Enhancing ethanol production from thermophilic and mesophilic solid digestate using ozone combined with aqueous ammonia pretreatment

Enhancing ethanol production from thermophilic and mesophilic solid digestate using ozone combined with aqueous ammonia pretreatment

Bioresource Technology 207 (2016) 52–58 Contents lists available at ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/b...

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Bioresource Technology 207 (2016) 52–58

Contents lists available at ScienceDirect

Bioresource Technology journal homepage: www.elsevier.com/locate/biortech

Enhancing ethanol production from thermophilic and mesophilic solid digestate using ozone combined with aqueous ammonia pretreatment Dianlong Wang a,b,d, Jiang Xi c, Ping Ai a,b,⇑, Liang Yu d, Hong Zhai a, Shuiping Yan a,b, Yanlin Zhang a,b a

College of Engineering, Huazhong Agricultural University, Wuhan 430070, China Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070, China c Biogas Institute of Ministry of Agriculture, Chengdu 610041, China d Department of Biological Systems Engineering, Washington State University, Pullman, WA 99164, USA b

h i g h l i g h t s  Recover residual organic carbon from solid digestate to produce ethanol.  Pre-treat recalcitrant solid digestate using combined ozone with aqueous ammonia.  Elucidate a combined and successive utilization of lignocelluloses.

a r t i c l e

i n f o

Article history: Received 5 December 2015 Received in revised form 29 January 2016 Accepted 30 January 2016 Available online 4 February 2016 Keywords: Solid digestate Anaerobic digestion Methane Ethanol fermentation Pretreatment

a b s t r a c t Pretreatment with ozone combined with aqueous ammonia was used to recover residual organic carbon from recalcitrant solid digestate for ethanol production after anaerobic digestion (AD) of rice straw. Methane yield of AD at mesophilic and thermophilic conditions, and ethanol production of solid digestate were investigated. The results showed that the methane yield at thermophilic temperature was 72.2% higher than that at mesophilic temperature under the same conditions of 24 days and 17% solid concentration. And also the ethanol production efficiency of solid digestate after thermophilic process was 24.3% higher than that of solid digestate after mesophilic process. In this study, the optimal conditions for integrated methane and ethanol processes were determined as 55 °C, 17% solid concentration and 24 days. 58.6% of glucose conversion, 142.8 g/kg of methane yield and 65.2 g/kg of ethanol yield were achieved, and the highest net energy balance was calculated as 6416 kJ/kg. Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction Anaerobic digestion (AD) technology has been widely applied for treating various wastes, such as crop straws, animal manure and municipal wastes, etc. (Liu et al., 2015). Methane or hydrogen can be obtained to meet the increasing need for energy (Jariyaboon et al., 2015; Razaviarani and Buchanan, 2015; Risberg et al., 2013). Although biogas is a promising renewable energy alternative, the sustainable development of AD mainly depends on the ability to deal with the excessive digestate (Dahlin et al., 2015). This is because an improper handling of digestate would lead to serious environmental problems.

⇑ Corresponding author at: Room B306, College of Engineering, Huazhong Agricultural University, No.1, Shizishan Street, Hongshan District, Wuhan 430070, PR China. Tel./fax: +86 27 87288723. E-mail address: [email protected] (P. Ai). http://dx.doi.org/10.1016/j.biortech.2016.01.119 0960-8524/Ó 2016 Elsevier Ltd. All rights reserved.

