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.     

Pretreatment of corn stover by soaking in aqueous ammonia

Soaking in aqueous ammonia (SAA) was investigated as a pretreatment method for corn stover. In this method, the feedstock was soaked in aqueous ammonia over an extended period (10–60 d) at room temperature. It was done without agitation at atmospheric pressure. SAA treatment removed 55–74% of the lignin, but retained nearly 100% of the glucan and 85% of the xylan. The xylan remaining in the corn stover after SAA treatment was hydrolyzed along with the glucan by xylanase present in the Spezyme CP enzyme. In the simultaneous saccharification and fermentation (SSF) test of SAA-treated corn stover, using S. cerevisiae (D₅A), an ethanol yield of 73% of theoretical maximum was obtained on the basis of the glucan content in the treated corn stover. The accumulation of xylose in the SSF appears to inhibit the cellulase activity on glucan hydrolysis, which limits the yield of ethanol. In the simultaneous saccharification and co-fermentation (SSCF) test, using recombinant E. coli (KO11), both the glucan and xylose were effectively utilized, resulting in on overall ethanol yield of 77% based on the glucan and xylan content of the substrate. When the SSCF process is used, the fact that the xylan fraction is retained during pretreatment is a desirable feature since the overall bioconversion can be carried out in a single step without separate recovery of xylose from the pretreatment liquid.     

Thermotolerant yeast for simultaneous saccharification and fermentation of cellulose to ethanol

Ten promising microbial strains were screened for glucose fermentation over the temperature range of 37–47°C, and five temperature-tolerant yeasts (Saccharomyces cerevisiae SERI strain (D₅A),S. uvarum, andCandida generaacidothermophilium, brassicae, andlusitaniae), were chosen for SSF evaluation on Sigmacell-50 cellulose with Genencor 150 L cellulase enzyme.Brettanomyces clausenii (Y-1414) was included for comparison to previous studies both by itself and in mixed culture withS. cerevisiae (D₅A). Good conversion rates were achieved at temperatures as high as 43°C withC. brassicae andS. uvarum; mixed cultures of either of these yeasts with the thermotolerant cellobiose fermenting yeastC. lusitaniae achieved higher rates and yields than any of the three yeasts alone. However, the mixed culture ofB. clausenii andS. cerevisiae at 37°C achieved as high conversion rates and higher yields than any of the other yeasts tested.     

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.     

Enzymatic Hydrolysis and Ethanol Fermentation of High Dry Matter Wet-Exploded Wheat Straw at Low Enzyme Loading

Wheat straw was pretreated by wet explosion using three different oxidizing agents (H₂O₂, O₂, and air). The effect of the pretreatment was evaluated based on glucose and xylose liberated during enzymatic hydrolysis. The results showed that pretreatment with the use of O₂ as oxidizing agent was the most efficient in enhancing overall convertibility of the raw material to sugars and minimizing generation of furfural as a by-product. For scale-up of the process, high dry matter (DM) concentrations of 15–20% will be necessary. However, high DM hydrolysis and fermentation are limited by high viscosity of the material, higher inhibition of the enzymes, and fermenting microorganism. The wet-explosion pretreatment method enabled relatively high yields from both enzymatic hydrolysis and simultaneous saccharification and fermentation (SSF) to be obtained when performed on unwashed slurry with 14% DM and a low enzyme loading of 10 FPU/g cellulose in an industrial acceptable time frame of 96 h. Cellulose and hemicellulose conversion from enzymatic hydrolysis were 70 and 68%, respectively, and an overall ethanol yield from SSF was 68%.     

