Enzymatic saccharification and fermentation of xylose-optimized dilute acid-treated lignocellulosics

The cellulose reactivity of two lignocellulosic feedstocks, switchgrass and poplar, was evaluated under straight saccharification (SS) and simultaneous saccharification and fermentation (SSF) conditions following dilute sulfuric acid pretreatments designed for optimum xylose yields. The optimum pretreatment conditions, within the constraints of the experimental system (Parr batch reactor), were 1.2% acid, 180°C, and 0.5 min for switchgrass and 1% acid, 180°C, and 0.56 min for poplar. The cellulase enzyme preparation was from Trichoderma reesei and fermentations were done with Saccharomyces cerevisiae. Time courses for SS were monitored as the sum of glucose and cellobiose; those for SSF as the sum of glucose, cellobiose, and ethanol. Percentage conversions under SS conditions were 79.1% and 91.4% for the pretreated poplar and switchgrass feedstocks, respectively. Analogous values under SSF conditions were 73.0% and 90.3% for pretreated poplar and switchgrass, respectively.     

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.     

Simultaneous saccharification and fermentation of steam-pretreated spruce to ethanol

Ethanol production was studied in simultaneous saccharification and fermentation (SSF) of steam-pretreated spruce at 42°C, using a thermotolerant yeast. Three yeast strains of Kluyveromyces marxianus were compared in test fermentations. SSF experiments were performed with the best of these on 5% (w/w) of substrate at a cellulase loading of 37 filter paper units/g of cellulose, and a β-glucosidase loading of 38 IU/gof cellulose. The detoxification of the substrate and the lack of pH control in the experiments increased the final ethanol concentration. The final ethanol yield was 15% lower compared to SSF with Saccharomyces cerevisiae at 37°C, owing to the cessation of ethanol fermentation after the first 10 h.     

Conversion of lignocellulosics pretreated with liquid hot water to ethanol

Lignocellulosic materials pretreated using liquid hot water (LHW) (220°C, 5 MPa, 120 s) were fermented to ethanol by batch simultaneous saccharification and fermentation (SSF) usingSaccharomyces cerevisiae in the presence ofTrichoderma reesei cellulase. SSF of sugarcane bagasse (as received), aspen chips (smallest dimension 3 mm), and mixed hardwood flour (−60 +70 mesh) resulted in 90% conversion to ethanol in 2–5 d at enzyme loadings of 15–30 FPU/g. In most cases, 90% of the final conversion was achieved within 75 h of inoculation. Comminution of the pretreated substrates did not affect the conversion to ethanol. The hydrolysate produced from the LHW pretreatment showed slight inhibition of batch growth ofS. cerevisiae. Solids pretreated at a concentration of 100 g/L were as reactive as those pretreated at a lower concentration, provided that the temperature was maintained at 220°C.     

Lactic Acid Production from a Whole Slurry of Acid-Pretreated Spent Coffee Grounds by Engineered Saccharomyces cerevisiae

Spent coffee grounds (SCG) generated after coffee extraction are the main byproduct of the coffee industry. Valorization of the SCG has been increasingly focused following considerable attention in coffee consumption. Lactic acid bacteria fermentation is the primary source of generation of lactic acid, a monomer of polylactic acid that has various industrial applications; however, because of the low tolerance of lactic acid bacteria to toxic compounds, it is necessary to apply Saccharomyces cerevisiae to produce lactic acid whose tolerance to toxic compounds is higher. In this study, we evaluated the feasibility of using SCG as substrate for the production of lactic acid by S. cerevisiae strain expressing heterologous lactate dehydrogenase. The fermentation profiles of the engineered yeast showed that lactic acid production was promoted by xylose addition. From simultaneous saccharification and fermentation (SSF) using a whole slurry of acid-pretreated SCG, containing high amounts of hemicellulose fractions, lactic acid (0.11 g) and ethanol (0.10 g) per g SCG were obtained after 24 h of SSF, of which yields were 413% and 221% higher, respectively, than those of washed pretreated SCG. Thus, fermentation of whole slurry SCG by engineered S. cerevisiae is a suitable way of lactic acid production, selectively.

