Ethanol production of semi-simultaneous saccharification and fermentation from mixture of cotton gin waste and recycled paper sludge

Ethanol production from the steam-exploded mixture of 75% cotton gin waste and 25% recycled paper sludge in various conditions was investigated by semi-simultaneous saccharification and fermentation (SSSF) consisting of a pre-hydrolysis and a simultaneous saccharification and fermentation (SSF). Four cases were studied: 24-h pre-hydrolysis + 48-h SSF (SSSF 24), 12-h pre-hydrolysis + 60-h SSF (SSSF 12), 72-h SSF, and 48-h hydrolysis + 24-h fermentation (SHF). The ethanol concentration, yield, and productivity of SSSF 24 were higher than those of the other operations. A model of SSF was used to simulate the data for four components in SSF. The analysis of the reaction rates of cellobiose, glucose, cell, and ethanol using the model and the parameters from the experiments showed that there was a transition point of the rate-controlling step at which the cell growth control in the initial 2 h was changed to the cellobiose reaction control in later period during ethanol production of SSF from the mixture.     

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.     

Production of xylitol by Saccharomyces cerevisiae using waste xylose mother liquor and corncob residues

Exorbitant outputs of waste xylose mother liquor (WXML) and corncob residue from commercial-scale production of xylitol create environmental problems. To reduce the wastes, a Saccharomyces cerevisiae strain tolerant to WXML was conferred with abilities to express the genes of xylose reductase, a xylose-specific transporter and enzymes of the pentose phosphate pathway. This strain showed a high capacity to produce xylitol from xylose in WXML with glucose as a co-substrate. Additionally, a simultaneous saccharification and fermentation (SSF) process was designed to use corncob residues and cellulase instead of directly adding glucose as a co-substrate. Xylitol titer and the productivity were, respectively, 91.0 g l^-1 and 1.26 ± 0.01 g l^-1 h^-1 using 20% WXML, 55 g DCW l^-1 delignified corncob residues and 11.8 FPU g_cellulose^-1 cellulase at 35° during fermentation. This work demonstrates the promising strategy of SSF to exploit waste products to xylitol fermentation process.     

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.     

Utilization of dry distiller’s grain and solubles as nutrient supplement in the simultaneous saccharification and ethanol fermentation at high solids loading of corn stover

Dry distiller’s grain and solubles (DDGS) is a major by-product of corn-based ethanol production and is usually used as animal feed. Here, it was added to the simultaneous saccharification and ethanol fermentation (SSF) carried out at high solids loading of steam explosion pretreated corn stover using a mutant strain Saccharomyces cerevisiae DQ1. The performance of SSF process with DDGS was comparable to those using the expensive yeast extract supplementation. With 30% (w/w) solids plus the addition of cellulase and 1 g DDGS l⁻¹, the final ethanol reached 55 g l⁻¹ (7% v/v). The results indicated that the expensive supplement of yeast extract could be replaced by DDGS.     

Challenges in enzymatic hydrolysis and fermentation of pretreated Arundo donax revealed by a comparison between SHF and SSF

The perennial herbaceous crop Arundo donax is a potential feedstock for second-generation bioethanol production. In the present work, two different process options were investigated for the conversion of two differently steam-pretreated batches of A. donax. The pretreated raw material was converted to ethanol with a xylose-consuming Saccharomyces cerevisiae strain, VTT C-10880, by applying either separate hydrolysis and fermentation (SHF) or simultaneous saccharification and fermentation (SSF). The highest overall ethanol yield and final ethanol concentration were achieved using SHF (0.27 g g^?1 and 20.6 g L^?1 compared to 0.24 g g^?1 and 19.0 g L^?1 when SSF was used). The performance of both SHF and SSF was improved by complementing the cellulolytic enzymes with hemicellulases. The higher amount of acetic acid in one of the batches was shown to strongly affect xylose consumption in the fermentation. Only half of the xylose was consumed when batch 1 (high acetic acid) was fermented, compared to that 94% of the xylose was consumed in fermentation of batch 2 (lower acetic acid). Furthermore, the high amount of xylooligomers present in the pretreated materials considerably inhibited the enzymatic hydrolysis. Both the formation of xylooligomers and acetic acid thus need to be considered in the pretreatment process in order to achieve efficient conversion of A. donax to ethanol.     

An industrial level system with nonisothermal simultaneous solid state saccharification,fermentation and separation for ethanol production

To alleviate the problems of low substrate loading, nonisothermal, end-product inhibition of ethanol during the simultaneous saccharification and fermentation, a nonisothermal simultaneous solid state saccharification, fermentation, and separation (NSSSFS) process was investigated; one novel pilot scale nonisothermal simultaneous solid state enzymatic saccharification and fermentation coupled with CO₂ gas stripping loop system was invented and tested. The optimal pretreatment condition of steam-explosion was 1.5 MPa for 5 min in industrial level. In the NSSSFS, enzymatic saccharification and fermentation proceeded at around 50 °C and 37 °C, respectively, and were coupled together by the hydrolyzate loop; glucose from enzymatic saccharification was timely consumed by yeast, and the formed ethanol was separated online by CO₂ gas stripping coupled with adsorption of activated carbon; the solids substrate loading reached 25%; ethanol yields from 18.96% to 30.29% were obtained in fermentation depending on the materials tested. Based on the pilot level of 300 L fermenter, a novel industrial-level of 110 m³ solid state enzymatic saccharification, fermentation and ethanol separation plant had been successfully established and operated. The NSSSFS was a novel and feasible engineering solution to the inherent problems of simultaneous saccharification and fermentation, which would be used in large scale and in industrial production of ethanol.     

