Ethanol Production from Sugarcane Bagasse by Zymomonas mobilis Using Simultaneous Saccharification and Fermentation (SSF) Process

Considerable efforts have been made to utilize agricultural and forest residues as biomass feedstock for the production of second-generation bioethanol as an alternative fuel. Fermentation utilizing strains of Zymomonas mobilis and the use of simultaneous saccharification and fermentation (SSF) process has been proposed. Statistical experimental design was used to optimize the conditions of SSF, evaluating solid content, enzymatic load, and cell concentration. The optimum conditions were found to be solid content (30%), enzymatic load (25 filter paper units/g), and cell concentration (4 g/L), resulting in a maximum ethanol concentration of 60 g/L and a volumetric productivity of 1.5 g L^?1?h^?1.     

Research on the Conditions of Enzymatic Saccharification for Sugarcane Bagasse

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Lime Pretreatment and Fermentation of Enzymatically Hydrolyzed Sugarcane Bagasse

Sugarcane bagasse was subjected to lime (calcium hydroxide) pretreatment and enzymatic hydrolysis for second-generation ethanol production. A central composite factorial design was performed to determine the best combination of pretreatment time, temperature, and lime loading, as well as to evaluate the influence of enzymatic loadings on hydrolysis conversion. The influence of increasing solids loading in the pretreatment and enzymatic hydrolysis stages was also determined. The hydrolysate was fermented using Saccharomyces cerevisiae in batch and continuous mode. In the continuous fermentation, the hydrolysates were concentrated with molasses. Lime pretreatment significantly increased the enzymatic digestibility of sugarcane bagasse without the need for prior particle size reduction. In the optimal pretreatment conditions (90 h, 90 °C, 0.47 g?lime/g bagasse) and industrially realistic conditions of hydrolysis (12.7 FPU/g of cellulase and 7.3 CBU/g of β-glucosidase), 139.6 kg?lignin/ton raw bagasse and 126.0 kg hemicellulose in the pretreatment liquor per ton raw bagasse were obtained. The hydrolysate from lime pretreated sugarcane bagasse presented low amounts of inhibitors, leading to ethanol yield of 164.1 kg?ethanol/ton raw bagasse.     

Statistical Optimization of Recycled-Paper Enzymatic Hydrolysis for Simultaneous Saccharification and Fermentation Via Central Composite Design

A central composite design of the response surface methodology (RSM) was employed to study the effects of temperature, enzyme concentration, and stirring rate on recycled-paper enzymatic hydrolysis. Among the three variables, temperature and enzyme concentration significantly affected the conversion efficiency of substrate, whereas stirring rate was not effective. A quadratic polynomial equation was obtained for enzymatic hydrolysis by multiple regression analysis using RSM. The results of validation experiments were coincident with the predicted model. The optimum conditions for enzymatic hydrolysis were temperature, enzyme concentration, and stirring rate of 43.1 °C, 20 FPU g⁻¹ substrate, and 145 rpm, respectively. In the subsequent simultaneous saccharification and fermentation (SSF) experiment under the optimum conditions, the highest 28.7 g ethanol l⁻¹ was reached in the fed-batch SSF when 5% (w/v) substrate concentration was used initially, and another 5% added after 12 h fermentation. This ethanol output corresponded to 77.7% of the theoretical yield based on the glucose content in the raw material.     

The Operable Modeling of Simultaneous Saccharification and Fermentation of Ethanol Production from Cellulose

An operable batch model of simultaneous saccharification and fermentation (SSF) for ethanol production from cellulose has been developed. The model includes four ordinary differential equations that describe the changes of cellobiose, glucose, yeast, and ethanol concentrations with respect to time. These equations were used to simulate the experimental data of the four main components in the SSF process of ethanol production from microcrystalline cellulose (Avicel PH101). The model parameters at 95% confidence intervals were determined by a MATLAB program based on the batch experimental data of the SSF. Both experimental data and model simulations showed that the cell growth was the rate-controlling step at the initial period in a series of reactions of cellulose to ethanol, and later, the conversion of cellulose to cellobiose controlled the process. The batch model was extended to the continuous and fed-batch operating models. For the continuous operation in the SSF, the ethanol productivities increased with increasing dilution rate, until a maximum value was attained, and rapidly decreased as the dilution rate approached the washout point. The model also predicted a relatively high ethanol mass for the fed-batch operation than the batch operation.     