     

Nonisothermal simultaneous saccharification and fermentation for direct conversion of lignocellulosic biomass to ethanol

The enzymatic reaction in the simultaneous saccharification and fermentation (SSF) is operated at a temperature much lower than its optimum level. This forces the enzyme activity to be far below its potential, consequently raising the enzyme requirement. To alleviate this problem, a nonisothermal simultaneous saccharification and fermentation process (NSSF) was investigated. The NSSF is devised so that saccharification and fermentation occur simultaneously, yet in two separate reactors that are maintained at different temperatures. Lignocellulosic biomass is retained inside a column reactor and hydrolyzed at the optimum temperature for the enzymatic reaction (50°C). The effluent from the column reactor is recirculated through a fermenter, which runs at its optimum temperature (20-30°C). The cellulase enzyme activity is increased by a factor of 2-3 when the hydrolysis temperature is raised from 30 to 50°C. The NSSF process has improved the enzymatic reaction in the SSF to the extent that it reduces the overall enzyme requirement by 30-40%. The effect of temperature on β-glucosidase activity was the most significant among the individual cellulase compounds. Both ethanol yield and productivity in the NSSF are substantially higher than those in the SSF at the enzyme loading of 5 IFPU/g glucan. With 10 IFPU/g glucan, improvement in productivity was more discernible for the NSSF. The terminal yield attainable in 4 d with the SSF was reachable in 40 h with the NSSF.     

Deficiency of Cellulase Activity Measurements for Enzyme Evaluation

Switchgrass was used as a model feedstock to determine the influence of pretreatment conditions and biomass quality on enzymatic hydrolysis using different enzyme products. Dilute sulfuric acid and soaking in aqueous ammonia pretreatments were used to produce biomass with varied levels of hemicellulose and lignin sheathing. Pretreated switchgrass solids were tested with simple enzymatic hydrolysis and simultaneous saccharification and fermentation (SSF) with three commercial enzyme products: Accellerase 1000 (Genencor), Spezyme CP (Genencor)/Novozyme 188 (Novozymes), and Celluclast/Novozyme 188 (Novozymes). Enzymes were loaded on a common activity basis (FPU/g cellulose and CBU/g cellulose). Despite identical enzyme loadings, glucose yields were significantly different for both acid and alkaline pretreatments but differences diminished as hydrolysis progressed for acid-pretreated biomass. Cellobiose concentrations in Accellerase treatments indicated an initial β-glucosidase limitation that became less significant over time. SSF experiments showed that differences in glucose and ethanol yields could not be attributed to enzyme product inhibition. Yield discrepancies of glucose or ethanol in acid pretreatment, alkaline pretreatment, and acid pretreatment/SSF were as much as 15%, 19%, and 5%. These results indicate that standardized protocols for measuring enzyme activity may not be adequate for assessing activity using pretreated biomass substrates.     

The simultaneous saccharification and fermentation of pretreated woody crops to ethanol

Four promising woody crops (Populusmaximowiczii x nigra (NE388), P.trichocarpa x deltoides (Nll), P.tremuloides, and SweetgumLiquidambar styraciflua) were pretreated by dilute sulfuric acid and evaluated in the simultaneous saccharification and fermentation (SSF) process for ethanol production. The yeastSaccharomyces cerevisiae was used in the fermentations alone, and in mixed cultures with β -glucosidase producingBrettanomyces dausenii. Commercial Genencor 150L cellulase enyme was either employed alone or supplemented with β- glucosidase. All SSFs were run at 37 …C for 8 d and compared to saccharifications at 45…C under the same enzyme loadings.S. cerevisiae alone achieved the highest ethanol yields and rates of hydrolysis at the higher enzyme loadings, whereas the mixed culture performed better at the lower enzyme loadings without β -glucosidase supplementation. The best overall rates of fermentation (3 d) and final theoretical ethanol yields (86–90%) were achieved with P.maximowiczii x nigra (NE388) and SweetgumLiquidambar styraciflua, followed by P.tremuloides and P.trichocarpa xdeltoides (N1l) with slightly slower rates and lower yields. Although there were some differences in SSF performance, all these pretreated woody crops show promise as substrates for ethanol production.     

