Effect of substrate and cellulase concentration on simultaneous saccharification and fermentation of steam-pretreated softwood for ethanol production

Economic optimization of the production of ethanol by simultaneous saccharification and fermentation (SSF) requires knowledge about the influence of substrate and enzyme concentration on yield and productivity. Although SSF has been investigated extensively, the optimal conditions for SSF of softwoods have yet not been determined. In this study, SO2-impregnated and steam-pretreated spruce was used as substrate for the production of ethanol by SSF. Commercial enzymes were used in combination with the yeast Saccharomyces cerevisiae. The effects of the concentration of substrate (2% to 10% w/w) and of cellulases (5 to 32 FPU/g cellulose) were investigated. SSF was found to be sensitive to contamination because lactic acid was produced. The ethanol yield increased with increasing cellulase loading. The highest ethanol yield, 68% of the theoretical based on the glucose and mannose present in the original wood, was obtained at 5% substrate concentration. This yield corresponds to 82% of the theoretical based on the cellulose and soluble glucose and mannose present at the start of SSF. A higher substrate concentration caused inefficient fermentation, whereas a lower substrate concentration, 2%, resulted in increased formation of lactic acid, which lowered the yield. Compared with separate hydrolysis and fermentation, SSF gave a higher yield and doubled the productivity.     

Impact of an acid fungal protease in high gravity fermentation for ethanol production using indian sorghum as a feedstock

This study evaluated the conventional jet cooking liquefaction process followed by simultaneous saccharification and fermentation (SSF) at 30% and 35% dry solids (DS) concentration of Indian sorghum feedstock for ethanol production, with addition of acid fungal protease or urea. To evaluate the efficacy of thermostable α‐amylase in liquefaction at 30% and 35% DS concentration of Indian sorghum, liquefact solubility, higher dextrins, and fermentable sugars were analyzed at the end of the process. The liquefact was further subjected to SSF using yeast. In comparison with urea, addition of an acid fungal protease during SSF process was observed to accelerate yeast growth (μ), substrate consumption (Q_s), ultimately ethanol yield based on substrate (Y_p/s) and ethanol productivity based on fermentation time (Q_p). The fermentation efficiency and ethanol recovery were determined for both concentrations of Indian sorghum and found to be increased with use of acid fungal protease in SSF process. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 329–336, 2013     

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.     

SSF of steam-pretreated wheat straw with the addition of saccharified or fermented wheat meal in integrated bioethanol production

Background

Integration of second-generation (2G) bioethanol production with existing first-generation (1G) production may facilitate commercial production of ethanol from cellulosic material. Since 2G hydrolysates have a low sugar concentration and 1G streams often have to be diluted prior to fermentation, mixing of streams is beneficial. Improved ethanol concentrations in the 2G production process lowers energy demand in distillation, improves overall energy efficiency and thus lower production cost. There is also a potential to reach higher ethanol yields, which is required in economically feasible ethanol production. Integrated process scenarios with addition of saccharified wheat meal (SWM) or fermented wheat meal (FWM) were investigated in simultaneous saccharification and (co-)fermentation (SSF or SSCF) of steam-pretreated wheat straw, while the possibility of recovering the valuable protein-rich fibre residue from the wheat was also studied.

Results

The addition of SWM to SSF of steam-pretreated wheat straw, using commercially used dried baker’s yeast, S. cerevisiae, resulted in ethanol concentrations of about 60 g/L, equivalent to ethanol yields of about 90% of the theoretical. The addition of FWM in batch mode SSF was toxic to baker’s yeast, due to the ethanol content of FWM, resulting in a very low yield and high accumulation of glucose. The addition of FWM in fed-batch mode still caused a slight accumulation of glucose, but the ethanol concentration was fairly high, 51.2 g/L, corresponding to an ethanol yield of 90%, based on the amount of glucose added.In batch mode of SSCF using the xylose-fermenting, genetically modified S. cerevisiae strain KE6-12, no improvement was observed in ethanol yield or concentration, compared with baker’s yeast, despite the increased xylose utilization, probably due to the considerable increase in glycerol production. A slight increase in xylose consumption was seen when glucose from SWM was fed at a low feed rate, after 48 hours, compared with batch SSCF. However, the ethanol yield and concentration remained in the same range as in batch mode.

