Simultaneous Saccharification And Fermentation Experiments Research Articles

Physiological and Molecular Characterization of Yeast Cultures Pre-Adapted for Fermentation of Lignocellulosic Hydrolysate

Economically feasible bioethanol process from lignocellulose requires efficient fermentation by yeast of all sugars present in the hydrolysate. However, when exposed to lignocellulosic hydrolysate, Saccharomyces cerevisiae is challenged with a variety of inhibitors that reduce yeast viability, growth, and fermentation rate, and in addition damage cellular structures. In order to evaluate the capability of S. cerevisiae to adapt and respond to lignocellulosic hydrolysates, the physiological effect of cultivating yeast in the spruce hydrolysate was comprehensively studied by assessment of yeast performance in simultaneous saccharification and fermentation (SSF), measurement of furaldehyde reduction activity, assessment of conversion of phenolic compounds and genome-wide transcription analysis. The yeast cultivated in spruce hydrolysate developed a rapid adaptive response to lignocellulosic hydrolysate, which significantly improved its fermentation performance in subsequent SSF experiments. The adaptation was shown to involve the induction of NADPH-dependent aldehyde reductases and conversion of phenolic compounds during the fed-batch cultivation. These properties were correlated to the expression of several genes encoding oxidoreductases, notably AAD4, ADH6, OYE2/3, and YML131w. The other most significant transcriptional changes involved genes involved in transport mechanisms, such as YHK8, FLR1, or ATR1. A large set of genes were found to be associated with transcription factors (TFs) involved in stress response (Msn2p, Msn4p, Yap1p) but also cell growth and division (Gcr4p, Ste12p, Sok2p), and these TFs were most likely controlling the response at the post-transcriptional level.

Cellobiose와 xylose를 동시에 대사하는 Pichia stipitis 돌연변이를 활용한 동시당화발효

In order to economically produce biofuels in simultaneous saccharification and fermentation (SSF) of cellulosic biomass, it is required to construct microbial host efficiently utilizing various sugars. Previously, a mutant Pichia stipitis YN14 strain co-fermenting cellobiose and xylose was isolated. In this study, ethanol production performance of P. stipitis YN14 was compared with the parental P. stipitis CBS 6054 through the series of SSF experiments. In SSF of cellulose, the mutant strain employing Celluclast 1.5 L as the cellulolytic enzyme produced 6.1 g/L of ethanol, which was 5.5-times and 1.2-times higher than the parental strain with Celluclast 1.5 L and Cellic CTec2, respectively. In addition, the mutant YN14 strain with Celluclast 1.5 L showed 26.6 g/L of ethanol production in simultaneous saccharification and cofermentation (SSCF) of cellulose and xylose, which was 75% and 60% higher than the parental CBS 6054 strain with Celluclast 1.5 L and Cellic CTec2, respectively. Our results propose that efficient ethanol production from the cellulosic biomass would be accomplished by utilization of yeast strain fermenting cellobiose and xylose simultaneously.

Simultaneous saccharification and fermentation of oil palm front for the production of 2,3-butanediol

The present study aims to develop a process for the production of 2,3-butanediol by Simultaneous Saccharification and Fermentation (SSF) of Oil Palm Front (OPF) biomass. The study compares SSF with Separate Hydrolysis and Fermentation (SHF) of oil palm biomass and batch fermentation using glucose. The results showed that SSF is one of the most attractive techniques for the microbial production of 2,3-butanediol using lignocellulosic biomass. The enzymatic digestibility and fermentative efficiency of alkali pre-treated OPF biomass was checked and the role of various experimental parameters like enzyme loading and inoculum loading were optimized. SSF experiments could give 30.74 g/l of BDO in shake flask and 12.53 g/l in 500 ml bioreactor with a productivity of 0.32 and 0.13 g/l/h respectively.