Simultaneous saccharification and fermentation of cellulose: effect of β- -glucosidase activity and ethanol inhibition of cellulases

Compared with saccharification in the absence of yeast, simultaneous saccharification and fermentation (SSF) using Trichoderma cellulases and Saccharomyces cerevisiae enhanced cellulose hydrolysis rates by 13–30%. The optimum temperature for SSF was 35°C. The requirement for β- -glucosidase (β- -glucoside glucohydrolase, EC 3.2.1.21) in SSF was lower than for saccharification: maximal ethanol production was attained when the ratio of the activity of β- -glucosidase to filter paper activity was 1.0. Ethanol inhibited cellulases uncompetitively, with an inhibition constant of 30.5 gl ⁻¹, but its effect was less severe than that of an equivalent concentration of cellobiose or glucose. No irreversible denaturation of cellulases [1,4-(1,3;1,4)-β- -glucan 4-glucanohydrolase, EC 3.2.1.4] by ethanol was observed.     

Optimization of fermentation conditions for the production of ethanol from sago starch by co-immobilized amyloglucosidase and cells of Zymomonas mobilis using response surface methodology

Statistical experimental design was used to optimize the conditions of simultaneous saccharification and fermentation (SSF), viz. temperature, pH and time of fermentation of ethanol from sago starch with co-immobilized amyloglucosidase (AMG) and Zymomonas mobilis MTCC 92 by submerged fermentation. Maximum ethanol concentration of 55.3 g/l was obtained using a starch concentration of 150 g/l. The optimum conditions were found to be a temperature of 32.4 °C, pH of 4.93 and time of fermentation of 17.24 h. Thus, by using SSF process with co-immobilized AMG and Z. mobilis cells MTCC 92, the central composite design (CCD) was found to be the most favourable strategy investigated with respect to ethanol production and enzyme recovery.     

Simultaneous saccharification and fermentation (SSF) of <Emphasis Type="Italic">Jatropha curcas</Emphasis> shells: utilization of co-products from the biodiesel production process

Jatropha curcas has great potential as an oil crop for use in biodiesel applications, and the outer shell is rich in lignocellulose that may be converted to ethanol, giving rise to the concept of a biorefinery. In this study, two dilute pretreatments of 0.5% H₂SO₄ and 1.0% NaOH were performed on Jatropha shells with subsequent simultaneous saccharification and fermentation (SSF) of the pretreated water-insoluble solids (WIS) to evaluate the effect of inhibitors in the pretreatment slurry. A cellulase loading of 15 FPU/g WIS, complimented with an excess of cellobiase (19.25 U/g), was used for SSF of either the washed WIS or the original slurry to determine the effect of inhibitors. Ethanol and glucose were monitored during SSF of 20 g of pretreated biomass. The unwashed slurry showed to have a positive effect on SSF efficiency for the NaOH-pretreated biomass. Maximum efficiencies of glucan conversion to ethanol in the WIS were 40.43% and 41.03% for the H₂SO₄- and NaOH-pretreated biomasses, respectively.     

Simultaneous saccharification and fermentation of cellulose: effect of β-d-glucosidase activity and ethanol inhibition of cellulases

Compared with saccharification in the absence of yeast, simultaneous saccharification and fermentation (SSF) using Trichoderma cellulases and Saccharomyces cerevisiae enhanced cellulose hydrolysis rates by 13–30%. The optimum temperature for SSF was 35°C. The requirement for β-d-glucosidase (β-d-glucoside glucohydrolase, EC 3.2.1.21) in SSF was lower than for saccharification: maximal ethanol production was attained when the ratio of the activity of β-d-glucosidase to filter paper activity was ～1.0. Ethanol inhibited cellulases uncompetitively, with an inhibition constant of 30.5 gl ^?1, but its effect was less severe than that of an equivalent concentration of cellobiose or glucose. No irreversible denaturation of cellulases [1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] by ethanol was observed.     

Effects of growth stage on enzymatic saccharification and simultaneous saccharification and fermentation of bamboo shoots for bioethanol production

Bamboo is a fast-growing renewable biomass that is widely distributed in Asia. Although bamboo is recognised as a useful resource, its utilization is limited and further development is required. Immature bamboo shoots harvested before branch spread were found to be a good biomass resource to achieve a high saccharification yield. The saccharification yield of the shoots increased (up to 98% for immature Phyllostachys bambusoides) when xylanase was used in addition to cellulase. Simultaneous saccharification and fermentation (SSF) processing converted immature shoots of P. bambusoides and Phyllostachys pubescens to ethanol with an ethanol yield of 169 and 139 g kg⁻¹, respectively (98% and 81%, respectively, of the theoretical yields based on hexose conversion) when 12 FPU g⁻¹ enzyme and the yeast Saccharomyces cerevisiae were used.     