Conclusion

Ethanol concentrations of about 6% (w/v) were obtained, which will result in a significant reduction in the cost of downstream processing, compared with SSF of the lignocellulosic substrate alone. As an additional benefit, it is also possible to recover the protein-rich residue from the SWM in the process configurations presented, providing a valuable co-product.

     

BIOETHANOL PRODUCTION FROM LEAFY BIOMASS OF MANGO (Mangifera indica) INVOLVING NATURALLY ISOLATED AND RECOMBINANT ENZYMES

The present study describes the usage of dried leafy biomass of mango (Mangifera indica) containing 26.3% (w/w) cellulose, 54.4% (w/w) hemicellulose, and 16.9% (w/w) lignin, as a substrate for bioethanol production from Zymomonas mobilis and Candida shehatae. The substrate was subjected to two different pretreatment strategies, namely, wet oxidation and an organosolv process. An ethanol concentration (1.21 g/L) was obtained with Z. mobilis in a shake-flask simultaneous saccharification and fermentation (SSF) trial using 1% (w/v) wet oxidation pretreated mango leaves along with mixed enzymatic consortium of Bacillus subtilis cellulase and recombinant hemicellulase (GH43), whereas C. shehatae gave a slightly higher (8%) ethanol titer of 1.31 g/L. Employing 1% (w/v) organosolv pretreated mango leaves and using Z. mobilis and C. shehatae separately in the SSF, the ethanol titers of 1.33 g/L and 1.52 g/L, respectively, were obtained. The SSF experiments performed with 5% (w/v) organosolv-pretreated substrate along with C. shehatae as fermentative organism gave a significantly enhanced ethanol titer value of 8.11 g/L using the shake flask and 12.33 g/L at the bioreactor level. From the bioreactor, 94.4% (v/v) ethanol was recovered by rotary evaporator with 21% purification efficiency.     

Characterization of the steam-exploded spent Shiitake mushroom medium and its efficient conversion to ethanol

Spent Shiitake mushroom medium was subjected to steam explosion followed by simultaneous saccharification and fermentation (SSF) using Meicelase and Saccahromyces cerevisiae AM12. Water extraction of the medium exposed to steam at 20 atm for 5 min enhanced the saccharification rate by about 20% compared to steam-exploded medium before water extraction and resulted in the production of 23.8 g/l ethanol from a substrate concentration of 100 g/l. This corresponded to 87.6% of the theoretical ethanol yield, i.e., 15.9 g ethanol was obtained from 100 g of spent Shiitake mushroom medium. Spent Shiitake mushroom medium subjected to steam explosion and then water extraction appears to be a candidate for efficient bioconversion to ethanol.     

Ethanol from lignocellulosic materials by a simultaneous saccharification and fermentation process (SFS) with Kluyveromyces marxianus CECT 10875

Simultaneous saccharification and fermentation (SSF) process for ethanol production from various lignocellulosic woody (poplar and eucalyptus) and herbaceous (Sorghum sp. bagasse, wheat straw and Brassica carinata residue) materials has been assayed using the thermotolerant yeast strain Kluyveromyces marxianus CECT 10875. Biomass samples were previously treated in a steam explosion pilot plant to provide pretreated biomass with increased cellulose content relative to untreated materials and to enhance cellulase accessibility. SSF experiments were performed in laboratory conditions at 42 °C, 10% (w/v) substrate concentration and 15 FPU/g substrate of commercial cellulase. The results indicate that it is possible to reach SSF yields in the range of 50–72% of the maximum theoretical SSF yield, based on the glucose available in pretreated materials, in 72–82 h. Maximum ethanol contents from 16 to 19 g/l were obtained in fermentation media, depending on the material tested.     