Modeling simultaneous saccharification and fermentation of softwood

Simultaneous saccharification and fermentation (SSF) of wood has been modeled for the past 15–20 years, but the substrates used for model evaluation have so far not included pretreated softwood. In the present study, data from lab-scale batch SSF of SO₂-impregnated, steam-pretreated spruce chips were used to evaluate a model found in the literature. The model, which was somewhat modified, consists of a number of nonlinear, coupled ordinary differential equations, which were solved numerically. Some parameter values were fitted to data by use of least-squares minimization. A difficulty in parameter estimation was the lack of cellobiose measurements, something that was relieved by adding assumptions about parameter relations. The simulated concentration profiles agreed well with the measured concentrations of glucose and ethanol. It is therefore concluded that the basic model features apply to softwood SSF. The model predicts rate saturation with respect to enzyme concentration at concentrations above 60 FPU/g cellulose, although this was not observed in the experimental data, which only comprised enzyme concentrations up to 32 FPU/g cellulose.     

A Novel Technique that Enables Efficient Conduct of Simultaneous Isomerization and Fermentation (SIF) of Xylose

Of the sugars recovered from lignocellulose, d-glucose can be readily converted into ethanol by baker’s or brewer’s yeast (Saccharomyces cerevisiae). However, xylose that is obtained by the hydrolysis of the hemicellulosic portion is not fermentable by the same species of yeasts. Xylose fermentation by native yeasts can be achieved via isomerization of xylose to its ketose isomer, xylulose. Isomerization with exogenous xylose isomerase (XI) occurs optimally at a pH of 7–8, whereas subsequent fermentation of xylulose to ethanol occurs at a pH of 4–5. We present a novel scheme for efficient isomerization of xylose to xylulose at conditions suitable for the fermentation by using an immobilized enzyme system capable of sustaining two different pH microenvironments in a single vessel. The proof-of-concept of the two-enzyme pellet is presented, showing conversion of xylose to xylulose even when the immobilized enzyme pellets are suspended in a bulk solution whose pH is sub-optimal for XI activity. The co-immobilized enzyme pellets may prove extremely valuable in effectively conducting “simultaneous isomerization and fermentation” (SIF) of xylose. To help further shift the equilibrium in favor of xylulose formation, sodium tetraborate (borax) was added to the isomerization solution. Binding of tetrahydroxyborate ions to xylulose effectively reduces the concentration of xylulose and leads to increased xylose isomerization. The formation of tetrahydroxyborate ions and the enhancement in xylulose production resulting from the complexation was studied at two different bulk pH values. The addition of 0.05 M borax to the isomerization solution containing our co-immobilized enzyme pellets resulted in xylose to xylulose conversion as high as 86% under pH conditions that are suboptimal for XI activity. These initial findings, which can be optimized for industrial conditions, have significant potential for increasing the yield of ethanol from xylose in an SIF approach.     

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.     

Pretreatment of Whole-Crop Harvested, Ensiled Maize for Ethanol Production

To have all-year-round available feedstock, whole-crop maize is harvested premature, when it still contains enough moisture for the anaerobic ensiling process. Silage preparation is a well-known procedure for preserving plant material. At first, this method was applied to obtain high-quality animal feed. However, it was found that such ensiled crops are very suitable for bioenergy production. Maize silage, which consists of hardly degradable lignocellulosic material, hemicellulosic material, and starch, was evaluated for its potential as a feedstock in the production of bioethanol. It was pretreated at low severity (185 °C, 15 min) giving very high glucan (∼100%) and hemicellulose recoveries (<80%)—as well as very high ethanol yield in simultaneous saccharification and fermentation experiments (98% of the theoretical production based on available glucan in the medium). The theoretical ethanol production of maize silage pretreated at 185 °C for 15 min without oxygen or catalyst was 392 kg ethanol per ton of dry maize silage.     