Ethanol production from sugarcane bagasse by fed‐batch simultaneous saccharification and fermentation at high solids loading

AbstractTo make the bioethanol conversion process economically viable, improving solids loading (>15% [w/v]) in different unit operations is crucial. In this study, alkali pretreatment of sugarcane bagasse was carried out at high solids loading. Temperature and pH were optimised via batch simultaneous saccharification and fermentation (SSF) experiments using Saccharomyces cerevisiae Y‐2034 or Kluyveromyces marxianus NCYC‐587. The effects of enzyme addition mode and solids loading on ethanol production were studied in fed‐batch SSF experiments. It was demonstrated that the optimised pH and temperature for both S. cerevisiae Y‐2034 and K. marxianus NCYC‐587 were 5.2 and 37°C. The mode of enzyme addition “one‐time dose” improved ethanol productivity better than the “batch addition” mode. With fresh substrates added, solids loading strongly increased from 19% to 33% (w/v), corresponded to a small decline in the ethanol yield. At the final solids loading of 33% (w/v), the highest ethanol concentration of 75.57 ± 2.14 g/L was obtained, with ethanol productivity of 0.63 ± 0.02 g/(L h) and a yield of 66.17 ± 1.87%.

Reusable Floating Beads with Immobilized Xylose-Fermenting Yeast Cells for Simultaneous Saccharification and Fermentation of Lime-Pretreated Rice Straw.

Novel bioreactor beads for simultaneous saccharification and fermentation (SSF) of lime-pretreated rice straw (RS) into ethanol were prepared. Genetically modified Saccharomyces cerevisiae cells expressing genes encoding xylose reductase, xylitol dehydrogenase, and xylulokinase were immobilized in calcium alginate beads containing inorganic lightweight filler particles to reduce specific gravity. For SSF experiments, the beads were floated in slurry composed of lime-pretreated RS and enzymes and incubated under CO2 atmosphere to reduce the pH for saccharification and fermentation. Following this reaction, beads were readily picked up from the upper part of the slurry and were directly transferred to the next vessel with slurry. After 240 h of incubation, ethanol production by the beads was equivalent to that by free cells, a trend that was repeated in nine additional runs, with slightly improved ethanol yields. Slurry with pre-saccharified lime-pretreated RS was subjected to SSF with floating beads for 168 h. Although higher cell concentrations in beads resulted in more rapid initial ethanol production rates, with negligible diauxic behavior for glucose and xylose utilization, no improvement in the ethanol yield was observed. A fermentor-scale SSF experiment with floating beads was successfully performed twice, with repeated use of the beads, resulting in the production of 40.0 and 39.7 g/L ethanol. There was no decomposition of the beads during agitation at 60 rpm. Thus, this bioreactor enables reuse of yeast cells for efficient ethanol production by SSF of lignocellulosic feedstock, without the need for instruments for centrifugation or filtration of whole slurry.

Boosting bioethanol production from Eucalyptus wood by whey incorporation

The mixture of Eucalyptus globulus wood (EGW) and cheese whey powder (CWP) was proposed for intensification of simultaneous saccharification and fermentation (SSF) at high temperature and solid loadings using the industrial Saccharomyces cerevisiae Ethanol Red® strain. High ethanol concentration (93 g/L), corresponding to 94% ethanol yield, was obtained at 35 °C from 37% of solid mixture using cellulase and β-galactosidase enzymes (24.2 FPU/g and 20.0 U/g, respectively). The use of CWP mixed with pretreated EGW increased the ethanol concentration in 1.5-fold, in comparison with SSF experiments without CWP for both Ethanol Red® and CEN.PK113-7D strains. Moreover, 1.4-fold higher ethanol concentration was obtained with Ethanol Red®, in comparison with CEN.PK113-7D strain. Ethanol Red® strain was genetically engineered for β-galactosidase production in order to advance towards a fully integrated process. This work shows the feasibility of attaining high ethanol concentrations in second generation bioprocesses by a multi-waste valorization approach.

Bioethanol Production from Several Tropical Wood Species using Simultaneous Saccharification and Fermentation Processes