Continuous Production of Ethanol from Starch Using Glucoamylase and Yeast Co-Immobilized in Pectin Gel

This work presents a continuous simultaneous saccharification and fermentation (SSF) process to produce ethanol from starch using glucoamylase and Saccharomyces cerevisiae co-immobilized in pectin gel. The enzyme was immobilized on macroporous silica, after silanization and activation of the support with glutaraldehyde. The silica–enzyme derivative was co-immobilized with yeast in pectin gel. This biocatalyst was used to produce ethanol from liquefied manioc root flour syrup, in three fixed bed reactors. The initial reactor yeast load was 0.05 g wet yeast/ml of reactor (0.1 g wet yeast/g gel), used in all SSF experiments. The enzyme concentration in the reactor was defined by running SSF batch assays, using different amount of silica–enzyme derivative, co-immobilized with yeast in pectin gel. The chosen reactor enzyme concentration, 3.77 U/ml, allowed fermentation to be the rate-limiting step in the batch experiment. In this condition, using initial substrate concentration of 166.0 g/l of total reducing sugars (TRS), 1 ml gel/1 ml of medium, ethanol productivity of 8.3 g/l/h was achieved, for total conversion of starch to ethanol and 91% of the theoretical yield. In the continuous runs, feeding 163.0 g/l of TRS and using the same enzyme and yeast concentrations used in the batch run, ethanol productivity was 5.9 g ethanol/l/h, with 97% of substrate conversion and 81% of the ethanol theoretical yield. Diffusion effects in the extra-biocatalyst film seemed to be reduced when operating at superficial velocities above 3.7 × 10⁻⁴ cm/s.     

Continuous fermentation of cellulosic biomass to ethanol

Experimental results are presented for continuous conversion of pretreated hardwood flour to ethanol. A simultaneous saccharification and fermentation (SSF) system comprised ofTrichoderma reesei cellulase supplemented with additional β-glucosidase and fermentation bySaccharomyces cerevisiae was used for most experiments, with data also presented for a direct microbial conversion (DMC) system comprised ofClostridium thermocellum. Using a batch SSF system, dilute acid pretreatment of mixed hardwood at short residence time(10 s, 220°C, 1% H₂SO₄) was compared to poplar wood pretreated at longer residence time (20 min, 160°C, 0.45% H₂SO₄). The short residence time pretreatment resulted in a somewhat (10–20%) more reactive substrate, with the reactivity difference particularly notable at low enzyme loadings and/or low agitation. Based on a preliminary screening, inhibition of SSF by byproducts of short residence time pretreatment was measurable, but minor. Both SSF and DMC were carried out successfully in well-mixed continuous systems, with steady-state data obtained at residence times of 0.58–3 d for SSF as well as 0.5 and 0.75 d for DMC. The SSF system achieved substrate conversions varying from 31% at a 0.58-d residence time to 86% at a 2-d residence time. At comparable substrate concentrations (4–5 g/l) and residence times (0.5–0.58 d), substrate conversion in the DMC system (77%) was significantly higher than that in the SSF system (31%). Our results suggest that the substrate conversion in SSF carried out in CSTR is relatively insensitive to enzyme loading in the range 7–25 U/g cellulose and to substrate concentration in the range of 5–60 g/L cellulose in the feed.     

Sugarcane Bagasse Mild Alkaline/Oxidative Pretreatment for Ethanol Production by Alkaline Recycle Process

In order to decrease the alkali and water consumptions in the sugarcane bagasse alkaline/oxidative pretreatment for ethanol production, an alkaline recycle process was carried out. Two recycles of NaOH/H₂O₂ pretreatment did not decrease the pretreatment and enzymatic hydrolysis efficiencies and the consumptions of NaOH and water would be saved by 26% and 40%, respectively. A simultaneous saccharification and fermentation (SSF) culture with pretreated bagasse as substrate was developed giving 25 g ethanol l⁻¹ with a yield of 0.2 g g⁻¹ bagasse and productivity of 0.52 g l⁻¹ h⁻¹.     