Adaptation of the xylose fermenting yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> F12 for improving ethanol production in different fed-batch SSF processes

An efficient fermenting microorganism for bioethanol production from lignocellulose is highly tolerant to the inhibitors released during pretreatment and is able to ferment efficiently both glucose and xylose. In this study, directed evolution was employed to improve the xylose fermenting Saccharomyces cerevisiae F12 strain for bioethanol production at high substrate loading. Adapted and parental strains were compared with respect to xylose consumption and ethanol production. Adaptation led to an evolved strain more tolerant to the toxic compounds present in the medium. When using concentrated prehydrolysate from steam-pretreated wheat straw with high inhibitor concentration, an improvement of 65 and 20% in xylose consumption and final ethanol concentration, respectively, were achieved using the adapted strain. To address the need of high substrate loadings, fed-batch SSF experiments were performed and an ethanol concentration as high as 27.4 g/l (61% of the theoretical) was obtained with 11.25% (w/w) of water insoluble solids (WIS).     

Optimization of fermentation conditions for the production of ethanol from sago starch using response surface methodology

The quantitative effects of temperature, pH and time of fermentation were investigated on simultaneous saccharification and fermentation (SSF) of ethanol from sago starch with glucoamylase (AMG) and Zymomonas mobilis ZM4 using a Box–Wilson central composite design protocol. The SSF process was studied using free enzyme and free cells and it was found that with sago starch, maximum ethanol concentration of 70.68 g/l was obtained using a starch concentration of 140 g/l, which represents an ethanol yield of 97.08%. The optimum conditions for the above yield were found to be a temperature of 36.74 °C, pH of 5.02 and time of fermentation of 17 h. Thus by using the central composite design, it is possible to determine the accurate values of the fermentation parameters where maximum production of ethanol occurs.     

A short review on SSF – an interesting process option for ethanol production from lignocellulosic feedstocks

Simultaneous saccharification and fermentation (SSF) is one process option for production of ethanol from lignocellulose. The principal benefits of performing the enzymatic hydrolysis together with the fermentation, instead of in a separate step after the hydrolysis, are the reduced end-product inhibition of the enzymatic hydrolysis, and the reduced investment costs. The principal drawbacks, on the other hand, are the need to find favorable conditions (e.g. temperature and pH) for both the enzymatic hydrolysis and the fermentation and the difficulty to recycle the fermenting organism and the enzymes. To satisfy the first requirement, the temperature is normally kept below 37°C, whereas the difficulty to recycle the yeast makes it beneficial to operate with a low yeast concentration and at a high solid loading. In this review, we make a brief overview of recent experimental work and development of SSF using lignocellulosic feedstocks. Significant progress has been made with respect to increasing the substrate loading, decreasing the yeast concentration and co-fermentation of both hexoses and pentoses during SSF. Presently, an SSF process for e.g. wheat straw hydrolyzate can be expected to give final ethanol concentrations close to 40 g L^-1 with a yield based on total hexoses and pentoses higher than 70%.     

Simultaneous saccharification and ethanol fermentation of oxalic acid pretreated corncob assessed with response surface methodology

Response surface methodology was used to evaluate optimal time, temperature and oxalic acid concentration for simultaneous saccharification and fermentation (SSF) of corncob particles by Pichia stipitis CBS 6054. Fifteen different conditions for pretreatment were examined in a 2³ full factorial design with six axial points. Temperatures ranged from 132 to 180 °C, time from 10 to 90 min and oxalic acid loadings from 0.01 to 0.038 g/g solids. Separate maxima were found for enzymatic saccharification and hemicellulose fermentation, respectively, with the condition for maximum saccharification being significantly more severe. Ethanol production was affected by reaction temperature more than by oxalic acid and reaction time over the ranges examined. The effect of reaction temperature was significant at a 95% confidence level in its effect on ethanol production. Oxalic acid and reaction time were statistically significant at the 90% level. The highest ethanol concentration (20 g/l) was obtained after 48 h with an ethanol volumetric production rate of 0.42 g ethanol l⁻¹ h⁻¹. The ethanol yield after SSF with P. stipitis was significantly higher than predicted by sequential saccharification and fermentation of substrate pretreated under the same condition. This was attributed to the secretion of β-glucosidase by P. stipitis. During SSF, free extracellular β-glucosidase activity was 1.30 pNPG U/g with P. stipitis, while saccharification without the yeast was 0.66 pNPG U/g.     

