Production of ethanol from waste paper using immobilized yeasts

This work is aimed at a selection of yeast strains suitable for simultaneous saccharification and fermentation of waste paper. The waste paper, as a lignocellulosic material, represents an unconventional source for the production of ethanol which is a promising alternative fuel. The yeast strains Saccharomyces cerevisiae and Pichia kudriavzevii produced the highest amounts of ethanol at 30 °C and were also resistant at 40 °C during the first 92 h of fermentation. These two strains were immobilized by entrapment into poly(vinyl alcohol) hydrogel lens-shaped particles LentiKats^®. The immobilized S. cerevisiae was a better ethanol producer and retained higher metabolic activity in repeated batch fermentations than P. kudriavzevii. The immobilized S. cerevisiae was also suitable for a long-term storage, with 23% decrease in the ethanol production ability after 1-year storage of yeast cells.     

Characteristics of an Immobilized Yeast Cell System Using Very High Gravity for the Fermentation of Ethanol

The characteristics of ethanol production by immobilized yeast cells were investigated for both repeated batch fermentation and continuous fermentation. With an initial sugar concentration of 280?g/L during the repeated batch fermentation, more than 98% of total sugar was consumed in 65?h with an average ethanol concentration and ethanol yield of 130.12?g/L and 0.477?g ethanol/g consumed sugar, respectively. The immobilized yeast cell system was reliable for at least 10 batches and for a period of 28?days without accompanying the regeneration of Saccharomyces cerevisiae inside the carriers. The multistage continuous fermentation was carried out in a five-stage column bioreactor with a total working volume of 3.75?L. The bioreactor was operated for 26?days at a dilution rate of 0.015?h^?1. The ethanol concentration of the effluent reached 130.77?g/L ethanol while an average 8.18?g/L residual sugar remained. Due to the high osmotic pressure and toxic ethanol, considerable yeast cells died without regeneration, especially in the last two stages, which led to the breakdown of the whole system of multistage continuous fermentation.     

Comparison of Alcoholic Fermentation Performance of the Free and Immobilized Yeast on Water Hyacinth Stem Pieces in Medium with Different Glucose Contents

Ethanol fermentation with Saccharomyces cerevisiae cells was performed in medium with different glucose concentrations. As the glucose content augmented from 200 to 250 g/L, the growth of the immobilized cells did not change while that of the free cells was reduced. At higher glucose concentration (300, 350, and 400 g/L), the cell proliferation significantly decreased and the residual sugar level sharply augmented for both the immobilized and free yeast. The specific growth rate of the immobilized cells was 27–65 % higher than that of the free cells, and the final ethanol concentration in the immobilized yeast cultures was 9.7–18.5 % higher than that in the free yeast cultures. However, the immobilized yeast demonstrated similar or slightly lower ethanol yield in comparison with the free yeast. High fermentation rate of the immobilized yeast was associated with low unsaturation degree of fatty acids in cellular membrane. Adsorption of S. cerevisiae cells on water hyacinth stem pieces in the nutritional medium decreased the unsaturation degree of membrane lipid and the immobilized yeast always exhibited lower unsaturation degree of membrane lipid than the free yeast in ethanol fermentation.     

Ethanol Production by Fermentation Using Immobilized Cells of Saccharomyces cerevisiae in Cashew Apple Bagasse

In this work, cashew apple bagasse (CAB) was used for Saccharomyces cerevisiae immobilization. The support was prepared through a treatment with a solution of 3% HCl, and delignification with 2% NaOH was also conducted. Optical micrographs showed that high populations of yeast cells adhered to pre-treated CAB surface. Ten consecutive fermentations of cashew apple juice for ethanol production were carried out using immobilized yeasts. High ethanol productivity was observed from the third fermentation assay until the tenth fermentation. Ethanol concentrations (about 19.82–37.83 g L^?1 in average value) and ethanol productivities (about 3.30–6.31 g L^?1 h^?1) were high and stable, and residual sugar concentrations were low in almost all fermentations (around 3.00 g L^?1) with conversions ranging from 44.80% to 96.50%, showing efficiency (85.30–98.52%) and operational stability of the biocatalyst for ethanol fermentation. Results showed that cashew apple bagasse is an efficient support for cell immobilization aiming at ethanol production.     

