Unraveling the structure of sugarcane bagasse after soaking in concentrated aqueous ammonia (SCAA) and ethanol production by Scheffersomyces (Pichia) stipitis
© Chandel et al.; licensee BioMed Central Ltd. 2013
Received: 15 March 2013
Accepted: 2 July 2013
Published: 15 July 2013
Fuel ethanol production from sustainable and largely abundant agro-residues such as sugarcane bagasse (SB) provides long term, geopolitical and strategic benefits. Pretreatment of SB is an inevitable process for improved saccharification of cell wall carbohydrates. Recently, ammonium hydroxide-based pretreatment technologies have gained significance as an effective and economical pretreatment strategy. We hypothesized that soaking in concentrated aqueous ammonia-mediated thermochemical pretreatment (SCAA) would overcome the native recalcitrance of SB by enhancing cellulase accessibility of the embedded holocellulosic microfibrils.
In this study, we designed an experiment considering response surface methodology (Taguchi method, L8 orthogonal array) to optimize sugar recovery from ammonia pretreated sugarcane bagasse (SB) by using the method of soaking in concentrated aqueous ammonia (SCAA-SB). Three independent variables: ammonia concentration, temperature and time, were selected at two levels with center point. The ammonia pretreated bagasse (SCAA-SB) was enzymatically hydrolysed by commercial enzymes (Celluclast 1.5 L and Novozym 188) using 15 FPU/g dry biomass and 17.5 Units of β-glucosidase/g dry biomass at 50°C, 150 rpm for 96 h. A maximum of 28.43 g/l reducing sugars corresponding to 0.57 g sugars/g pretreated bagasse was obtained from the SCAA-SB derived using a 20% v/v ammonia solution, at 70°C for 24 h after enzymatic hydrolysis. Among the tested parameters, pretreatment time showed the maximum influence (p value, 0.053282) while ammonia concentration showed the least influence (p value, 0.612552) on sugar recovery. The changes in the ultra-structure and crystallinity of native SCAA-SB and enzymatically hydrolysed SB were observed by scanning electron microscopy (SEM), x-ray diffraction (XRD) and solid-state 13C nuclear magnetic resonance (NMR) spectroscopy. The enzymatic hydrolysates and solid SCAA-SB were subjected to ethanol fermentation under separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) by Scheffersomyces (Pichia) stipitis NRRL Y-7124 respectively. Higher ethanol production (10.31 g/l and yield, 0.387 g/g) was obtained through SSF than SHF (3.83 g/l and yield, 0.289 g/g).
SCAA treatment showed marked lignin removal from SB thus improving the accessibility of cellulases towards holocellulose substrate as evidenced by efficient sugar release. The ultrastructure of SB after SCAA and enzymatic hydrolysis of holocellulose provided insights of the degradation process at the molecular level.
KeywordsSugarcane bagasse Taguchi design Soaking in concentrated aqueous ammonia Scheffersomyces (Pichia) stipitis Bioethanol
The increased demand for energy, regular price hikes of gasoline, geo-political factors, and environmental damage have alarmed the scientific community, economists and governments to evaluate the potential of cellulosic ethanol as a sustainable, economic and ecofriendly alternative of gasoline [1–3]. Sugarcane bagasse (SB) is an excellent raw material for second generation ethanol production in countries like Brazil, India and China where it is generated in huge amount every year . In Brazil, around 163–169 million tons of SB was generated in the 2012/13 harvest . Almost 50% of SB is used for energy generation in industry but the rest remains unused. The judicious conversion of the left over SB into second generation ethanol may have sustainable, economic and strategic benefits; however it needs intensive technological and multidisciplinary efforts . SB is primarily comprised of carbohydrate polymers (hemicellulose and cellulose) and lignin linked into a highly complex and recalcitrant matrix. Because of this, pretreatment is an inevitable process to render the carbohydrate fraction accessible for cellulolytic enzyme action to release fermentable sugars followed by their bioconversion into ethanol by microbial fermentation. Together, pretreatment and saccharification contributes 50-60% of the total cost incurred for bioethanol production from lignocellulosics . In the past, several pretreatment methods such as auto-hydrolysis, liquid hot water, super critical fluids, alkali, acid, organic solvents, and biological pretreatments have been applied to SB  to make it more amenable to saccharification. Despite several developments in pretreatment processes for SB, complete mechanisms involved in biomass deconstruction remain to be elucidated thus complicating the decision of which pretreatment to apply .