Ethanol Production from Residual Wood Chips of Cellulose Industry: Acid Pretreatment Investigation,Hemicellulosic Hydrolysate Fermentation,and Remaining Solid Fraction Fermentation by SSF Process

Current research indicates the ethanol fuel production from lignocellulosic materials, such as residual wood chips from the cellulose industry, as new emerging technology. This work aimed at evaluating the ethanol production from hemicellulose of eucalyptus chips by diluted acid pretreatment and the subsequent fermentation of the generated hydrolysate by a flocculating strain of Pichia stipitis. The remaining solid fraction generated after pretreatment was subjected to enzymatic hydrolysis, which was carried out simultaneously with glucose fermentation [saccharification and fermentation (SSF) process] using a strain of Saccharomyces cerevisiae. The acid pretreatment was evaluated using a central composite design for sulfuric acid concentration (1.0–4.0 v/v) and solid to liquid ratio (1:2–1:4, grams to milliliter) as independent variables. A maximum xylose concentration of 50 g/L was obtained in the hemicellulosic hydrolysate. The fermentation of hemicellulosic hydrolysate and the SSF process were performed in bioreactors and the final ethanol concentrations of 15.3 g/L and 28.7 g/L were obtained, respectively.     

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.     

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.     

The Application of Ultrasound in the Enzymatic Hydrolysis of Switchgrass

In a series of experiments, untreated and ammonium hydroxide pretreated Klenow lowland variety switchgrasses are converted to reducing sugars using low-frequency (20 kHz) ultrasound and commercially available cellulase enzyme. Results from experiments using untreated and pretreated switchgrasses with and without ultrasound are presented and discussed. In untreated switchgrass experiments, the combination of ultrasound and enzymes resulted in an increase of 7.5% in reducing sugars compared to experiments using just enzymes. In experiments using ammonium hydroxide pretreated switchgrass, the combination of ultrasound and enzymes resulted in an increase of 9.3% in reducing sugars compared to experiments using just enzymes. Experimental evidence indicates that there is a synergistic effect from the combination of ultrasound and enzymes which lowers the diffusion-limiting barrier to enzyme/substrate binding and results in an increase in reaction rate. Scanning electron microscopic images provide evidence that ultrasound-induced pitting increases substrate surface area and affects reaction rate and yield.     

Ethanol Production from the Organic Fraction Obtained After Thermal Pretreatment of Municipal Solid Waste

In this work, the use of organic fraction from municipal solid waste (MSW) as substrate for ethanol production based on enzymatic hydrolysis was evaluated. MSW was subjected to a thermal pretreatment (active hygienization) at 160?°C from 5 to 50 min. The organic fiber obtained after 30 min was used as substrate in a simultaneous saccharification and fermentation (SSF) and fed-batch SSF process using cellulases and amylases. In a fed-batch mode with 25% (w/w) substrate loading, final ethanol concentration of 30 g/L was achieved (60% of theoretical). In these conditions, more than 160 L of ethanol per ton of dry matter could be produced from the organic fraction of MSW.     

Analysis of biomass cellulose in simultaneous saccharification and fermentation processes

A direct method for determining the cellulose content of biomass residues resulting from simultaneous saccharifiaction and fermentation (SSF) experiment has been developed and evaluated. The method improves on classical cellulose assays by incorporating the enzymatic removal of yeast glucans from the biomass residue prior to acid hydrolysis and subsequent quantification of cellulose-derived glucose. An appropriate cellulasefree, commercially available, yeast-lysing enzyme preparation fromCytophaga was identified. A freeze-drying step was identified as necessary to render the SSF yeast cells susceptible to enzymatic lysis. The method was applied to the analysis of cellulose and yeast-associated glucans in SSF residues from three pretreated feedstocks; hybrid poplar, switchgrass, and cornstover. Cellulose assays employing the lysing-enzyme preparation demonstrated relative errors up to 7.2% when yeast-associated glucans were not removed prior to analysis of SSF residues. Enzymatic lysis of SSF yeast cells may be viewed as a general preparatory procedure to be used prior to subsequent chemical and physical analysis of SSF residues. Oregon State University Agricultural Experiment Station Technical Publication Number 10977.     