Fourier transform infrared imaging and microscopy studies of Pinus radiata pulps regarding the simultaneous saccharification and fermentation process

The distribution and chemical patterns of lignocellulosic components at microscopic scale and their effect on the simultaneous saccharification and fermentation process (SSF) in the production of bioethanol from Pinus radiata pulps were analyzed by the application of diverse microscopical techniques, including scanning electronic microscopy (SEM), confocal laser scanning microscopy (CLSM) and attenuated total reflectance (ATR) – Fourier transform infrared microspectroscopy. This last technique was accompanied with multivariate methods, including principal component analysis (PCA) and multivariate curve resolution with alternating least squares (MCR-ALS) to evaluate the distribution patterns and to generate pure spectra of the lignocellulosic components of fibers. The results indicate that the information obtained by the techniques is complementary (ultrastructure, confocality and chemical characterization) and that the distribution of components affects the SSF yield, identifying lignin coalescence droplets as a characteristic factor to increase the SSF yield. Therefore, multivariate analysis of the infrared spectra enabled the in situ identification of the cellulose, lignin and lignin-carbohydrates arrangements. These techniques could be used to investigate the lignocellulosic components distribution and consequently their recalcitrance in many applications where minimal sample manipulation and microscale chemical information is required.     

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.     

Effect of reduction in yeast and enzyme concentrations in a simultaneous-saccharification-and-fermentation-based bioethanol process

The ethanol production cost in a simultaneous saccharification and fermentation-based bioethanol process is influenced by the requirements for yeast production and for enzymes. The main objective of this study was to evaluate—technically and economically—the influence of these two factors on the production cost. A base case with 5 g/L of baker’s yeast and an initial concentration of water-insoluble solids of 5% resulted in an experimental yield of 85%. When these data were implemented in Aspen Plus, yeast was assumed to be produced from sugars in the hydrolysate, reducing the overall ethanol yield to 69%. The ethanol production cost was 4.80 SEK/L (2.34 US$/gal). When adapted yeast was used at 2 g/L, an experimental yield of 74% was achieved and the estimated ethanol production cost was the same as in the base case. A 50% reduction in enzyme addition resulted in an increased production cost, to 5.06 SEK/L (2.47 US$/gal) owing to reduced ethanol yield.     

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.     

Hybrid process for the conversion of lignocellulosic materials

Because of the recalcitrant nature of lignocellulosic materials, it is important to pretreat the biomass in order to obtain a suitable material for the bioconversion. In this study, two different types of pretreatments were performed. The first experiment used a 2-gal Parr reactor operated at 140, 150,160, and 170‡C with sulfuric acid concentrations varying from 0.5 to 2%. A second pretreatment was performed with a two-stage low-temperature process. The first-stage pretreatment was performed at 100 or 120‡C with sulfuric acid concentrations of 0.5, 2, and 5% followed by a secondstage pretreatment at 120‡C with 2% acid concentration. The best residues for enzymatic hydrolysis and simultaneous saccharification and fermentations (SSF) came from the higher temperature pretreatment with the Parr reactor. However, a large portion of the xylose fraction was degraded to furfural and glucose was degraded to HMF. On the contrary, the two-stage low temperature pretreatment resulted in a very low percentage of xylose degradation, and no glucose degradation. The residues from this two-stage pretreatment performed satisfactorily toward the production of ethanol by SSFs. This study discusses the results obtained from these experiments.     

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.     

Selection of thermotolerant yeasts for simultaneous saccharification and fermentation (SSF) of cellulose to ethanol

A total of 27 yeast strains belonging to the groupsCandida, Saccharomyces, andKluyveromyces were screened for their ability to grow and ferment glucose at temperatures ranging 32-45°C. K. marxianus andK. fragilis were found to be the best ethanol producing organisms at the higher temperature tested and, so, were selected for subsequent simultaneous saccharification and fermentation (SSF) studies.     

Pretreatment of switchgrass by ammonia fiber explosion (AFEX)

The effects of ammonia fiber explosion (AFEX) pretreatment of switch grass using its major process variables are reported. The optimal pretreatment conditions for switchgrass were found to be near 100°C reactor temperature, and ammonia loading of 1:1 kg of ammonia: kg of dry matter with 80% moisture content (dry weight basis [dwb]) at 5 min residence time. Hydrolysis results of AFEX-treated and untreated samples showed 93% vs 16% glucan conversion, respectively. The ethanol yield of optimized AFEX-treated switchgrass was measured to be about 0.2 g ethanol/g dry biomass, which is 2.5 times more than that of the untreated sample.     