Many large-scale biogas plants that use crop straws as feedstock have a low degradation rate compared with those that use animal manure. There is a high residual organic carbon in solid digestate after AD (Pokój et al., 2015). For example, Sambusiti et al. (2015) studied the recovery of methane from solid digestate in a mesophilic full-scale AD plant with mixed biomass. The solid digestate still contained 17.5% of cellulose, 20.3% of hemicellulose and 24.1% of lignin after AD. Wang et al. (2013) reported that the anaerobic digested corn stover still had 25.1% of cellulose, 1.1% hemicellulose and 21.7% of lignin after 20 days. These studies have demonstrated that the solid digestate can be further reused due to highly residual lignocelluloses. Usually, anaerobic digestate can be used as fertilizer via composting to replace inorganic fertilizers and improve soil quality (Bustamante et al., 2012; Tambone et al., 2015). Although solid digestate has high fertilizing potential, its full year production needs large storage and heavy transportation due to the limitation

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of crop growth stage and soil type. Moreover, the increasing number of biogas plants has led to an oversupply of digestate. Therefore, one of the important challenges for AD development is how to handle large amounts of solid digestate. In recent years, some researchers have proposed a new biorefinery way that would use solid digestate to produce ethanol. Teater et al. (2011) assessed the solid digestate for ethanol production and indicated that solid digestate is a suitable feedstock for biorefinery compared with switchgrass and corn stover. After an AD process, most hemicellulose was consumed, and crystal structure of lignocelluloses was broken. Yue et al. (2011) compared continuous stirring-tank reactor (CSTR) and plug flow reactor (PFR) to use solid digestate for ethanol production and indicated that CSTR was a preferred reactor type. Since it is hard to reuse solid digestate in high conversion efficiency (Tambone et al., 2009), pretreatment is necessary to break down the recalcitrant structure in solid digestate. Ozone pretreatment has been proved to delignify with minimal effects on hemicellulose and cellulose. Different mechanisms including selective reaction with carbon–carbon double bonds and glycosidic bond cleavage have been proposed for lignin degradation by ozone pretreatment (Bule et al., 2013). Ozone pretreatment can result in condensation of lignin structures due to lignin–ozone interaction. Aqueous ammonia pretreatment was also extensively studied. It was found that the major effect of aqueous ammonia pretreatment is to remove lignin. Aqueous ammonia pretreatment can remove 60% of lignin from rice straw while achieving 70% of enzymatic digestibility (Ko et al., 2009). A fiber expansion theory was proposed that aqueous ammonia pretreatment resulted in the increased access for cellulosic enzyme due to the insertion of ammonia molecules (Gao et al., 2012). In addition, studies have shown that ammonia can selectively act with lignin bonds, as well as ester and ether bonds, especially C–O–C bonds, causing the selective removal of lignin in biomass. Above all, aqueous ammonia is inexpensive and recyclable in industrial applications. Jurado et al. (2013) used aqueous ammonia soaking to treat solid digestate of swine manure fiber, resulting in a 40–80% enhancement of methane yield from digested manure fibers. This research demonstrated that aqueous ammonia can be used to treat the recalcitrant solid digestate for the enhancement of methane productivity. Yu et al. (2014) studied the combined pretreatment of ozone and aqueous ammonia soaking to improve enzymatic hydrolysis of grass, achieving 90% sugar recovery. Therefore, ozone combined with aqueous ammonia pretreatment may be a promising pretreatment method. However, to our best knowledge, the investigation on solid digestate using ozone and aqueous ammonia pretreatment to enhance ethanol production is limited. This investigation will be the theoretical basis for clarifying the combined and successive utilization path of organic carbon. The aim of this study was to evaluate a two-stage conversion of waste biomass to methane and ethanol under both mesophilic (37 °C) and thermophilic (55 °C) conditions, and demonstrate overall improvement in energy yield upon pretreatment of the solid digestate with a combined ozone and aqueous ammonia soaking. The pretreatment and enzymatic hydrolysis of solid digestate after mesophilic and thermophilic AD were investigated. The ethanol production from the treated solid digestate was studied to clarify the residual organic carbon recovery. Furthermore, preliminary energetic balances were calculated for the integrated process of AD and ethanol fermentation to provide a useful insight in terms of the partitioning of energy into the methane versus ethanol product streams. Finally, the changes of crystallinity were measured in the entire process to reveal recovery mechanism of organic carbon in solid digestate.