The study was conducted to determine the best method of hydrolysis (saccharification) and fermentation for bioethanol production using simultaneous saccharification and fermentation process. Three different tropical wood species namely gmelina wood (Gmelina sp.), pine wood (Pinus merkusii) and oil palm (Elaeis guineensis Jacq.) were pre-treated using kraft process and then converted into bioethanol using simultaneous saccharification and fermentation (SSF) processes. The pulp produced by kraft process was analized to determine their chemical properties before treatments. SSF was performed in 500 ml fermentors with total slurry of 200 ml. The substrate and nutrient media were autoclaved (121ºC and 20 min). The samples diluted to 2.5% (w/v) of total slurry were used as substrate. The enzyme preparation used commercial cellulase enzyme. The amount of cellulase added was 4 and 8% (w/w) of dry mass of samples. All SSF process was conducted by inoculating yeast Saccharomyces cerevisiae into fermentor in the amount of 10% (v/v) 1.5 x 109 CFU/cc. The SSF experiments run for 96 h, and the data were investigated periodically every 24 h. The results showed total sugar and reducing sugar tended to decrease with time of inoculation whereas ethanol concentration increases significantly. The growth of yeast Saccharomyces cerevisiae tended to incease in initial inoculation and decrease by the end of inoculation. The best method of hydrolysis (saccharification) and fermentation using SSF process for all tropical wood species tested were using cellulase 8% of dry mass (DM) and 10% (v/v) of Saccharomyces cerevisiae which produced bioethanol with concentration of 0.98; 0.57 and 0.51% for gmelina, pine and oil palm respectively and produced yields 11.21, 5.85 and 3.20%, in that order.

Production of optically pure D-lactic acid from brown rice using metabolically engineered Lactobacillus plantarum.

Simultaneous saccharification and fermentation (SSF) of D-lactic acid was performed using brown rice as both a substrate and a nutrient source. An engineered Lactobacillus plantarum NCIMB 8826 strain, in which the ʟ-lactate dehydrogenase gene was disrupted, produced 97.7g/L D-lactic acid from 20% (w/v) brown rice without any nutrient supplementation. However, a significant amount of glucose remained unconsumed and the yield of lactic acid was as low as 0.75 (g/g-glucose contained in brown rice). Interestingly, the glucose consumption was significantly improved by adapting L. plantarum cells to the low-pH condition during the early stage of SSF (8-17h). As a result, 117.1g/L D-lactic acid was produced with a high yield of 0.93 and an optical purity of 99.6% after 144h of fermentation. SSF experiments were repeatedly performed for ten times and D-lactic acid was stably produced using recycled cells (118.4-129.8g/L). On average, D-lactic acid was produced with a volumetric productivity of 2.18g/L/h over 48h.

Kinetic modeling and dynamic analysis of simultaneous saccharification and fermentation of cellulose to bioethanol

Kinetic modeling and dynamic analysis of the simultaneous saccharification and fermentation (SSF) of cellulose to ethanol was carried out in this study to determine the key reaction kinetics parameters and product inhibition features of the process. To obtain the more reliable kinetic parameters which can be applied for a wide range of operating conditions, batch SSF experiments were carried out at three enzyme loadings (10, 15 and 20FPU/g cellulose) and two levels of initial concentrations of fermentable sugars (glucose and mannose). Results indicated that the maximum ethanol yield and concentration were achieved at high level of sugar concentrations with intermediate enzyme loading (15FPU/g cellulose). Dynamic analysis of the acquired experimental results revealed that cellulase inhibition by cellobiose plays the most important role at high level of enzyme loading and low level of initial sugar concentrations. The inhibition of glucose becomes significant when high concentrations of sugars were present in the feedstock. Experimental results of SSF process also reveal that an efficient mixing between the phases helps to improve the ethanol yield significantly.

Simultaneous saccharification and fermentation of cactus pear biomass—evaluation of using different pretreatments

Lignocellulosic biomasses of two species of cactus pear (Opuntia ficus indica and Nopalea cochenillifera) were tested for the production of ethanol. Their ability to grow in water-stressed regions makes them eligible for reducing the production cost of biofuels in drought regions. The biomass was pretreated using three different strategies: alkaline hydrogen peroxide, alkaline using NaOH, and acid using H2SO4 followed by alkaline delignification with NaOH. Analyses of the composition, crystallinity, and enzymatic hydrolysis were performed on the material before and after pretreatment. An experimental design was used to evaluate the influence of temperature and initial cellulose concentration on the process of simultaneous saccharification and fermentation (SSF) using the pretreated material, which showed the best performance in previous tests, and two strains of Saccharomyces cerevisiae (PE-2 and LNF CA-11). Biomass characterization showed a content of 31.55% cellulose, 17.12% hemicelluloses, and 10.25% lignins for N. cochenillifera and 34.86% cellulose, 19.97% hemicelluloses, and 15.72% lignins for O. ficus indica. The alkaline pretreatment provided the best enzymatic saccharification yields. The SSF experiments resulted in the greatest cellulose-to-ethanol yields using strain PE-2 with the pretreated N. cochenillifera (93.81%) at 40°C and an initial cellulose concentration of 4%. N. cochenillifera showed better yields than O. ficus indica, and the PE-2 strain performed better than CA-11. These results suggest a good potential for cactus pear cladodes in the production of cellulosic ethanol.