Improved Ethanol Production by Mixed Immobilized Cells of Kluyveromyces marxianus and Saccharomyces cerevisiae from Cheese Whey Powder Solution Fermentation

Ethanol productions from cheese whey powder (CWP) solution were investigated by using free or immobilized cells of Kluyveromyces marxianus in monocultures or mixed cultures with free or immobilized cells of K. marxianus and Saccharomyces cerevisiae. K. marxianus free cells produced 3.8% v/v ethanol in monocultures, while S. cerevisiae immobilized cells produced 5.3% v/v ethanol in mixed cultures. The percentage of theoretical yield was found to be higher in mixed cultures than that in monocultures. The maximum ethanol fermentation efficiency was achieved (79.9% of the theoretical value) using mixed cultures of immobilized cells of K. marxianus and S. cerevisiae. The beads were relatively stable without significant reduction in activity for about eight batches of fermentation.     

Microbial Fed-batch Production of 1,3-Propanediol Using Raw Glycerol with Suspended and Immobilized Klebsiella pneumoniae

The production of 1,3-propanediol (1,3-PD) was investigated with Klebsiella pneumoniae DSM 4799 using raw glycerol without purification obtained from a biodiesel production process. Fed-batch cultures with suspended cells revealed that 1,3-PD production was more effective when utilizing raw glycerol than pure glycerol (productivity after 47 h of fermentation, 0.84 g?L^?1?h^?1 versus 1.51 g?L^?1?h^?1 with pure and raw glycerol, respectively). In addition, more than 80 g/L of 1,3-PD was produced using raw glycerol; this is the highest 1,3-PD concentration reported thus far for K. pneumoniae using raw glycerol. Repeated fed-batch fermentation with cell immobilization in a fixed-bed reactor was performed to enhance 1,3-PD production. Production of 1,3-PD increased with the cycle number (1.06 g?L^?1?h^?1 versus 1.61 g?L^?1?h^?1 at the first and fourth cycle, respectively) due to successful cell immobilization. During 46 cycles of fed-batch fermentation taking place over 1,460 h, a stable and reproducible 1,3-PD production performance was observed with both pure and raw glycerol. Based on our results, repeated fed batch with immobilized cells is an efficient fermentor configuration, and raw glycerol can be utilized to produce 1,3-PD without inhibitory effects caused by accumulated impurities.     

Ethanol Production from Starch Hydrolyzates using <Emphasis Type="Italic">Zymomonas mobilis</Emphasis> and Glucoamylase Entrapped in Polyvinylalcohol Hydrogel

The glucoamylase from Aspergillus niger, immobilized into poly(vinylalcohol) hydrogel lens-shaped capsules LentiKats®, was used for simultaneous saccharification and fermentation (SSF) with Zymomonas mobilis in free form. This system was stable in both the repeated batch and continuous mode of SSF. The microorganism was found to adsorb on the capsules with immobilized enzyme. This increased the ethanol productivity of the repeated batch system with 5% w/v of immobilized glucoamylase almost 2.1 times (7.2 g l^?1 h^?1) compared to free enzyme–free microorganism system (3.5 g l^?1 h^?1). The continuous SSF with the immobilized glucoamylase (11.5% w/v) tested for 15 days had productivity 10 g l^?1 h^?1, which is comparable to continuous experiments on semi-defined glucose medium (10 g l^?1 h^?1). These two systems were stable in both glucoamylase activity and microorganism productivity.     

Animal Bone Char Solubilization with Itaconic Acid Produced by Free and Immobilized Aspergillus terreus Grown on Glycerol-Based Medium

Cells of Aspergillus terreus, free and immobilized in polyurethane foam, were employed in itaconic acid fermentation processes on glycerol-based media. The purpose was to assess their suitability for animal bone char solubilization and the development of a biotechnological alternative to P fertilizers chemically produced from rock phosphate. Animal bones constitute a renewable source of P that can replace the traditionally used finite, nonrenewable rock phosphate as a P source. Glycerol was an excellent substrate for growth (10.2 g biomass L^?1) and itaconic acid production (26.9 g?L^?1) by free fungal cells after 120-h fermentation. Simultaneously, A. terreus solubilized the insoluble phosphate to a yield of 23 to 50 %, depending on the particle size and concentration. Polyurethane foam cut into cubes of 0.5–0.6 cm per side, with 0.3 mm pore size and applied at 2.0 g?L^?1 proved to be an excellent cell carrier. In repeated batch fermentation, the immobilized mycelium showed a high capacity to solubilize animal bone char, which resulted on average in 168.8 mg?L^–1 soluble phosphate per 48-h cycle and 59.4 % yield (percent of total phosphate) registered in the fourth batch.     