2. Methods 2.1. Materials Rice straw was collected from fields in a suburb of Wuhan, China. After collection, the straw was air dried, ground using a hammer mill and passed through a 2 mm aperture standard screen. The ground straw was then sealed in plastic bags and stored at room temperature for further use. The total solid (TS) and volatile solid (VS) of rice straw were 87.62% and 78.34%, respectively. The rice straw was composed of 38.29% cellulose, 27.23% hemicellulose, 2.42% lignin and 3.93% ash. The inoculum was taken from a mesophilic anaerobic digester operated for two years. The TS and VS of inoculum were 12.95% and 6.62%, respectively. Prior to use for thermophilic AD, the inoculum was gradually acclimated to thermophilic conditions in water bath through increasing 1.0 °C per day from 37 °C to 55 °C (Broughton et al., 1998). 2.2. Experimental methods 2.2.1. Anaerobic digestion AD experiments were conducted at mesophilic (37 °C) and thermophilic (55 °C) conditions. The solid concentrations of 7% and 17% and the hydraulic retention time of 17 days and 24 days were considered. The batch experiments of rice straw were implemented using 0.5 L AD reactor. During each run, 25 g rice straw (28.53 g fresh rice straw) was added and inoculated with 10 g sludge (77.16 g fresh sludge). To avoid acidification, 2 g NaHCO3 was added into the reactors. Then deionized water was added to make the final total solids concentrations of 7% and 17%. All reactors were capped with rubber stoppers and put into a water bath. Before the fermentation test, the reactors were flushed with nitrogen to remove oxygen from the headspace and maintain an anaerobic environment. To minimize errors, each run was conducted in duplicate. The biogas volume was measured by drainage method, and the biogas was collected by a 1 mL plastic syringe for the gas composition analysis. 2.2.2. Ozone combined with aqueous ammonia pretreatment Ozone was produced by an Ozone Generator (XKH-YA10G, Wuhan, Hubei, China) with ozone concentration (16.67 mg/L) at a flow rate of 10 L/min. The solid digestate (10 g) was adjusted to a moisture content of 40% w/w and put into an enclosed stainless steel reactor. The reactor was operated in a semi-continuous mode. The ozone pretreatment was implemented for 45 min. The ozone dose was 0.75 g O3/g-TS. The treated samples were dried at 50 °C and stored for further analysis and use. After ozone pretreatment, the solid digestate (7.5 g) was subsequently treated using 26–28% (w/w) ammonium hydroxide solution under the solid to liquid ratio of 1:10 at 50 °C for 6 h. After completion of soaking, the solid digestate was separated from the liquid by a vacuum filtration with 0.1 mm mesh, and the filter cake was washed with 1 L distilled water to neutralize pH. Then the treated samples were dried at 50 °C. 2.2.3. Enzymatic hydrolysis process 3 g dry solid fiber and 47 mL acetic acid-sodium acetate buffer (0.2 M, pH = 4.8) were mixed into a 100 mL shake flask, which made the dry matter concentration 6.0%. All flasks were added with 0.5 g mixed-cellulases containing b-glucanase P 6  104 U, cellulase P 600 U and xylanase P 10  104 U (Imperial Jade Biotechnology Co., Ltd). During the enzymatic hydrolysis, the flasks were shaken at 150 rpm at 50 °C for 48 h. The amounts of hydrolyzed sugars were determined by high performance liquid chromatography (HPLC).