Techno-economic evaluation of producing ethanol from softwood: comparison of SSF and SHF and identification of bottlenecks

The aim of the study was to evaluate, from a technical and economic standpoint, the enzymatic processes involved in the production of fuel ethanol from softwood. Two base case configurations, one based on simultaneous saccharification and fermentation (SSF) and one based on separate hydrolysis and fermentation (SHF), were evaluated and compared. The process conditions selected were based mainly on laboratory data, and the processes were simulated by use of Aspen plus. The capital costs were estimated using the Icarus Process Evaluator. The ethanol production costs for the SSF and SHF base cases were 4.81 and 5.32 SEK/L or 0.57 and 0.63 USD/L (1 USD = 8.5SEK), respectively. The main reason for SSF being lower was that the capital cost was lower and the overall ethanol yield was higher. A major drawback of the SSF process is the problem with recirculation of yeast following the SSF step. Major economic improvements in both SSF and SHF could be achieved by increasing the income from the solid fuel coproduct. This is done by lowering the energy consumption in the process through running the enzymatic hydrolysis or the SSF step at a higher substrate concentration and by recycling the process streams. Running SSF with use of 8% rather than 5% nonsoluble solid material would result in a 19% decrease in production cost. If after distillation 60% of the stillage stream was recycled back to the SSF step, the production cost would be reduced by 14%. The cumulative effect of these various improvements was found to result in a production cost of 3.58 SEK/L (0.42 USD/L) for the SSF process.     

A comparison of the production of ethanol between simultaneous saccharification and fermentation and separate hydrolysis and fermentation using unpretreated cassava pulp and enzyme cocktail

The processes of separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) were employed using Saccharomyces cerevisiae for the production of ethanol from cassava pulp without any pretreatment. A combination of amylase, cellulase, cellobiase, and glucoamylase produced the highest levels of ethanol production in both the SHF and the SSF method. A temperature of 37 °C, a pH of 5.0, and an inoculum size of 6% were the optimum conditions for SSF. For the batch process at a pulp concentration of 20%, ethanol production levels from SHF and SSF were the highest, at 23.51 and 34.67 g L(-1) respectively, but in the fed-batch process, the levels of ethanol production from SHF and SSF rose to 29.39 and 43.25 g L(-1) respectively, which were 25% and 24.7% higher than those of the batch process. Thus SSF using the fed-batch provided a more efficient method for the utilization of cassava pulp.     

Indirect method for quantification of cellular biomass in a solidscontaining medium used as pre-culture for cellulase production

A process that combines the advantages of solid state fermentation (SSF) and submerged fermentation (SmF) could increase the efficiency of cellulase production required in the cellulosic ethanol industry. Due to the difficulty of measuring cellular biomass in the presence of solids, we developed a novel methodology for indirect quantification of biomass during production of the preculture for a combined fermentation process. Cultivation of Aspergillus niger was initiated as SSF using sugar cane bagasse as a solid substrate. Experiments were conducted in the absence of bagasse to determine growth kinetic parameters. Changes in glucose and biomass concentrations were measured. and the data were used for simulation employing a simple unstructured model. Parameters were estimated by applying a combination of Simulated Annealing (SA) and Levenberg-Marquardt (LM) algorithms to search for minimization of the error between model estimates and experimental data. Growth kinetics followed the Contois model, with a maximum specific growth rate (μ_max) of 0.042/h, a yield coefficient for biomass formation (Y_x/s) of 0.30 g/g and a death constant (k_D) of 0.005/h.These parameters were used to simulate cellular growth in the solids-containing medium. The proposed model accurately described the experimental data and succeeded in simulating the cell concentration profile. The selected pre-culture conditions (24 h as SSF followed by 48 h as SmF) were applied for cellulase production using the combined fermentation process and resulted in an endoglucanase activity (1,052 ± 34 U/L) greater than that obtained using the conventional SmF procedure (824 ± 44 U/L). Besides the standardization of pre-culture conditions, this methodology could be very useful in systems where direct measurement of cell mass is not possible.     