Production of 2,3- butanediol from pretreated corn cob byKlebsiella oxytoca in the presence of fungal cellulase

A simple and effective method of treatment of lignocellulosic material was used for the preparation of corn cob for the production of 2,3-butanediol byKlebsiella oxytoca ATCC 8724 in a simultaneous saccharification and fermentation process. During the treatment, lignin, and alkaline extractives were solubilized and separated from cellulose and hemicellulose fractions by dilute ammonia (10%) steeping. Hemicellulose was then hydrolyzed by dilute hydrochloric acid (1%, wJv) hydrolysis at 100°C at atmospheric pressure and separated from cellulose fraction. The remaining solid, with 90% of cellulose, was then used as the substrate. A butanediol concentration of 25 g/L and an ethanol concentration of 7 g/L were produced byK. oxytoca from 80 g/L of corn cob cellulose with a cellulase dosage of 8.5 IFPU/g corn cob cellulose after 72 h of SSF. With only dilute acid hydrolysis, a butanediol production rate of 0.21 g/L/h was obtained that is much lower than the case in which corn cob was treated with ammonia steeping prior to acid hydrolysis. The butanediol production rate for the latter was 0.36 g/L/h.     

Effect of surfactants and zeolites on simultaneous saccharification and fermentation of steam-exploded poplar biomass to ethanol

In this work, the effect of the addition of different concentrations of Tween-80 and three different zeolite-like products on enzymatic hydrolysis, ethanol fermentation, and simultaneous saccharification and fermentation (SSF) process has been investigated. The ability of these products to enhance the effectiveness of the SSF process to ethanol of steam-exploded poplar biomass using the thermotolerant strainKluyveromyces marxianus EMS-26 has been tested. Tween-80 (0.4 g/L) increased enzymatic hydrolysis yield by 20% when compared to results obtained in hydrolysis in absence of the additive. Zeolite-like products (ZESEP-56 and ZECER-56) (2.5 g/L) improved rates of conversion and ethanol yields in the fermentation of liquid fraction recovered from steam-exploded poplar. The periods required for the completion of fermentation were approx 10 h in the presence of zeolite-like products and 24 h in the absence of additives. The probable mode of action is through lowered levels of inhibitory substances because of adsorption by the additive.     

New method for simultaneous determination of Fe(II) and Fe(III) in water using flow injection technique

The method exploits the possibilities of flow injection gradient titration in a system of reversed flow with spectrophotometric detection. In the developed approach a small amount of titrant (EDTA) is injected into a stream of sample containing a mixture of indicators (sulfosalicylic acid and 1,10-phenanthroline). In acid environment sulfosalicylic acid forms a complex with Fe(III), whereas 1,10-phenanthroline forms a complex with Fe(II). Measurements are performed at wavelength λ = 530 nm when radiation is absorbed by both complexes. After injection EDTA replaces sulfosalicylic acid and forms with Fe(III) more stable colourless complex. As a result, a characteristic “cut off” peak is registered with a width corresponding to the Fe(III) concentration and with a height corresponding to the Fe(II) concentration. Calibration was performed by titration of four two-component standard solutions of the Fe(II)/Fe(III) concentrations established in accordance with 2² factorial plan. The method was tested with the use of synthetic samples and then it was applied to the analysis of water samples taken from artesian wells. Under optimized experimental conditions Fe(II) and Fe(III) were determined with precision less than 0.8 and 2.5% (RSD) and accuracy less than 3.2 and 5.1% (relative error) within the concentration ranges of 0.1-3.0 and 0.9-3.5 mg L⁻¹ of both analytes, respectively.