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2.2.4. Ethanol fermentation Angel yeast Saccharomyces cerevisiae (Angel Biotechnology Group, Hubei) was used in this fermentative experiment. The inoculum to solution ratio was 1:20. All flasks were shaken in a thermostatic shaking water bath at 37 °C for 48 h. Samples were taken from both the beginning and the end of this process for ethanol and sugars analysis. 2.3. Analytical methods Total solids and volatile solids were measured using standard methods (APHA, 1998). The pH was measured using pH meter (FE20 LAB). Fiber composition was measured using the Laboratory Analytical Procedure (LAP) developed by the National Renewable Energy Laboratory (NREL) (Sluiter et al., 2008). The biogas composition was detected by a GC9790II gas chromatography (Fuli Analytical Instrument Co., Ltd., Taizhou, Zhejiang, China) equipped with a thermal conductivity detector (TCD), a 1.5 m stainless steel packed column with 5A molecular sieve and a Hayesep Q packed column (Lanzhou Atech Technologies Co., Ltd., Lanzhou, Gansu, China). The temperatures of the injector, detector and oven were maintained at 55 °C, 100 °C and 50 °C, respectively. Argon was used as the carrier gas at a flow rate of 30 mL min 1. Crystallinity, a physical property expressed as crystallinity index (Cr I), was examined by measuring X-ray diffraction using a powder X-ray diffract meter (Bruker D8 Advance, Karlsruhe, Germany) with Cu Ka radiation at 40 kV and 40 mA (Fu et al., 2015). Glucose in the hydrolysate was analyzed using an Agilent1220 chromatography system (Agilent Technologies, Palo alto, California, USA) equipped with a Zorbax carbohydrate analytical column (4.6  150 mm) and a refractive index detector. The ethanol concentration was detected by a GC9790II gas chromatography equipped with a flame ionized detector (FID) and a KB-Wax column (Kromat Corporation, New Jersey, USA). The temperatures of the injector, detector and oven were maintained at 250 °C, 250 °C and 130 °C, respectively. Nitrogen was used as the carrier gas. The total glucose conversion and ethanol production efficiency were calculated based on the previous research (Yue et al., 2011), respectively. 3. Results and discussion

Fig. 1. Biogas production during anaerobic digestion (a. 7% solid concentration; b. 17% solid concentration).

The methane content was also provided to calculate methane yield. The results showed that the average methane content of thermophilic digestion was about 70%, significantly (p < 0.05) higher than that of mesophilic digestion. After 24 days of AD, the methane yields at the solid concentration of 7% and 17% were 214.8 mL/g and 185.5 mL/g under thermophilic condition, respectively. Under the aforementioned conditions, the methane yields of thermophilic digestion were 21.5% and 72.2% higher than those of mesophilic digestion, respectively.

3.1. Biogas production and methane yield Fig. 1 shows the accumulated biogas production of rice straw during AD processes. The biogas and methane yields based on total solids in rice straw are shown in Fig. 2. The experimental data and one-way analysis of variance (ANOVA) showed that AD temperature had a significant (p < 0.05) effect on biogas production when solid concentrations increased from 7% to 17%. A maximum biogas yield of 304.2 mL/g was obtained at thermophilic condition (55 °C) for 24 days with solid concentration of 7% instead of 17% (biogas yield of 277.2 mL/g). The solid concentration of 17% also did not produce more biogas than that of 7% probably because of lower mass transfer rate between substrate and microorganism. Under mesophilic condition, higher solid concentrations resulted in a significant (p < 0.05) decrease in biogas yield for 17 days and 24 days. This is because the anaerobic sludge at the digestion temperature of 37 °C and the solid concentration of 17% was acidified to pH 6.0 that led to a decrease in the biogas yield (Sheets et al., 2015). Compared with that of mesophilic digestion, the biogas yield of thermophilic digestion was higher. Although thermophilic digestion required more heating, it can be compensated by higher methane yield than that of mesophilic digestion (Böske et al., 2015).