Ammonium hydroxide mediated pretreatment is a state-of-the-art technology which specifically acts on the lignin-carbohydrate complex by cleaving ester linkages via ammonolysis to remove lignin and increase the accessibility of the remaining holocellulose fraction to cellulase action . Ammonia pretreatment in any form such as ammonia fiber expansion (AFEX), ammonia freeze explosion and soaking in aqueous ammonia (SAA) has been proven as an effective strategy for the delignification of various lignocellulosic substrates [5–13]. Key for the success of AFEX is selection of influential parameters in their appropriate ranges to maximize lignin removal while minimizing carbohydrate degradation [5, 9]. After pretreatment, the ammonia solution can be recovered for subsequent use as a delignification agent to further economize the process .
The Taguchi method is a statistical method that utilizes the fundamental principles of statistics, randomization and replication for the design and analysis of factorial experiments. This method allows the investigation of the influence of several parameters on a process while saving time by reducing the number of experiments required .
The delignified material can be separately hydrolysed by cellulolytic enzymes and the resultant sugar syrup can be fermented directly into ethanol by an appropriate microorganism (SHF). Another option is to perform the enzymatic hydrolysis and fermentation of all released sugars (pentose and hexose) into ethanol simultaneously (SSF). The latter has shown several advantages such as process intensification, less processing time, low contamination risk, cost reduction and high ethanol titers . The most important feature of SSF is to overcome the enzyme inhibition by the released sugars during enzymatic hydrolysis as the released sugars are simultaneously converted into ethanol by the microorganism. However, the difference in optimum temperature for microbial fermentation and enzymatic hydrolysis is a major concern of SSF [3, 15].
Here, we report the ammonia pretreatment optimization of SB and the efficiency of all pretreated bagasse samples for sugar recovery during enzymatic hydrolysis. Ethanol production was evaluated under SHF and SSF using the sugar hydrolysate of optimized ammonia pretreated bagasse or delignified SB by S. stipitis NRRL Y-7124. Structural and crystallinity changes in native, ammonia pretreated and enzymatically digested SB were also analyzed by SEM, XRD and solid-state 13C NMR spectroscopy.
Results and Discussion
Optimization of ammonia pretreatment and enzymatic digestibility
Factors and their levels for soaking in concentrated aqueous ammonia solution (SCAA) of sugarcane bagasse
Ammonia concentration (% v/v)
Taguchi orthogonal array design of experiment with central point (L 8 ) and the data collection
Ammonia concentration (% v/v)
Recovery of reducing sugars (g/L)
Analysis of variance (ANOVA), F-test and interactions
Time × Temperature
Ammonia concentration (% v/v)
Time × Ammonia concentration
Temperature x Ammonia concentration
In addition to appropriate hydrolysis conditions, a fine cocktail of cellulolytic enzymes comprising cellulases, β-glucosidases, xylanases is required to obtain sugar monomers from SCAA-SB . We obtained low sugar recovery from ammonia-pretreated bagasse samples probably due to low solid (SB) loadings in ammonia solutions (1:10). Ammonium hydroxide solubilizes the hemicellulose and cellulose considerably in addition to significant removal of lignin from the substrate . For the pretreatment of SB, process parameters such as ammonia concentration, temperature and time that were used in this study were previously studied with various lignocellulosic materials [7, 13, 18]. The important aspect of AFEX pretreatment at mild temperatures (40-90°C) for longer reaction times is known to preserve most of the carbohydrate in the samples . For instance, rice straw after pretreatment with aqueous ammonia (21% w/w, 10 h, 69°C) showed 71.1% enzymatic hydrolysis using 15 FPU of cellulase/g-glucan and 30 CBU of β-glucosidase/g-glucan). The sugar recovery from various AFEX-pretreated substrates depends upon the raw material and pretreatment conditions employed. For example, Kim et al.  pretreated barley hull (15 wt.% ammonia, 24-72 h, 75°C) and reported 50-66% lignin removal which showed saccharification yield (83% glucan, 63% xylan solubilisation) with 15 FPU/g –glucan enzyme loadings. These ammonia-assisted delignification studies were based on varying range of diluted ammonia solution (15-29% ammonia), temperature (50-90°C), time (12-72 h) and solid to liquid ratio (1: 10).