Design and Evaluation of an Optimal Controller for Simultaneous Saccharification and Fermentation Process

Ethanol from corn is produced using dry grind corn process in which simultaneous saccharification and fermentation (SSF) is one of the most critical unit operations. In this work an optimal controller based on a previously validated SSF model was developed by formulating the SSF process as a Bolza problem and using gradient descent methods. Validation experiments were performed to evaluate the performance of optimal controller under different process disturbances that are likely to occur in practice. Use of optimal control algorithm for the SSF process resulted in lower peak glucose concentration, similar ethanol yields (13.38±0.36% v/v and 13.50±0.15% v/v for optimally controlled and baseline experiments, respectively). Optimal controller improved final ethanol concentrations as compared to process without optimal controller under conditions of temperature (13.35±1.28 and 12.52±1.19% v/v for optimal and no optimal control, respectively) and pH disturbances (12.65±0.74 and 11.86±0.49% v/v for optimal and no optimal control, respectively). Cost savings due to lower enzyme usage and reduced cooling requirement were estimated to be up to $1 million for a 151 million L/yr (40 million gal/yr) dry grind plant.     

Inhibitors Compounds on Sugarcane Bagasse Saccharification: Effects of Pretreatment Methods and Alternatives to Decrease Inhibition

Considering bioethanol production, extensive research has been performed to decrease inhibitors produced during pretreatments, to diminish energy input, and to decrease costs. In this study, sugarcane bagasse was pretreated with NaOH, H₂SO₄, and water. The higher concentration of phenols, 3.3 g/L, was observed in biomass liquid fraction after alkaline pretreatment. Acid pretreatment was responsible to release considerable acetic acid concentration, 2.3 g/L, while water-based pretreatment was the only to release formic acid, 0.02 g/L. Furans derivatives were not detected in liquid fractions regardless of pretreatment. Furthermore, washing step removed most of the phenols from pretreated sugarcane bagasse. Saccharification of alkali-pretreated biomass plus polyethylene glycol (PEG) at 0.4% (w/v) enhanced 8 and 26% the glucose and the xylose release, respectively, while polyvinylpyrrolidone (PVP) also at 0.4% (w/v) increased the release by 10 and 31% of these sugars, respectively, even without washing and filtration steps. Moreover, these polymers cause above 50% activation of endoglucanase and xylanase activities which are crucial for biomass hydrolysis.

     

Aqueous Ammonia Soaking of Switchgrass Followed by Simultaneous Saccharification and Fermentation

Simultaneous saccharification and fermentation (SSF) of switchgrass was performed following aqueous ammonia pretreatment. Switchgrass was soaked in aqueous ammonium hydroxide (30%) with different liquid–solid ratios (5 and 10 ml/g) for either 5 or 10 days. The pretreatment was carried out at atmospheric conditions without agitation. A 40–50% delignification (Klason lignin basis) was achieved, whereas cellulose content remained unchanged and hemicellulose content decreased by approximately 50%. The Sacccharomyces cerevisiae (D₅A)-mediated SSF of ammonia-treated switchgrass was investigated at two glucan loadings (3 and 6%) and three enzyme loadings (26, 38.5, and 77 FPU/g cellulose), using Spezyme CP. The percentage of maximum theoretical ethanol yield achieved was 72. Liquid–solid ratio and steeping time affected lignin removal slightly, but did not cause a significant change in overall ethanol conversion yields at sufficiently high enzyme loadings. These results suggest that ammonia steeping may be an effective method of pretreatment for lignocellulosic feedstocks.     

Optimization of Saccharification and Fermentation Process in Bioethanol Production from Oil Palm Fronds

Oil Palm Frond (OPF) is one of lignocellulosic biomass, which can be utilized as raw material for bioethanol production. Bioethanol is produced as alternative energy to substitute gasoline. There are four steps in bioethanol production from OPF, i.e pretreatement, saccharification, fermentation and purification process. In this study, optimization of saccharification and fermentation process for OPF was investigated. Two methods and the variations of enzyme concentration were carried out in the saccharification and fermentation process. Separate hydrolysis and fermentation process (SHF) and simultaneous saccharification and fermentation process (SSF) were conducted to produce ethanol optimally. Variations of enzyme concentration used in this process were 10, 20, 30 and 40 FPU/g substrate. The result shows that the highest ethanol concentration can be obtained in SSF process with 30 FPU/g substrate of enzyme concentration. The process produced 59.20 g/L ethanol (95.95% yield ethanol) at 96 h of SSF process.     