Modeling of a simultaneous saccharification and fermentation process for ethanol production from lignocellulosic wastes by kluyveromyces marxianus

This paper presents the modeling of the main dynamics of a Simultaneous Saccharification and Fermentation (SSF) process using lignocellulosic wastes as substrate. SSF experiments were carried out using the yeast Kluyveromyces marxianus as the inoculum and oil palm wastes as the substrate, in order to obtain glucose and ethanol concentration data. The experimental data were used for the parameter identification and model validation. The resulting model predictsthe dynamic behavior of glucose and ethanol concentrations very closely. Performing a sensitivity analysis, parameters which have a higher effect in the modelpredictions are recognized, so the model can be re-optimized in particular cases with low computational requirements. The re-optimization strategy improves the model capacity to predict the dynamics of the SSF process.

Physiological characterization of thermotolerant yeast for cellulosic ethanol production

The conversion of lignocellulose into fermentable sugars is considered a promising alternative for increasing ethanol production. Higher fermentation yield has been achieved through the process of simultaneous saccharification and fermentation (SSF). In this study, a comparison was performed between the yeast species Saccharomyces cerevisiae and Kluyveromyces marxianus for their potential use in SSF process. Three strains of S. cerevisiae were evaluated: two are widely used in the Brazilian ethanol industry (CAT-1 and PE-2), and one has been isolated based on its capacity to grow and ferment at 42 °C (LBM-1). In addition, we used thermotolerant strains of K. marxianus. Two strains were obtained from biological collections, ATCC 8554 and CCT 4086, and one strain was isolated based on its fermentative capacity (UFV-3). SSF experiments revealed that S. cerevisiae industrial strains (CAT-1 and PE-2) have the potential to produce cellulosic ethanol once ethanol had presented yields similar to yields from thermotolerant strains. The industrial strains are more tolerant to ethanol and had already been adapted to industrial conditions. Moreover, the study shows that although the K. marxianus strains have fermentative capacities similar to strains of S. cerevisiae, they have low tolerance to ethanol. This characteristic is an important target for enhancing the performance of this yeast in ethanol production.

Optimization of corn stover biorefinery for coproduction of oligomers and second generation bioethanol using non-isothermal autohydrolysis

Corn stover was used for manufacturing 2nd generation bioethanol following a biorefinery scheme based on fractionation by autohydrolysis and further Simultaneous Saccharification and Fermentation (SSF) of pretreated solids. Autohydrolysis was performed under a wide range of severities to identify conditions leading simultaneously to a liquid phase containing hemicellulosic saccharides (accounting for up to 68% of initial xylan) and to a solid phase with high enzymatic susceptibility. SSF experiments were carried out under a variety of experimental conditions to assess the effects of the major operational variables. The glucan conversion into ethanol reached values up to 86%, with a bioethanol concentration of 37.8g/L. Fed-batch operation in the SSF stage allowed the utilization of higher solid loadings, allowing an increase in the bioethanol concentration up to 51.6g/L, or to reduce the amount of enzymes needed for reaching a given conversion.

Lactic Acid Production from Repeated-Batch and Simultaneous Saccharification and Fermentation of Cassava Starch Wastewater by Amylolytic Lactobacillus Plantarum MSUL 702

The present study is aimed at determining the performance of an amylolytic Lactobacillus plantarum MSUL 702 for lactic acid production from the repeated-batch and simultaneous saccharification and fermentation (SSF) of a synthetic cassava starch wastewater (SCW). An ability of the bacteria to treat the SCW in terms of chemical oxygen demand (COD) and total kjeldahl nitrogen (TKN) removal efficiencies was also investigated during the fermentation processes. The SSF experiments were performed for five consecutive batches under a non-sterile condition and at a room temperature. The highest lactic acid concentration and viable lactic acid bacteria at 28.71g/L and 9.26 log CFU/mL, respectively, were obtained in the 48h of the first batch fermentation. The highest COD and TKN removal efficiencies at 98% and 85%, respectively, were obtained in the 48h of the second batch fermentation. The bacteria could retain the high lactic acid production and treatment efficiency up to four consecutive batches.