Cell wall characterization and structural analysis
Cell wall composition
The physico-chemical analysis of native sugarcane bagasse cell wall revealed the following composition (on percentage base): total lignin, 27.53% (24.46% Klason lignin + 3.07% acid soluble lignin); 41.96% cellulose; 19.20% hemicellulose; 2.90% extractives; 8.16% moisture and 1.5% structural ash. This SB composition is comparable with other reports [2, 4, 18]. However, direct comparison of compositional data is not feasible since the chemical composition of sugarcane bagasse like any other lignocellulosic material depends on several factors such as the variety, location, and agricultural practices used to grow the crop and the employed analytical method for cell wall composition analysis . After pretreatment of SB with 20% (v/v) NH4OH for 24 h at 70°C, 41.51% lignin removal was observed. The final cell wall composition (% base) of SCAA-bagasse was as follows: lignin, 16.10; cellulose, 67.42; hemicellulose, 6.0; ash, 2.5 and moisture, 8.16. Extractives were found in negligable amount. Cellulose was found to be increased by 62.20% in SCAA-bagasse samples. On the contrary, a considerable amount of hemicellulose (68.75%) was lost during the pretreatment. Loss of hemicellulose was expected in addition to removal of lignin during the SCAA of sugarcane bagasse. However, loss of hemicellulose and removal of lignin during ammonia hydroxide mediated pretreatment depend upon the reaction conditions and lignocellulosic material used. Prior and Day  reported the composition of sugarcane bagasse (21.1% lignin, 56.6% glucan, 24% xylan and 1.2% arabinan) after ammonia hydroxide pretreatment (0.5 g NH4OH of 28% v/v per gram bagasse, 160°C, 60 min). Aita et al.  observed 55% delignification, 9% glucan loss and 30% removal of hemicellulose from ammonia pretreated (bagassae 1: NH4OH 0.5: water 8, 160°C, 60 min, 0.9-1.1 MPa) high fiber sugarcane bagasse. Aqueous ammonium hydroxide pretreatment of barley hull (15% w/w hydrated ammonia, 24-72 h, 75°C) showed 50-66% lignin removal while maintained 65-76% xylan and negligible loss of glucan . Ko et al.  reported 60.6 ± 0.3% lignin removal and retaining of 86.9 ± 1.1% glucan in aqueous-ammonia soaking pretreatment of rice straw (70°C, 12 h and 20% w/w hydrated ammonia) which eventually showed 70.4 ± 1.1% enzymatic digestibility. Complete delignification from biomass is extremely difficult due to the mechanistic location of lignin within the lignin-carbohydrate complex, strong poly-ring bonds of C-O-C, C-C and hydrophobicity [6, 11]. It is speculated that the aqueous ammonia-mediated pretreatment at low temperature show a considerable loss of hemicellulose and lower lignin removal [11, 12] which is also evident with the results obtained in the present study.