Fermentation of Sugarcane Bagasse and Chicken Manure to Calcium Carboxylates under Thermophilic Conditions

Sugarcane bagasse and chicken manure were anaerobically fermented to carboxylic acids using a mixed culture of marine microorganisms at 55 °C. Using the MixAlco process— an example of consolidated bioprocessing— the resulting carboxylate salts can be converted to mixed alcohol fuels or gasoline. To enhance digestibility, sugarcane bagasse was lime pretreated with 0.1 g Ca(OH)₂/g dry biomass at 100 °C for 2 h. Four-stage countercurrent fermentation of 80% sugarcane bagasse/20% chicken manure was performed at various volatile solids (VS) loading rates and liquid residence times. Calcium carbonate was used as a buffer during fermentation. The highest acid productivity of 0.79 g/(L day) occurred at a total acid concentration of 21.5 g/L. The highest conversion (0.59 g VS digested/g VS fed) and yield (0.18 g total acids/g VS fed) occurred at a total acid concentration of 15.5 g/L. The continuum particle distribution model (CPDM) predicted the experimental total acid concentrations and conversions at an average error of 10.14% and 12.68%, respectively. CPDM optimizations show that high conversion (>80%) and total acid concentration of 21.3 g/L are possible with 300 g substrate/(L liquid), 30 days liquid residence time, and 3 g/(L day) solid loading rate. Thermophilic fermentation has a higher acetate content (∼63 wt%) than mesophilic fermentation (∼39 wt%).     

Parameter Estimation for Simultaneous Saccharification and Fermentation of Food Waste Into Ethanol Using Matlab Simulink

The increase in waste disposal and energy costs has provided an incentive to convert carbohydrate-rich food waste streams into fuel. For example, dining halls and restaurants discard foods that require tipping fees for removal. An effective use of food waste may be the enzymatic hydrolysis of the waste to simple sugars and fermentation of the sugars to ethanol. As these wastes have complex compositions which may change day-to-day, experiments were carried out to test fermentability of two different types of food waste at 27 degrees C using Saccharomyces cerevisiae yeast (ATCC4124) and Genencor's STARGEN enzyme in batch simultaneous saccharification and fermentation (SSF) experiments. A mathematical model of SSF based on experimentally matched rate equations for enzyme hydrolysis and yeast fermentation was developed in Matlab Simulink. Using Simulink parameter estimation 1.1.3, parameters for hydrolysis and fermentation were estimated through modified Michaelis-Menten and Monod-type equations with the aim of predicting changes in the levels of ethanol and glycerol from different initial concentrations of glucose, fructose, maltose, and starch. The model predictions and experimental observations agree reasonably well for the two food waste streams and a third validation dataset. The approach of using Simulink as a dynamic visual model for SSF represents a simple method which can be applied to a variety of biological pathways and may be very useful for systems approaches in metabolic engineering in the future.     

Optimization of Dilute Sulfuric Acid Pretreatment to Maximize Combined Sugar Yield from Sugarcane Bagasse for Ethanol Production

Increasing fermentable sugar yields per gram of biomass depends strongly on optimal selection of varieties and optimization of pretreatment conditions. In this study, dilute acid pretreatment of bagasse from six varieties of sugarcane was investigated in connection with enzymatic hydrolysis for maximum combined sugar yield (CSY). The CSY from the varieties were also compared with the results from industrial bagasse. The results revealed considerable differences in CSY between the varieties. Up to 22.7 % differences in CSY at the optimal conditions was observed. The combined sugar yield difference between the best performing variety and the industrial bagasse was 34.1 %. High ratio of carbohydrates to lignin and low ash content favored the release of sugar from the substrates. At mild pretreatment conditions, the differences in bioconversion efficiency between varieties were greater than at severe condition. This observation suggests that under less severe conditions the glucose recovery was largely determined by chemical composition of biomass. The results from this study support the possibility of increasing sugar yields or improving the conversion efficiency when pretreatment optimization is performed on varieties with improved properties.     