Ethanol production from olive oil extraction residue pretreated with hot water

The olive pulp fraction contained in the residue generated in olive oil extraction by a two-step centrifugation process can be upgraded by using the cellulose fraction to produce ethanol and recovering high value phenols (tyrosol and hydroxytyrosol). Olive pulp was pretreated in a laboratory scale stirred autoclave at different temperatures (150–250°C). Pretreatment was evaluated regarding cellulose recovery, enzymatic hydrolysis effectiveness ethanol production by a simultaneous saccharification and fermentation process (SSF), and phenols recovery in the filtrate. The pretreatment of olive pulp using water at temperatures between 200°C and 250°C enhanced enzymatic hydrolysis. Maximum ethanol production (11.9 g/L) was obtained after pretreating pulp at 210°C in a SSF fed-batch procedure. Maximum hydroxytyrosol recovery was obtained in the liquid fraction when pretreated at 230°C.     

Dilute-sulfuric acid pretreatment of corn stover in pilot-scale reactor

Corn stover is a domestic feedstock that has potential to produce significant quantities of fuel ethanol and other bioenergy and biobased products. However, comprehensive yield and carbon mass balance information and validated kinetic models for dilute-sulfuric acid (H₂SO₄) pretreatment of corn stover have not been available. This has hindered the estimation of process economics and also limited the ability to perform technoeconomic modeling to guide research. To better characterize pretreatment and assess its kinetics, we pretreated corn stover in a continuous 1 t/d reactor. Corn stover was pretreated at 20% (w/w) solids concentration over a range of conditions encompassing residence times of 3–12 min, temperatures of 165–195°C, and H₂SO₄ concentrations of 0.5–1.4% (w/w). Xylan conversion yield and carbon mass balance data were collected at each run condition. Performance results were used to estimate kinetic model parameters assuming biphasic hemicellulose hydrolysis and a hydrolysis mechanism incorporating formation of intermediate xylo-oligomers. In addition, some of the pretreated solids were tested in a simultaneous saccharification and fermentation (SSF) process to measure the reactivity of their cellulose component to enzymatic digestion by cellulase enzymes. Monomeric xylose yields of 69–71% and total xylose yields (monomers and oligomers) of 70–77% were achieved with performance level depending on pretreatment severity. Cellulose conversion yields in SSF of 80–87% were obtained for some of the most digestible pretreated solids.     

Recirculation of process streams in fuel ethanol production from softwood based on simultaneous saccharification and fermentation

The effect of process stream recirculation on ethanol production from steam- pretreated softwood based on simultaneous saccharification and fermentation (SSF) was investigated for two process configurations. In the first configuration, a part of the stillage stream after distillation was recycled and, in the second configuration, the liquid after SSF was recycled. The aim was to minimize the energy consumption in the distillation of the fermentation broth and in the evaporation of the stillage, as well as the use of fresh water. However, recirculation leads to an increased concentration of nonvolatiles in the first configuration, and of both volatiles and nonvolatiles in the second configuration. These substances might be inhibitory to the enzymes and the yeast in SSF. When 60% of the fresh water was replaced by stillage, the ethanol yield and the productivity were the same as for the configuration without recirculation. The ethanol production cost was reduced by 17%. In the second configuration, up to 40% of the fresh water could be replaced without affecting the final ethanol yield, although the initial ethanol productivity decreased. The ethanol production cost was reduced by 12%. At higher degrees of recirculation, fermentation was clearly inhibited, resulting in a decrease in ethanol yield while hydrolysis seemed unaffected.