3.2. Solid digestate production and its composition The solid digestate production and its composition are shown in Table 1. A high solid digestate of 650–850 g/kg was produced. The amount of solid digestate produced from thermophilic digestion decreased compared with that from mesophilic digestion. The solid digestate also contained higher fiber content than that of raw rice straw. The cellulosic and heimicellulosic contents in solid digestate decreased with an increased digestion time from 17 days to 24 days. Moreover, the cellulosic and heimicellulosic contents of solid digestate after thermophilic digestion were lower than those after mesophilic digestion. Although the mesophilic digestion at the solid concentration of 17% did not achieve high biogas productivity, the degradation of cellulose and hemicelluloses was not low. This is because too much volatile fatty acids was produced to be used by methanogens in the first 3 days due to the inhibition of acidification (Chen et al., 2015). Fig. 3 shows the remaining cellulose and heimicellulose in solid digestate after AD. At 55 °C and 24 days, the highest biogas and methane production were achieved. However, the remaining celluloses in the solid digestate after thermophilic AD at 7% and 17%


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Fig. 2. Biogas, methane yield and methane content during anaerobic digestion (a. 7% solid concentration; b. 17% solid concentration). Fig. 3. Remaining fiber in solid digestate after anaerobic digestion (a. 7% solid concentration; b. 17% solid concentration).

solid concentration were still 43.0% and 40.3%, respectively. Thus, the solid digestate contains high cellulosic content available for further reuse.

3.3. Hydrolysis of solid digestate using ozone and aqueous ammonia pretreatment The mass changes and lignocellulosic content of solid digestate after pretreatment are also shown in Table 1. After pretreatment, the mass loss of the solid digestate produced from the digestion time of 17 days was higher than that of 24 days. This indicated that longer digestion time resulted in more degradation of readily biodegradable fraction (non-fiber and fiber fraction) (Pokój et al., 2015). Therefore, the solid digestate from longer digestion time may be more recalcitrant to degradation. The cellulosic content of solid digestate significantly (p < 0.05) increased after pretreatment. The hemicellulose content in percentage did not have signif-

icant changes. However, the lignin content was increased, not consistent with the delignification theory of aqueous ammonia. That was because aqueous ammonia can selectively remove lignin. After AD, the easily degradable lignin was removed, but the remaining lignin may be recalcitrant. Therefore, the lignin in solid digestate cannot be decreased. This also demonstrated that solid digestate was recalcitrant to chemical pretreatment. Therefore, for the cellulose’s recovery in solid digestate, ozone and aqueous ammonia pretreatment mainly focused on destroying the structure of solid digestate and increasing the surface area. The glucose concentration and total glucose conversion during enzymatic hydrolysis are shown in Fig. 4. The glucose concentration during enzymatic hydrolysis of the pretreated solid digestate from the digestion time of 17 days was higher than that of 24 days. However, the operational temperature of AD did not have a significant effect on the glucose concentration during enzymatic

Table 1 Changes in solid digestate during anaerobic digestion and pretreatment. Anaerobic digestion

Solid digestate

Pretreated solid digestate

Solid con. [%]

Temp. [°C]

Time [d]

Production [g/kg-raw feed]

Cell. [%]

Hemi. [%]

Lignin [%]

Production [g/kg-raw feed]

Cell. [%]

Hemi. [%]

Lignin [%]


37 55 37 55 37 55 37 55

17 17 24 24 17 17 24 24

780.29 690.86 757.14 663.14 855.43 721.71 811.14 694.57

23.64 19.86 19.47 17.73 24.34 20.21 21.90 15.85

17.26 14.84 13.25 13.02 11.27 11.69 13.67 11.69

6.08 8.48 7.42 4.20 5.90 4.67 3.84 6.83

522.27 509.39 478.51 458.01 565.72 492.69 509.40 471.38

32.55 29.59 32.10 26.39 33.91 26.33 26.39 21.71

16.70 15.83 16.01 14.02 16.43 12.97 14.02 13.31

8.26 9.00 8.25 10.92 7.29 11.17 10.92 10.98



D. Wang et al. / Bioresource Technology 207 (2016) 52–58

Fig. 4. Glucose concentration and total glucose conversion in enzymatic hydrolysis (a. 7% solid concentration; b. 17% solid concentration).

hydrolysis of the pretreated solid digestate. The total glucose conversion with respect to the pretreated solid digestate was calculated to evaluate the pretreatment performance for enzymatic hydrolysis. The results showed that the total glucose conversion from thermophilic digested digestate was significantly (p < 0.05) higher than that from mesophilic digested digestate. Therefore, thermophilic digested digestate can be considered as a better option to produce ethanol.