Lime Pretreatment of Sugarcane Bagasse for Bioethanol Production

The pretreatment of sugarcane bagasse with lime (calcium hydroxide) is evaluated. The effect of lime pretreatment on digestibility was studied through analyses using central composite design (response surface), considering pretreatment time, temperature, and lime loading as factors. The responses evaluated were the yield of glucose from pretreated bagasse after enzymatic hydrolysis. Experiments were performed using the bagasse as it comes from an alcohol/sugar factory (non-screened bagasse) and bagasse in the size range from 0.248 to 1.397 mm (screened bagasse) (12-60 mesh). It was observed that the particle size presented influence in the release of fermentable sugars after enzymatic hydrolysis using low loading of cellulase and β-glucosidase (3.5 FPU/g dry pretreated biomass and 1.0 IU/g dry pretreated biomass, respectively).     

Fumaric Acid Production from Alkali-Pretreated Corncob by Fed-Batch Simultaneous Saccharification and Fermentation Combined with Separated Hydrolysis and Fermentation at High Solids Loading

Production of fumaric acid from alkali-pretreated corncob (APC) at high solids loading was investigated using a combination of separated hydrolysis and fermentation (SHF) and fed-batch simultaneous saccharification and fermentation (SSF) by Rhizopus oryzae. Four different fermentation modes were tested to maximize fumaric acid concentration at high solids loading. The highest concentration of 41.32 g/L fumaric acid was obtained from 20 % (w/v) APC at 38 °C in the combined SHF and fed-batch SSF process, compared with 19.13 g/L fumaric acid in batch SSF alone. The results indicated that a combination of SHF and fed-batch SSF significantly improved production of fumaric acid from lignocellulose by R. oryzae than that achieved with batch SSF at high solids loading.     

Preparation and Evaluation of Sodium Carboxymethylcellulose from Sugarcane Bagasse for Applications in Coal-Water Slurry

A study on the feasibility of preparing cellulose from sugarcane bagasse, by means of chemical procedures including acid hydrolysis and alkaline treatment was conducted. The extracted purified cellulose was further used to prepare a cost-effective additive via alkalization and etherification for Coal-water slurry (CWS). The degree of substitute (DS) and intrinsic viscosity of the prepared sodium carboxymethyl cellulose (SCMC1) were determined and its structure was also characterized by means of FT-IR and TGA, with another sample of SCMC2 produced from microcrystalline cellulose and a commercial SCMC3 as references. Results showed SCMC1 had a DS of 0.857 which was 32.7% and 44.7% higher than SCMC2 and SCMC3, respectively. The higher intrinsic viscosity of SCMC1 indicated it had a higher molecular mass. The SCMC samples were used as additives to prepare CWS of which the rheological behavior and static stability were measured to evaluate their applied performances. The data showed that CWS with SCMC1 had a lower apparent viscosity and higher static stability than others, which was due to the higher DS and higher molecular mass of SCMC1. For SCMC1 could provide stronger electrostatic repulsive forces and steric repulsive forces between the coal particles via adsorption.     

Efficient Production of Ethanol from Empty Palm Fruit Bunch Fibers by Fed-Batch Simultaneous Saccharification and Fermentation Using Saccharomyces cerevisiae

The concentration of ethanol produced from lignocellulosic biomass should be at least 40 g l^?1 [about 5 % (v/v)] to minimize the cost of distillation process. In this study, the conditions for the simultaneous saccharification and fermentation (SSF) at fed-batch mode for the production of ethanol from alkali-pretreated empty palm fruit bunch fibers (EFB) were investigated. Optimal conditions for the production of ethanol were identified as temperature, 30 °C; enzyme loading, 15 filter paper unit g^?1 biomass; and yeast (Saccharomyces cerevisiae) loading, 5 g l^?1 of dry cell weight. Under these conditions, an economical ethanol concentration was achieved within 17 h, which further increased up to 62.5 g l^?1 after 95 h with 70.6 % of the theoretical yield. To our knowledge, this is the first report to evaluate the economic ethanol production from alkali-pretreated EFB in fed-batch SSF using S. cerevisiae.     

Continuous Ethanol Production from Cassava Through Simultaneous Saccharification and Fermentation by Self-Flocculating Yeast Saccharomyces Cerevisiae CHFY0321