Bioethanol Production from Uncooked Raw Starch by Immobilized Surface-engineered Yeast Cells

Surface-engineered yeast Saccharomyces cerevisiae codisplaying Rhizopus oryzae glucoamylase and Streptococcus bovis α-amylase on the cell surface was used for direct production of ethanol from uncooked raw starch. By using 50 g/L cells during batch fermentation, ethanol concentration could reach 53 g/L in 7 days. During repeated batch fermentation, the production of ethanol could be maintained for seven consecutive cycles. For cells immobilized in loofa sponge, the concentration of ethanol could reach 42 g/L in 3 days in a circulating packed-bed bioreactor. However, the production of ethanol stopped thereafter because of limited contact between cells and starch. The bioreactor could be operated for repeated batch production of ethanol, but ethanol concentration dropped to 55% of its initial value after five cycles because of a decrease in cell mass and cell viability in the bioreactor. Adding cells to the bioreactor could partially restore ethanol production to 75% of its initial value.     

Repeated Batch Cell-Immobilized System for the Biotechnological Production of Xylitol as a Renewable Green Sweetener

The present paper studies the biotechnological production of xylitol using sugarcane bagasse hydrolysate in a repeated batch fermentation system with immobilized cells of Candida guilliermondii FTI20037. Immobilized cell system is considered as an attractive alternative to reuse the well-grown and adapted yeast cells in a new fresh fermentation media, without the need of the inoculum stage. In this work, seven repeated batches were performed in a fluidized bed bioreactor using immobilized cells in calcium alginate beads. According to the obtained results it was observed that the immobilized cells of C. guilliermondii can be reused for six successive batches maintaining an average xylitol yield (Y _p/s) of 0.7 g/L and a volumetric productivity (Q _p) of 0.42 g/L?h at the end of 432 h of fermentation. On the other hand, in the seventh batch (504 h), a decrease of 44 % in the final concentration of xylitol was observed. This reduction can be explained by the possible diffusion and accumulation of insoluble substances, found in the hemicellulosic hydrolysate, in the interior of the immobilization support resulting in substrate mass transfer limitations.     

Enzymatic Hydrolysis and Fermentation of Pretreated Cashew Apple Bagasse with Alkali and Diluted Sulfuric Acid for Bioethanol Production

The aim of this work was to optimize the enzymatic hydrolysis of the cellulose fraction of cashew apple bagasse (CAB) after diluted acid (CAB-H) and alkali pretreatment (CAB-OH), and to evaluate its fermentation to ethanol using Saccharomyces cerevisiae. Glucose conversion of 82?±?2 mg/g CAB-H and 730?±?20 mg/g CAB-OH was obtained when 2% (w/v) of solid and 30 FPU/g bagasse was used during hydrolysis at 45 °C, 2-fold higher than when using 15 FPU/g bagasse, 44?±?2 mg/g CAB-H, and 450?±?50 mg/g CAB-OH, respectively. Ethanol concentration and productivity, achieved after 6 h of fermentation, were 20.0?±?0.2 g L^?1 and 3.33 g L^?1 h^?1, respectively, when using CAB-OH hydrolyzate (initial glucose concentration of 52.4 g L^?1). For CAB-H hydrolyzate (initial glucose concentration of 17.4 g L^?1), ethanol concentration and productivity were 8.2?±?0.1 g L^?1 and 2.7 g L^?1 h^?1 in 3 h, respectively. Hydrolyzates fermentation resulted in an ethanol yield of 0.38 and 0.47 g/g glucose with pretreated CAB-OH and CAB-H, respectively. Ethanol concentration and productivity, obtained using CAB-OH hydrolyzate, were close to the values obtained in the conventional ethanol fermentation of cashew apple juice or sugar cane juice.     