Scanning electron microscopy (SEM)
X-ray diffraction (XRD)
Solid-state 13C NMR spectroscopy
For untreated SB sample, the lines in the 50 to 120 ppm region are primarily due to cellulose carbons with contributions from lignin and hemicellulose signals. C6 and C4 carbon from amorphous cellulose  are lines 3 at 63 ppm and 7 at 84 ppm, respectively, while C6 and C4 carbon in crystalline cellulose are lines 4 at 65 ppm and 8 at 88 ppm [25–27]. Lignin signals are scattered throughout all spectral regions, lines labeled as 2, 11, 12, 13, 14 and 15 in Figure 4a. For lignin signals [25, 27], the line marked by * is due to aliphatic lignin carbons not bound to oxygen . Hemicelluloses carbons contribute to lines, 1, 3, 6, 7, 9 and 17 . Figure 4b shows the spectrum of ammonia-treated bagasse which reveals the intensity decrease of lines 1 and 17, show the removal of hemicellulose. Since all spectra were normalized with respect to line 10 (C1 carbon of cellulose), the intensity decrease of lines *, 2, 11,12,13,14 and 15 indicates a reduction of the lignin to cellulose fraction in the bagasse sample after the ammonia treatment. Figure 4c shows the spectrum of the solid fraction obtained after enzyme hydrolysis of the ammonia treated bagasse sample. Assuming that the relative amount of lignin is constant during the enzyme hydrolysis, the increase of lignin signals, lines *,2,11,12,13,14 and 15, relative to cellulose ones, lines 3,4,5,6,7,8 and 10, is associated to the removal of cellulose [17, 25].
Ethanol fermentation (SHF)
Kinetic parameters for ethanol production by S. stipitis NRRL Y-7124 under SHF and SSF process after 72 h and 96 h respectively
Initial sugars (g/L)
Ethanol produced (g/L)
Cells produced (g/L)
Ethanol yield (g/g)
Fermentation efficiency (%)
Cells yield (g/g)
Ethanol productivity (g/L/h)
Cells productivity (g/L/h)
Simultaneous saccharification and fermentation (SSF)
In the present study, after 24 h of pre-hydrolysis with cellulases, sugar release was 27.95 g/l. The yeast was added after the 24 h pre-hydrolysis step to initiate ethanol fermentation under SSF. Pre-hydrolysis has been shown to increase sugar recovery prior to ethanol production. Jin et al.  reported improved ethanol metabolic yield (90.3%) after 24-h of pre-hydrolysis compared to 6-h of pre-hydrolysis (88% ethanol metabolic yield) from ammonium hydroxide pretreated corn stover. In the present study, maximum ethanol production (10.31 g/l, yield 0.387 g/g and 75.88% ethanol fermentation efficiency) was observed at 48 h of incubation followed by a gradual decrease in sugar level. Maximum ethanol productivity (0.214 g/l/h) was noticed during SSF. Previously, we reported ethanol production (15.73 ± 0.44 g/l) with the yield (0.42 ± 0.02 g/g) and productivity (0.218 ± 0.04 g/l/h) after the SSF of ammonia pretreated Saccharum spontaneum with S. stipitis NCIM 3498 . Cell growth during SSF was not detected due to the presence of solid substrates with the cells. Table 4 shows the kinetic parameters for ethanol production by S. stipitis NRRL Y-7124 under SSF process after 96 h of incubation. It is difficult to compare the results from previous studies due to difference in pretreatment conditions, enzymes used for hydrolysis, variation in fermentation conditions and microorganisms used for ethanol production.
Among the conditions studied for delignification of SB, we found pretreatment at 20% (v/v) NH4OH at 70°C for 24 h showed maximum lignin removal (41.51%) in addition to hemicellulose loss (68.75%) which increased the cellulose amount (62.2%) in the pretreated material. SCAA-SB showed maximum reducing sugars (28.43 g/l, 0.57 g/g bagasse) after 72 h of enzymatic hydrolysis. Temperature and pretreatment time contributed the maximum influence on enzymatic hydrolysis of ammonia hydroxide pretreated SB. SEM analysis indicated the cell wall disruption and heterogeneous nature of SB after delignification and enzymatic hydrolysis. XRD analysis also showed the change in crystallinity of SCAA-bagasse and enzymatically digested SB. Solid-state 13C NMR spectroscopy revealed changes in carbon chemical shift assignments in lignin, hemicellulose and cellulose fractions of bagasse. When SHF and SSF of pretreated bagasse was conducted using S. stipitis NRRL Y-7124, 3.83 g/l yield 0.289 g/g and 56.80% ethanol fermentation efficiency) and (10.31 g/l, yield 0.387 g/g and 75.88% ethanol fermentation efficiency) was obtained respectively.