In this study, a fermentor consisting of four linked stirred towers that can be used for simultaneous saccharification and fermentation (SSF) and for the accumulation of cell mass was applied to the continuous production of ethanol using cassava as the starchy material. For the continuous process with SSF, the pretreated cassava liquor and saccharification enzyme at total sugar concentrations of 175 g/L and 195 g/L were continuously fed to the fermentor with dilution rates of 0.014, 0.021, 0.031, 0.042, and 0.05 h⁻¹. Considering the maximum saccharification time, the highest volumetric productivity and ethanol yield were observed at a dilution rate of 0.042 h⁻¹. At dilution rates in the range of 0.014 h⁻¹ to 0.042 h⁻¹, high production rates were observed, and the yeast in the first to fourth fermentor showed long-term stability for 2 months with good performance. Under the optimal culture conditions with a feed sugar concentration of 195 g/L and dilution rate of 0.042 h⁻¹, the ethanol volumetric productivity and ethanol yield were 3.58 g/L∙h and 86.2%, respectively. The cell concentrations in the first to fourth stirred tower fermentors were 74.3, 71.5, 71.2, and 70.1 g dry cell/L, respectively. The self-flocculating yeast, Saccharomyces cerevisiae CHFY0321, developed by our group showed excellent fermentation results under continuous ethanol production.     

Use of Spectroscopic and Imaging Techniques to Evaluate Pretreated Sugarcane Bagasse as a Substrate for Cellulase Production Under Solid-State Fermentation

The enzymatic cocktail of cellulases is one of the most costly inputs affecting the economic viability of the biochemical route for biomass conversion into biofuels and other chemicals. Here, the influence of liquid hot water, dilute acid, alkali, and combined acid/alkali pretreatments on sugarcane bagasse (SCB) used for cellulase production was investigated by means of spectroscopic and imaging techniques. Chemical composition and structural characteristics, such as crystallinity (determined by X-ray diffraction), functional groups (Fourier transform infrared spectroscopy), and microstructure (scanning electron microscopy), were used to correlate SCB pretreatments with enzymatic biosynthesis by a strain of the filamentous fungus Aspergillus niger under solid-state fermentation. The combined acid/alkali pretreatment resulted in a SCB with higher cellulose content (86.7 %). However, the high crystallinity (74 %) of the resulting biomass was detrimental to microbial uptake and enzyme production. SCB pretreated with liquid hot water yielded the highest filter paper cellulase (FPase), carboxymethyl cellulase (CMCase), and xylanase activities (0.4, 14.9, and 26.1 U g^?1, respectively). The results showed that a suitable pretreatment for SCB to be used as a substrate for cellulase production should avoid severe conditions in order to preserve amorphous cellulose and to enhance the physical properties that assist microbial access.     

Extraction of Inulinase Obtained by Solid State Fermentation of Sugarcane Bagasse by Kluyveromyces marxianus NRRL Y-7571

Production of inulinase by solid state fermentation always involves an extraction step, which dictates enzyme recovery yield and is related to cultivation conditions and control of process parameters. This work is focused on the study of extraction conditions aiming to maximize yield of an inulinase obtained by solid state fermentation of sugar cane bagasse and Kluyveromyces marxianus NRRL Y-7571. Kinetics of extraction was followed varying the kind of solvent used. After determining the best solvent, an experimental design was carried out to study the effect of the solid/liquid ratio (1:10-1:20), extraction temperature (20-53 degrees C), and stirring rate (50-177 rpm). Results showed that maximum yield was obtained when sodium acetate buffer 0.1 M pH 4.8 was used, using a solid/liquid ratio of 1:10, at 53 degrees C and 150 rpm for 40 min.     

Selection of a Thermotolerant Kluyveromyces marxianus Strain with Potential Application for Cellulosic Ethanol Production by Simultaneous Saccharification and Fermentation

The development of technologies for cellulosic ethanol production by simultaneous saccharification and fermentation (SSF) depends on the use of microorganisms with high fermentative rates and thermotolerance. In this study, the ability of five Kluyveromyces marxianus strains to produce ethanol from glucose at 45 °C was investigated. The highest fermentative parameters were observed with K. marxianus NRRL Y-6860, which was then further studied. An initial evaluation of the oxygen supply on ethanol production by the selected yeast and a comparison of SSF process from acid pretreated rice straw between K. marxianus NRRL Y-6860 and Saccharomyces cerevisiae at 30 and 45 °C were carried out. Under the lowest evaluated conditions of aeration and agitation, K. marxianus NRRL Y-6860 produced 21.5 g/L ethanol from 51.3 g/L glucose corresponding to Y_P/S of 0.44 g/g and Q_P of 3.63 g/L h. In the SSF experiments, K. marxianus NRRL Y-6860 was more efficient than S. cerevisiae at both evaluated temperatures (30 and 45 °C), attained at the highest temperature an ethanol yield of 0.24 g/g and productivity of 1.44 g/L h.