Modeling and experimental studies on intermittent starch feeding and citrate addition in simultaneous saccharification and fermentation of starch to flavor compounds

Simultaneous saccharification and fermentation (SSF) is a combined process of saccharification of a renewable bioresource and fermentation process to produce products, such as lactic acid and ethanol. Recently, SSF has been extensively used to convert various sources of cellulose and starch into fermentative products. Here, we present a study on production of buttery flavors, namely diacetyl and acetoin, by growing Lactobacillus rhamnosus on a starch medium containing the enzyme glucoamylase. We further develop a structured kinetics for the SSF process, which includes enzyme and growth kinetics. The model was used to simulate the effect of pH and temperature on the SSF process so as to obtain optimum operating conditions. The model was experimentally verified by conducting SSF using an initial starch concentration of 100 g/L. The study demonstrated that the developed kinetic was able to suggest strategies for improved productivities. The developed model was able to accurately predict the enhanced productivity of flavors in a three stage process with intermittent addition of starch. Experimental and simulations demonstrated that citrate addition can also lead to enhanced productivity of flavors. The developed optimal model for SSF was able to capture the dynamics of SSF in batch mode as well as in a three stage process. The structured kinetics was also able to quantify the effect of multiple substrates present in the medium. The study demonstrated that structured kinetic models can be used in the future for design and optimization of SSF as a batch or a fed-batch process. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Microbial production of 2,3-butanediol from Jerusalem artichoke tubers by <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

2,3-Butanediol is one of the promising bulk chemicals with wide applications. Its fermentative production has attracted great interest due to the high end concentration. However, large-scale production of 2,3-butanediol requires low-cost substrate and efficient fermentation process. In the present study, 2,3-butanediol production by Klebsiella pneumoniae from Jerusalem artichoke tubers was successfully performed, and various technologies, including separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF), were investigated. The concentration of target products reached 81.59 and 91.63 g/l, respectively after 40 h in batch and fed-batch SSF processes. Comparing with fed-batch SHF, the fed-batch SSF provided 30.3% higher concentration and 83.2% higher productivity of target products. The results showed that Jerusalem artichoke tuber is a favorable substrate for 2,3-butanediol production, and the application of fed-batch SSF for its conversion can result in a more cost-effective process.     

Production of multiple extracellular enzyme activities by novel submerged culture of <Emphasis Type="Italic">Aspergillus kawachii</Emphasis> for ethanol production from raw cassava flour

Cassava is a starch-containing root crop that is widely used as a raw material in a variety of industrial applications, most recently in the production of fuel ethanol. In the present study, ethanol production from raw (uncooked) cassava flour by simultaneous saccharification and fermentation (SSF) using a preparation consisting of multiple enzyme activities from Aspergillus kawachii FS005 was investigated. The multi-activity preparation was obtained from a novel submerged fermentation broth of A. kawachii FS005 grown on unmilled crude barley as a carbon source. The preparation was found to consist of glucoamylase, acid-stable α-amylase, acid carboxypeptidase, acid protease, cellulase and xylanase activities, and exhibited glucose and free amino nitrogen (FAN) production rates of 37.7 and 118.7 mg/l/h, respectively, during A. kawachii FS005-mediated saccharification of uncooked raw cassava flour. Ethanol production from 18.2% (w/v) dry uncooked solids of raw cassava flour by SSF with the multi-activity enzyme preparation yielded 9.0% (v/v) of ethanol and 92.3% fermentation efficiency. A feasibility study for ethanol production by SSF with a two-step mash using raw cassava flour and the multi-activity enzyme preparation manufactured on-site was verified on a pilot plant scale. The enzyme preparation obtained from the A. kawachii FS005 culture broth exhibited glucose and FAN production rates of 41.1 and 135.5 mg/l/h, respectively. SSF performed in a mash volume of about 1,612 l containing 20.6% (w/v) dry raw cassava solids and 106 l of on-site manufactured A. kawachii FS005 culture broth yielded 10.3% (v/v) ethanol and a fermentation efficiency of 92.7%.     