Material and Methods
Preparation of raw substrate
Sugarcane bagasse was acquired from Usina Vale do Rosário (Morro Agudo, S.P), air-dried, and knife milled (Marconi Equipamentos, Model No. MA 680, Piracicaba-S.P, Brazil) and passed through a 20-mesh sieve. It was washed with running water to remove dust and dried at 45°C for further experiments.
Ammonia pretreatment optimization
For the pretreatment, sugarcane bagasse was treated with various concentration of aqueous ammonia in screw-capped laboratory bottles (Pyrex bottles) with no agitation. Solid-to-liquid ratio of 1:10 was applied. Taguchi experimental design (Taguchi method, L8 orthogonal array) was used to optimize the maximum delignification considering three independent variables (ammonia concentration, temperature and time) at two levels with center point (Table 1). A total number of 9 experiments were carried out in duplicate with center point (Table 2). The delignified SB obtained in each experiment was subjected to enzymatic hydrolysis and the sugars released after enzyme hydrolysis was the responsive variable (Table 2).
The experimental results were analyzed to extract independently the main effects of the factors; the analysis of variance (ANOVA) was then applied to determine which factors were statistically significant. The controlling factors were identified with the magnitude of effects qualified and the statistically significant effects determined. Accordingly, the optimal conditions were determined by combining the levels of factors that had the highest main effect value. All calculations were performed using software (Statistica 10, USA).
SCAA-bagasse samples were enzymatically hydrolysed to deploymerise the available carbohydrate into simpler sugars. Enzymatic hydrolysis of pretreated bagasse was performed in 150 mL Erlenmeyer flask containing (2 g d.wt. of ammonia pretreated bagasse) and 40 mL of citrate buffer (50 mM, pH: 4.8). Substrates with buffer were pre-incubated at room temperature for 1 h. Sodium azide was also added at a concentration of 0.005% to restrict any microbial growth during the course of enzymatic hydrolysis. The substrate soaked in citrate buffer was supplemented with cellulase loadings (15 FPU (Filter Paper Activity)/g of the dry biomass from Celluclast 1.5 L and 17.5 IU/g of the dry biomass of β-glucosidase from Novozym 188). Enzymatic hydrolysis was performed at 50°C at 150 rpm in incubator shaker (Innova 4000 Incubator Shaker, New Brunswick Scientific, Enfield, CT, USA). These enzymes were procured from Sigma Aldrich, USA. The enzymatic hydrolysis was performed up to 96 h. Samples were collected after every 24 h, centrifuged and analyzed to determine the total reducing sugars released.
Separate hydrolysis and fermentation (SHF)
Microorganism and inoculum preparation
Scheffersomyces stipitis NRRL Y-7124, native xylose-fermenting yeast, was used for the fermentation of enzymatic hydrolysates. Strains were maintained on YPD plates and stored at 4°C. Cells were grown in 150 mL Erlenmeyer flasks containing 50 mL of YPD (10 g/l yeast extract, 10 g/l peptone, 20 g/l glucose) in an orbital shaker incubator at 30°C, shaken at 200 rpm. Following 24 h growth, broth was centrifuged and inoculum was prepared corresponding to 1.0 g/l cells (d. wt). Inoculum was aseptically transferred into enzymatic hydrolysate (50 mL) supplemented with medium ingredients defined by Canilha et al .
Fermentation of enzymatic hydrolysates (50 mL) obtained from SCAA-SB (20% v/v NH4OH, 24 h, 70°C) was carried out in 150 mL Erlenmeyer flasks. The flasks were maintained in a rotator shaker (Innova 4000 Incubator Shaker, New Brunswick Scientific, Enfield, CT, USA) at 30°C and 200 rpm for 72 h. Four samples were collected in first 24 h (0, 8, 16 and 24 h) followed by every 24 h until 72 h of incubation.
