A sensitivity analysis was then conducted on five key items (from both CapEx and OpEx) of pretreatment/hydrolysis/condition unit, boiler and turbo-generator unit, sulfuric acid for pretreatment, enzyme protein for hydrolysis, and ethanol revenue to elucidate the impacts of them on the payback period (Table 5). An increase of 25% on ethanol production could reduce the payback period by 30% to 7 years, which is the largest reduction among these five key items. A capacity reduction of 25% on the boiler and turbo-generator can also decrease the payback period by 15% to 8.5 years. 25% reduction of sulfuric acid usage, enzyme protein usage, and size of the pretreatment/hydrolysis/condition unit could shorten the payback period by 8, 10, and 5%, respectively. According to the sensitivity analysis, the conclusion is similar with that from the mass and energy balance analysis. Increasing ethanol conversion from mono-sugars in the hydrolysate and improving the hydrolysis efficiency of mono-sugar release are two key steps to greatly reduce CapEx and OpEx, increase ethanol revenue, and significantly enhance the performance of the biorefinery.
Table 4. Economic assessment of a bioethanol plant with a capacity of 200,000 kg ethanol per year from Q. infectoria leaves in Kurdistan region of Iraq
Table 5. Sensitivity analysis of key CapEx, OpEx, and revenue items on the payback period of the biorefinery
Figure 5. Cash flow of the bioethanol plant with a capacity of 200,000 kg ethanol per year from Q. infectoria leaves