Optimized Fed-Batch Fermentation of Scheffersomyces stipitis for Efficient Production of Ethanol from Hexoses and Pentoses

Scheffersomyces stipitis was cultivated in an optimized, controlled fed-batch fermentation for production of ethanol from glucose–xylose mixture. Effect of feed medium composition was investigated on sugar utilization and ethanol production. Studying influence of specific cell growth rate on ethanol fermentation performance showed the carbon flow towards ethanol synthesis decreased with increasing cell growth rate. The optimum specific growth rate to achieve efficient ethanol production performance from a glucose-xylose mixture existed at 0.1 h^?1. With these optimized feed medium and cell growth rate, a kinetic model has been utilized to avoid overflow metabolism as well as to ensure a balanced feeding of nutrient substrate in fed-batch system. Fed-batch culture with feeding profile designed based on the model resulted in high titer, yield, and productivity of ethanol compared with batch cultures. The maximal ethanol concentration was 40.7 g/L. The yield and productivity of ethanol production in the optimized fed-batch culture was 1.3 and 2 times higher than those in batch culture. Thus, higher efficiency ethanol production was achieved in this study through fed-batch process optimization. This strategy may contribute to an improvement of ethanol fermentation from lignocellulosic biomass by S. stipitis on the industrial scale.     

Evaluation of Cashew Apple Juice for the Production of Fuel Ethanol

A commercial strain of Saccharomyces cerevisiae was used for the production of ethanol by fermentation of cashew apple juice. Growth kinetics and ethanol productivity were calculated for batch fermentation with different initial sugar (glucose + fructose) concentrations. Maximal ethanol, cell, and glycerol concentrations were obtained when 103.1 g L⁻¹ of initial sugar concentration was used. Cell yield (Y _X/S) was calculated as 0.24 (g microorganism)/(g glucose + fructose) using cashew apple juice medium with 41.3 g L⁻¹ of initial sugar concentration. Glucose was exhausted first, followed by fructose. Furthermore, the initial concentration of sugars did not influence ethanol selectivity. These results indicate that cashew apple juice is a suitable substrate for yeast growth and ethanol production.     

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.     

Improving the Performance of a Continuous Process for the Production of Ethanol from Starch

In a previous work, a continuous simultaneous saccharification and fermentation process to produce ethanol from cassava starch was studied, using a set of fixed-bed reactors. The biocatalyst consisted of glucoamylase immobilized in silica particles and co-immobilized with S. cerevisiae in pectin gel. Using 3.8 U mL^?1 _reactor and 0.05 g_{wet yeast} mL^?1 _reactor at start-up, starch hydrolysis was the rate-limiting step. Maximum ethanol productivity was 5.8 g_ethanol L^?1 h^?1, with 94.0% conversion of total reducing sugars (TRS) and 83.0% of the ethanol theoretical yield. In this work, the molar mass of the substrate and the biocatalyst particle size were reduced in an attempt to improve the bioreactor performance. The diameters of silica and pectin gel particles were reduced from 100 μm and 3–4 mm, respectively, to 60 μm and 1–1.5 mm, and the degree of substrate prehydrolysis by α-amylase was increased. The bioreactor performance was assessed for different loads of immobilized glucoamylase (2.1, 2.8, and 3.8 U mL^?1 _reactor), for the same initial cell concentration (0.05 g_{wet yeast.}mL^?1 _reactor). Feeding with 154.0 g L^?1 of TRS and using 3.8 U mL^?1 _reactor, fermentation became the rate-limiting step. Productivity reached 11.7 g L^?1 h^?1, with 97.0% of TRS conversion and 92.0% of the ethanol theoretical yield. The reactor was operated during 275 h without any indication of destabilization.     

Kinetics of ethanol production from carob pods extract by immobilizedSaccharomyces cerevisiae cells

Kinetics of ethanol production from carob pods extract by immobilizedS. cerevisiae cells in static and shake flask fermentation have been investigated. Shake flask fermentation proved to be a better fermentation system for the production of ethanol than static fermentation. The optimum values of ethanol concentration, ethanol productivity, ethanol yield, and fermentation efficiency were obtained at pH range 3.5–6.5 and temperature between 30–35°C. A maximum ethanol concentration (65 g/L), ethanol productivity (8.3 g/Lh), ethanol yield (0.44 g/g), and fermentation efficiency (95%) was achieved at an initial sugar concentration of 200, 150, 100, and 200 g/L, respectively. The highest values of specific ethanol production rate and specific sugar uptake rate were obtained at pH 6.5, temperature 40°C, and initial sugar concentration of 100 g/L. Other kinetic parameters, biomass concentration, biomass yield, and specific biomass production rate were maximum at pH 5.5, temperature 30°C, and initial sugar concentration 150 g/L. Under the same fermentation conditions non-sterilized carob pod extract gave higher ethanol concentration than sterilized medium. In repeated batch fermentations, the immobilizedS. cerevisiae cells in Ca-alginate beads retained their ability to produce ethanol for 5 d.     