Simultaneous saccharification and fermentation (SSF)
SSF was performed in 150 mL Erlenmeyer flasks containing 50 mL of fermentation medium prepared as above. For saccharification of SCAA-SB (20% v/v NH4OH, 24 h, 70°C), an appropriate amount of FPase and β-glucosidase was added as mentioned above. Sodium azide was not added in SSF. During first 24 h, enzymatic hydrolysis was carried out at 50°C and 150 rpm in a rotator shaker (Innova 4000 Incubator Shaker, New Brunswick Scientific, Enfield, CT, USA). After 24 h of enzymatic hydrolysis, S. stipitis was inoculated at a cell concentration of 1.0 g/l (d.wt). This reaction was carried out at 30°C, 200 rpm for 96 h.
Characterization of native, SCAA-SB and EH-SB
Cell wall compositional characterization
Native and SCAA-SB (20% v/v NH4OH, 24 h, 70°C) was characterized for moisture, lignin (Klason and acid soluble), hemicellulose and cellulose content following the method of Gouveia et al .
Scanning electron microscopy (SEM)
The SEM analysis of native, SCAA (20% v/v NH4OH, 24 h, 70°C) and enzymatically hydrolysed SB was performed as described earlier . Native, SCAA and enzymatically hydrolysed SB distributed on a 12 mm glass cover slip coated with poly-L-lysine (Sigma Diagnostics, S.P. Brazil). The dried section was mounted on aluminum stubs, sputter-coated (JEOL JFC-1600) with a gold layer, and used for scanning. The prepared samples were scanned and imaged with a Hitachi S520 scanning electron microscope (Hitachi, Tokyo, Japan).
X-ray diffraction (XRD)
Solid-state 13C nuclear magnetic resonance (NMR) spectroscopy
Solid-state 13C NMR analyses of native, SCAA-SB (20% v/v NH4OH, 24 h, 70°C) and enzyme digested SB were performed using a Varian Inova spectrometer operating at 13C and 1H frequencies of 88.02 and 350.50 MHz, respectively [17, 36]. A Varian 7-mm magic-angle spinning MAS double-resonance probe head was used. Spinning frequencies of 4.5 kHz were controlled by a Varian pneumatic system that ensures a rotation stability of about 2 Hz. Ramped cross-polarizations under magic angle spinning (CPMAS) combined with total suppression of spinning sidebands (TOSS) and heteronuclear 1H decoupling (CPMAS-TOSS) was used to acquire the 13C spectra. Typical π/2 pulse lengths of 4.0 μs (13C) and 4.5 μs (1H), cross-polarization time of 1 ms, acquisition time of 30 ms, and recycle delays of 2 s were used in all NMR analysis.
Soaking in concentrated aqueous ammonia
Soaking in aqueous ammonia
Ammonia fiber expansion
Soaking in concentrated aqueous ammonia- sugarcane bagasse
Scanning electron microscopy
Nuclear magnetic resonance
Cross-polarization under magic angle spinning and total suppression of spinning sidebands
Filter paper units
Separate hydrolysis and fermentation
Simultaneous saccharification and fermentation
Yeast peptone dextrose
High performance liquid chromatography.
Financial support provided by Bioenergy-Fundação de Amparo a Pesquisa do Estado de São Paulo (BIOEN/FAPESP-Project Numbers: 2008/57926-4, 2010/11258-0) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) is gratefully acknowledged. We thank Dr. Durval Rodrigues Jr. from Engineering School of Lorena, University of São Paulo, Dr. Igor Polikarpov from University of São Paulo -São Carlos for SEM and XRD analysis respectively. Authors also thank Dr. Eduardo R. de Azevedo and Mr. Oigres D. Bernardinelli from University of São Paulo -São Carlos for NMR analysis.
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