Model‐based estimation of optimal temperature profile during simultaneous saccharification and fermentation of Arundo donax

A kinetic model fitted to enzymatic hydrolysis of Arundo donax was coupled to a fermentation kinetic model derived from simultaneous saccharification and fermentation (SSF) experiments at different temperatures for the determination of optimal temperature profile (between 36 and 45°C) using iterative dynamic programming (IDP). A sensitivity analysis of enzyme kinetic model not only facilitated model reduction in terms of number of parameters, but also enabled artifacts from parameter estimations to be identified. In separate fermentation experiments conducted at 35, 40, 45, and 50°C using ∼40 g/L background glucose in fiber-free liquid fraction of Arundo it was found that growth was possible at 40°C, but the fermentation capacity was completely lost after 12 h at 50°C. The final ethanol concentration obtained after 120 h in isothermal SSF experiments at 36, 39, 42, and 45°C were 10.6, 13.7, 14.2, and 12.5 g/L, respectively. The predicted optimal temperature profile in SSF determined by iterative dynamic programming was (i) gradual decrease from 40 to 37.5°C until 16 h, (ii) a linear increase upto 45°C until 80 h, and (iii) gradual decrease by 1°C until 120 h. Experimental results were in good agreement with the model predictions. The ethanol concentration after 72 h obtained in the optimal case was 13.6 g/L in comparison to 9.1, 12.2, 12.6, and 11.6 g/L for ISO-SSF at 36, 39, 42, and 45°C, respectively. Moreover this value was 95.8% of the final value achieved at the end of 120 h, indicating that the process times could be significantly shortened by using non-isothermal SSF.

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 YP/S of 0.44 g/g and QP 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.

Fed-Batch Mode Optimization of SSF for Cellulosic Ethanol Production from Steam-Exploded Corn Stover

To achieve a desired ethanol concentration and maximize substrate concentration, the fed-batch simultaneous saccharification and fermentation (SSF) process was performed on steam-exploded corn stover using the yeast strain Saccharomyces cerevisiae Y5. The fed-batch SSF experiments were conducted with feed loading and scheduled feed time conditions that were optimized with response surface methodology (RSM). The overall ethanol yield (based on the raw material cellulose content) in 48 h was as high as 64.0%, which was achieved with a final substrate loading of 26%(w/w), enzyme loading of 7 FPU/g cellulose, and dry yeast loading weight of 2.0 g/L. No additional yeast cells or enzymes were added during solid substrate fermentation.

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 beta-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.

Comparação entre processos em SHF e em SSF de bagaço de cana-de-açúcar para a produção de etanol por Saccharomyces cerevisiae

In this work, four different process configurations, including three simultaneous saccharification and fermentation (SSF) schemes and one separate hydrolysis and fermentation (SHF) scheme, were compared, at 8% water-insoluble solids, regarding ethanol production from steam-pretreated and alkali-delignified sugar cane bagasse. Two configurations included a 16 h lasting enzymatic presaccharification prior to SSF, and the third one was a classical SSF without presaccharification. Cellulose conversion was higher for the delignified bagasse, and higher in SSF experiments than in SHF. The highest cellulose-to-ethanol conversion (around 60% in 24 h) and maximum ethanol volumetric productivities (0.29-0.30 g/L.h) were achieved in the presaccharification-assisted SSF.

Simultaneous saccharification and fermentation of lignocellulosic residues pretreated with phosphoric acid–acetone for bioethanol production

Bermudagrass, reed and rapeseed were pretreated with phosphoric acid–acetone and used for ethanol production by means of simultaneous saccharification and fermentation (SSF) with a batch and fed-batch mode. When the batch SSF experiments were conducted in a 3% low effective cellulose, about 16 g/L of ethanol were obtained after 96 h of fermentation. When batch SSF experiments were conducted with a higher cellulose content (10% effective cellulose for reed and bermudagrass and 5% for rapeseed), higher ethanol concentrations and yields (of more than 93%) were obtained. The fed-batch SSF strategy was adopted to increase the ethanol concentration further. When a higher water-insoluble solid (up to 36%) was applied, the ethanol concentration reached 56 g/L of an inhibitory concentration of the yeast strain used in this study at 38 °C. The results show that the pretreated materials can be used as good feedstocks for bioethanol production, and that the phosphoric acid–acetone pretreatment can effectively yield a higher ethanol concentration.