Purification of Alcohol Dehydrogenase from Saccharomyces cerevisiae Using Magnetic Dye-Ligand Affinity Nanostructures

Reactive Green 19 was covalently immobilized onto magnetic nanostructures for purification of alcohol dehydrogenase from Saccharomyces cerevisiae. The Reactive Green 19 immobilized magnetic nanostructures were characterized by Fourier transform infrared spectroscopy, electron spin resonance, atomic force microscope, and energy dispersive X-ray analysis. Particle size of nanostructures was found to be roughly 70 nm. Alcohol dehydrogenase adsorption experiments were investigated under different conditions in batch system (i.e., medium pH, alcohol dehydrogenase concentration, temperature, and ionic strength). Maximum alcohol dehydrogenase adsorption capacity was found to be 176.09 mg/g polymer while nonspecific alcohol dehydrogenase adsorption onto plain magnetic nanostructures was negligible (19.4 mg/g polymer). Alcohol dehydrogenase molecules were desorbed by using 1.0 M NaCl with 98.4 % recovery. Alcohol dehydrogenase from S. cerevisiae was purified 45.63-fold in single step with dye-immobilized magnetic nanostructures, and purity of alcohol dehydrogenase was shown by silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Impact of High Temperature on Ethanol Fermentation by Kluyveromyces marxianus Immobilized on Banana Leaf Sheath Pieces

Ethanol fermentation was carried out with Kluyveromyces marxianus cells at various temperatures (30, 35, 40, and 45 °C). Fermentation performance of the immobilized yeast on banana leaf sheath pieces and the free yeast were evaluated and compared. Generally, ethanol production of the immobilized and free yeast was stable in a temperature range of 30–40 °C. Temperature of 45 °C restricted yeast growth and lengthened the fermentation. The immobilized yeast demonstrated faster sugar assimilation and higher ethanol level in the fermentation broth in comparison with the free yeast at all fermentation temperatures. Change in fatty acid level in cellular membrane was determined to clarify the response of the free and immobilized yeast to thermal stress. The free cells of K. marxianus responded to temperature increase by increasing saturated fatty acid (C16:0 and C18:0) level and by decreasing unsaturated fatty acid (C18:1 and C18:2) level in cellular membrane. For fermentation at 40 °C with immobilized cells of K. marxianus, however, the changes were not observed in both saturated fatty acid (C16:0) and unsaturated fatty acid (C18:1 and C18:2) level.     

Bioethanol Production from Soybean Residue via Separate Hydrolysis and Fermentation

Bioethanol was produced using polysaccharide from soybean residue as biomass by separate hydrolysis and fermentation (SHF). This study focused on pretreatment, enzyme saccharification, and fermentation. Pretreatment to obtain monosaccharide was carried out with 20% (w/v) soybean residue slurry and 270 mmol/L H₂SO₄ at 121 °C for 60 min. More monosaccharide was obtained from enzymatic hydrolysis with a 16 U/mL mixture of commercial enzymes C-Tec 2 and Viscozyme L at 45 °C for 48 h. Ethanol fermentation with 20% (w/v) soybean residue hydrolysate was performed using wild-type and Saccharomyces cerevisiae KCCM 1129 adapted to high concentrations of galactose, using a flask and 5-L fermenter. When the wild type of S. cerevisiae was used, an ethanol production of 20.8 g/L with an ethanol yield of 0.31 g/g consumed glucose was obtained. Ethanol productions of 33.9 and 31.6 g/L with ethanol yield of 0.49 g/g consumed glucose and 0.47 g/g consumed glucose were obtained in a flask and a 5-L fermenter, respectively, using S. cerevisiae adapted to a high concentration of galactose. Therefore, adapted S. cerevisiae to galactose could enhance the overall ethanol fermentation yields compared to the wild-type one.