Fig. 5. Ethanol yield and ethanol production efficiency from solid digestate (a. 7% solid concentration; b. 17% solid concentration).

obtained 75.3% and 73.3% of ethanol production efficiency with 2% NaOH and 130 °C for 2–3 h pretreatment for CSTR and PFR AD fibers, respectively. In this study, thermophilic AD can obtain higher ethanol production efficiency than mesophilic AD. This is probably because the solid digestate from thermophilic digestion can be broken effectively to enhance enzymatic hydrolysis (Fernán dez-Rodríguez et al., 2013). 3.5. Mass and energy balances

3.4. Ethanol yield and ethanol production efficiency The ethanol yield and ethanol production rate are shown in Fig. 5. The ethanol was successfully produced from solid digestate. Moreover, the operational temperature of AD did not significantly (p > 0.05) affect ethanol yield. The decrease in digestion time resulted in an increase in ethanol yield, because longer anaerobic time caused more lignocelluloses degradation in AD process. Due to different characteristics and degradability of fiber in the processes of pretreatment and enzymatic hydrolysis, ethanol production efficiency was also used to evaluate the ethanol fermentation process of solid digestate. The calculation of ethanol production efficiency was based on the cellulosic content in the solid digestate. For the solid digestate produced under the thermophilic condition at 24 days with the solid concentrations of 7% and 17%, the ethanol production efficiency was 75.2% and 74.8%, respectively. The aforementioned values for the ethanol production efficiency showed no significant (p > 0.05) difference under the conditions of 7% and 17% solid concentration, and were 14.5% and 24.14% higher than those from the solid digestate produced under the mesophilic condition. Similar results were obtained in the previous studies. Yue et al. (2011)

Mass and energy balances were conducted based on the experimental data for methane and ethanol production (Table 2). In this study, the methane production was calculated based on dry rice straw. Under the conditions of 7% solid concentration and 24 days, the methane production from the mesophilic and thermophilic digestions was 136.1 g/kg and 165.4 g/kg, respectively, and the corresponding ethanol production was 78.0 g/kg and 70.1 g/kg, respectively. A similar net energy of 5558 kJ and 5535 kJ was obtained for the mesophilic and thermophilic digestions under the aforementioned conditions, respectively. These results indicated that the effect of AD temperature on the net energy balance was not significant (p > 0.05) at low concentration digestion when the digestion time was sufficient. At the solid concentration of 17%, the mesophilic AD tended to acidification in the first 3 days that resulted in a low methane production (He et al., 2012). The methane production at 17 days was 53.3 g/kg and the corresponding ethanol production was 123.3 g/kg. The methane production at 24 days was 82.9 g/kg and the corresponding ethanol production was 84.8 g/kg. The results showed that the integrated methane and ethanol production depended on the efficient operation of AD. If not efficient, the relative higher ethanol production could


D. Wang et al. / Bioresource Technology 207 (2016) 52–58 Table 4 Value of X-ray diffraction peaks and crystalline index value of rice straw.

not improve the net energy, for example, 3135 kJ at 17 days and 4204 kJ at 24 days. Among the different conditions, high temperature and solid concentration for the integrated methane and ethanol production would be a better choice. Although the energy input of thermophilic AD was 1.67 times than that of mesophilic AD, the net energy from thermophilic AD and ethanol fermentation approached or exceeded that from mesophilic AD and ethanol fermentation. Based on the net energy balance, the experimental batch at 55 °C, 17% and 24 days achieved the highest net energy of 6416 kJ. The results of the integrated methane and ethanol production were compared to data in previous literature shown in Table 3, suggesting similar methane and ethanol production was achieved. Moreover, the methane and ethanol production can be improved by increasing digestion temperature, solid concentration and digestion time. This is beneficial to have such information for practical application of the two-stage conversion of waste biomass to methane and the ethanol.