Conventional process for ethanol production from Indian broken rice and pearl millet

A conventional process for ethanol production involving liquefaction followed by simultaneous saccharification and fermentation (SSF) under the yeast fermentation conditions, was investigated at 30 and 35% dry solid (DS) of Indian broken rice and pearl millet feedstocks. The study followed the typical conventional process currently in use by the Indian Ethanol Industry. Liquefaction was carried out using a thermostable alpha amylase, and whereas SSF with a glucoamylase with additional side activities of pullulanase and protease under the yeast fermentation conditions. To measure the enzyme efficacy in the liquefaction process, fermentable sugar and liquefact solubility (brix) were monitored at the end of the liquefaction process. The liquefact was subjected to SSF with yeast. Addition of an acid fungal protease at a concentration of 0.1?kg per metric ton of grain during SSF was observed to accelerate yeast growth and ultimately, ethanol yield with both feedstocks. With both concentrations of feedstocks, the fermentation efficiency and ethanol recovery were determined. This study assesses the potential of these enzymes for ethanol production with higher dry solid concentration (≥30% w/w DS) of both these feedstocks in the conventional process to achieve higher plant throughput without compromising fermentation efficiency and ethanol recovery.     

Utilization of palm kernel cake for production of β-mannanase by Aspergillus niger FTCC 5003 in solid substrate fermentation using an aerated column bioreactor

The production of β-mannanase from palm kernel cake (PKC) as a substrate in solid substrate fermentation (SSF) was studied using a laboratory column bioreactor. The simultaneous effects of three independent variables, namely incubation temperature, initial moisture content of substrate and airflow rate, on β-mannanase production were evaluated by response surface methodology (RSM) on the basis of a central composite face-centered (CCF) design. Eighteen trials were conducted in which Aspergillus niger FTCC 5003 was cultivated on PKC in an aerated column bioreactor for seven days under SSF process. The highest level of β-mannanase (2117.89 U/g) was obtained when SSF process was performed at incubation temperature, initial moisture level and aeration rate of 32.5°C, 60% and 0.5 l/min, respectively. Statistical analysis revealed that the quadratic terms of incubation temperature and initial moisture content had significant effects on the production of β-mannanase (P < 0.01). A similar analysis also demonstrated that the linear effect of initial moisture level and an interaction effect between the initial moisture content and aeration rate significantly influenced the production of β-mannanase (P < 0.01). The statistical model suggested that the optimal conditions for attaining the highest level of β-mannanase were incubation temperature of 32°C, initial moisture level of 59% and aeration rate of 0.5 l/min. A β-mannanase yield of 2231.26 U/g was obtained when SSF process was carried out under the optimal conditions described above.     

The enzymatic hydrolysis and fermentation of pretreated wheat straw to ethanol

Autohydrolysis and ethanol-alkali pulping were used as pretreatment methods of wheat straw for its subsequent saccharification by Trichoderma reesei cellulase. The basic hydrolysis parameters, i.e., reaction time, pH, temperature, and enzyme and substrate concentration, were optimized to maximize sugar yields from ethanol-alkali modified straw. Thus, a 93% conversion of 2.5% straw material to sugar syrup containing 73% glucose was reached in 48 h using 40 filter paper units/g hydrolyzed substrate. The pretreated wheat straw was then fermented to ethanol at 43 degrees C in the simultaneous saccharification and fermentation (SSF) process using T. reesei cellulase and Kluyveromyces fragilis cells. From 10% (w/v) of chemically treated straw (dry matter), 2.4% (w/v) ethanol was obtained after 48 h. When the T. reesei cellulase system was supplemented with beta-glucosidase from Aspergillus niger, the ethanol yield in the SSF process increased to 3% (w/v) and the reaction time was shortened to 24 h.