Location [deg]

Intensity [Cps]

2h = 18°peak intensity [Cps]

Cr I [%]

Rice straw AD at 55 °C, 17% and 24 days Ozone pretreatment SAA pretreatment Enzymatic hydrolysis

22.58 22.54

1354 467

912 280

32.64 40.04

22.72 22.52 22.78

468 733 428

309 445 361

33.97 39.29 15.65

showed that the Cr I of the rice straw was 32.64%. After AD process, the Cr I of the solid digestate increased to 40.04%. This is because most of the hemicellulose in amorphous region was removed and hydrolyzed for enhancing biogas production (Kim et al., 2014; Lima et al., 2013), resulting in a rise of the Cr I and exposed crystalline cellulose. The intensity of crystalline and amorphous peaks become weaker in solid digestate compared to rice straw. After ozone pretreatment, the Cr I of the pretreated solid digestate reduced to 33.97% due to an increase in amorphous regions, resulting from swelling of crystalline cellulose. While soaking in aqueous ammonia, the Cr I of the sample was raised to 39.29%, resulting from the solubilization of the non-fiber and fiber components including partial lignin, xylan, and other components. The results in this study were in agreement with previous results that the Cr I of biomass increased after aqueous ammonia pretreatment (Fu et al., 2015; Yu et al., 2014). The appearance of crystalline peak is shown in Fig. S1 for aqueous ammonia pretreatment, indicating cellulose is exposed because of the pretreatment applied (Corredor,

3.6. Effect of pretreatment on solid digestate crystallinity To reveal the degradation mechanism of cellulose and hemicellulose for the two-stage conversion of waste biomass to methane and the ethanol using combined ozone and aqueous ammonia pretreatment, X-ray diffraction was used to determine the samples’ Cr I at the different stages in the whole process. The AD conditions for the samples were at the digestion temperature of 55 °C, the solid concentration of 17% and the digestion time of 24 days. The Cr I for each sample was calculated and given in Table 4. The results

Table 2 The energy products production from integrated processes per kg of rice straw.a Anaerobic digestion Solid con. [%] 7


Energy production [g]

Energy inputb [kJ] Anaerobic digestion

Temp. [°C]

Time [d]



37 55 37 55 37 55 37 55

17 17 24 24 17 17 24 24

99.7 118.3 136.1 165.4 53.3 120.2 82.9 142.8

96.1 84.5 78.0 70.1 123.3 96.2 84.8 65.2

2106 3510 2106 3510 867 1445 867 1445

Energy outputc [kJ] Ethanol ferment. 1526 1341 1238 1113 1958 1528 1347 1036

Net energy

Anaerobic digestion

Ethanol ferment.

Balance [kJ]

4993 5929 6820 8287 2669 6024 4154 7155

2566 2255 2082 1871 3291 2569 2264 1742

3927 3333 5558 5535 3135 5620 4204 6416


The calorific power of per kg dry rice straw was 17,600 kJ. The energy input of anaerobic digestion was calculated by MacLellan et al. (2013). The energy input of ethanol fermentation was calculated by Piccolo and Bezzo (2009). c The energy output of anaerobic digestion [kJ/kg] = methane production [g]  high heating value of methane [kJ/g], where the high heating value was 50 kJ/g (35.9 kJ/L). The energy output of ethanol fermentation [kJ/kg] = ethanol production [g]  high heating value of ethanol [kJ/g], where the high heating value was 26.7 kJ/g. d Methane density is 0.77 g/L. b

Table 3 Comparison of methane and ethanol production based on raw feeding substrate. Anaerobic digestion


Methane production [g/kg]

Ethanol production [g/kg]


CSTR PFR Dairy cow feces 40 °C, 20 d HRT 5% TS, 20 d HRT Swine manure:corn stover 20:80 40:60 80:20 Rice straw 37 °C, 7% TS, 24 d 55 °C, 7% TS, 24 d 55 °C, 17% TS, 17 d 55 °C, 17% TS, 24 d

2% NaOH, 130 °C, 3 h 2% NaOH, 130 °C, 2 h

164 125

26 23

Yue et al. (2011)

139 152 112

35 50 48

136 165 120 142

78 70 96 65

2% NaOH, 130 °C, 2 h

0.75 g O3/g-TS Soaking aqueous ammonia (26–28%) 50 °C, 6 h

MacLellan et al. (2013)

This study


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2008). Since aqueous ammonia pretreatment removed large amounts of the aforementioned unfavorable components from the treated samples, more crystalline cellulose was exposed to enzymatic hydrolysis. Thus, the Cr I of the samples was greatly decreased to 15.65% after enzymatic hydrolysis. This demonstrated that cellulose was effectively recovered from solid digestate using combined ozone and aqueous ammonia pretreatment. 4. Conclusions This study demonstrated that the residual organic carbon in recalcitrant solid digestate can be recovered to produce ethanol. The ozone combined with aqueous ammonia pretreatment was a better way to enhance ethanol production from solid digestate of mesophilic and thermophilic ADs. The two-stage conversion of rice straw at 55 °C, 17% and 24 days can obtain the highest net energy of 6416 kJ. Under these conditions, the methane and ethanol productions were 142.8 g/kg and 65.2 g/kg, respectively. This study also showed that anaerobic digestion coupling with ethanol fermentation was an effective biorefinery process, achieving a successive utilization of organic carbon. Acknowledgements The project was financed by the National Natural Science Foundation Program of China for (No. 51406064), Central Universities Fundamental Research Funds (No. 2015PY077) and the Agricultural Science and Technology Innovation Program (ASTIP) from Chinese Academy of Agricultural Sciences. The authors gratefully acknowledge their supports. And the authors are also grateful for financial support from China Scholarship Council. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2016.01. 119. References APHA, 1998. Standard Methods for the Examination of Water and Wastewater, 20th ed. American Public Health Association, Washington, DC. Böske, J., Wirth, B., Garlipp, F., Mumme, J., Van den Weghe, H., 2015. Upflow anaerobic solid-state (UASS) digestion of horse manure: thermophilic vs. mesophilic performance. Bioresour. Technol. 175, 8–16. Broughton, M.J., Thiele, J.H., Birch, E.J., Cohen, A., 1998. Anaerobic batch digestion of sheep tallow. Water Res. 32, 1423–1428. Bule, M.V., Gao, A.H., Hiscox, B., Chen, S., 2013. Structural modification of lignin and characterization of pretreated wheat straw by ozonation. J. Agric. Food. Chem. 61, 3916–3925. Bustamante, M.A., Alburquerque, J.A., Restrepo, A.P., de la Fuente, C., Paredes, C., Moral, R., Bernal, M.P., 2012. Co-composting of the solid fraction of anaerobic digestates, to obtain added-value materials for use in agriculture. Biomass Bioenergy 43, 26–35. Chen, X., Yuan, H., Zou, D., Liu, Y., Zhu, B., Chufo, A., Jaffar, M., Li, X., 2015. Improving biomethane yield by controlling fermentation type of acidogenic phase in twophase anaerobic co-digestion of food waste and rice straw. Chem. Eng. J. 273, 254–260. Corredor, D.Y., 2008. Pretreatment and Enzymatic Hydrolysis of Lignocellulosic Biomass. Kansas State University, Manhattan. Dahlin, J., Herbes, C., Nelles, M., 2015. Biogas digestate marketing: qualitative insights into the supply side. Resour. Conservat. Recycl. 104, 152–161, Part A.

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