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%.     

A comparison between simultaneous saccharification and fermentation and separate hydrolysis and fermentation using steam-pretreated corn stover

Two different process configurations, simultaneous saccharification and fermentation (SSF) and separate hydrolysis and fermentation (SHF), were compared, at 8% water-insoluble solids (WIS), regarding ethanol production from steam-pretreated corn stover. The enzymatic loading in these experiments was 10 FPU/g WIS and the yeast concentration in SSF was 1 g/L (dry weight) of a Saccharomyces cerevisiae strain. When the whole slurry from the pretreatment stage was used as it was, diluted to 8% WIS with water and pH adjusted, SSF gave a 13% higher overall ethanol yield than SHF (72.4% versus 59.1% of the theoretical). The impact of the inhibitory compounds in the liquid fraction of the pretreated slurry was shown to affect SSF and SHF in different ways. The overall ethanol yield (based on the untreated raw material) decreased when SSF was run in absence on inhibitors compared to SSF with inhibitors present. On the contrary, the presence of inhibitors decreased the overall ethanol yield in the case of SHF. However, the SHF yield achieves in the absence of inhibitors was still lower than the SSF yield achieves with inhibitors present.     

<Emphasis Type="SmallCaps">l</Emphasis>(+)-Lactic acid production from non-food carbohydrates by thermotolerant <Emphasis Type="Italic">Bacillus coagulans</Emphasis>

Lactic acid is used as an additive in foods, pharmaceuticals, and cosmetics, and is also an industrial chemical. Optically pure lactic acid is increasingly used as a renewable bio-based product to replace petroleum-based plastics. However, current production of lactic acid depends on carbohydrate feedstocks that have alternate uses as foods. The use of non-food feedstocks by current commercial biocatalysts is limited by inefficient pathways for pentose utilization. B. coagulans strain 36D1 is a thermotolerant bacterium that can grow and efficiently ferment pentoses using the pentose-phosphate pathway and all other sugar constituents of lignocellulosic biomass at 50°C and pH 5.0, conditions that also favor simultaneous enzymatic saccharification and fermentation (SSF) of cellulose. Using this bacterial biocatalyst, high levels (150–180 g l⁻¹) of lactic acid were produced from xylose and glucose with minimal by-products in mineral salts medium. In a fed-batch SSF of crystalline cellulose with fungal enzymes and B. coagulans, lactic acid titer was 80 g l⁻¹ and the yield was close to 80%. These results demonstrate that B. coagulans can effectively ferment non-food carbohydrates from lignocellulose to l(+)-lactic acid at sufficient concentrations for commercial application. The high temperature fermentation of pentoses and hexoses to lactic acid by B. coagulans has these additional advantages: reduction in cellulase loading in SSF of cellulose with a decrease in enzyme cost in the process and a reduction in contamination of large-scale fermentations.     

Ethanol production from lignocellulosic biomass of energy cane

Ethanol produced from lignocellulosic biomass is a renewable alternative to diminishing petroleum based liquid fuels. The release of many new sugarcane varieties by the United States Department of Agriculture to be used as energy crops is a promising feedstock alternative. Energy cane produces large amounts of biomass that can be easily transported, and production does not compete with food supply and prices because energy cane can be grown on marginal land instead of land for food crops. The purpose of this study was to evaluate energy cane for lignocellulosic ethanol production. Energy cane variety L 79-1002 was pretreated with weak sulfuric acid to remove lignin. In this study, 1.4 M sulfuric acid pretreated type II energy cane had a higher ethanol yield after fermentation by Klebsiella oxytoca without enzymatic saccharification than 0.8 M and 1.6 M sulfuric acid pretreated type II energy cane. Pretreated biomass was inoculated with K. oxytoca for cellulose fermentation and Pichia stipitis for hemicellulose fermentation under simultaneous saccahrification and fermentation (SSF) and separate hydrolysis and fermentation (SHF) conditions. For enzymatic saccharification of cellulose, the cellulase and ??-glucanase cocktail significantly increased ethanol production compared to the ethanol production of fermented acid pretreated energy cane without enzymatic saccharification. The results revealed that energy cane variety L 79-1002 produced maximum cellulosic ethanol under SHF (6995 mg/L) and produced 3624 mg/L ethanol from fermentation of hemicellulosic sugars.     

Comparison of SHF and SSF processes for the bioconversion of steam-exploded wheat straw

Two processes for ethanol production from wheat straw have been evaluated — separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF). The study compares the ethanol yield for biomass subjected to varying steam explosion pretreatment conditions: temperature and time of pretreatment was 200°C or 217°C and at 3 or 10 min. A rinsing procedure with water and NaOH solutions was employed for removing lignin residues and the products of hemicellulose degradation from the biomass, resulting in a final structure that facilitated enzymatic hydrolysis. Biomass loading in the bioreactor ranged from 25 to 100 g l⁻¹ (dry weight). The enzyme-to-biomass mass ratio was 0.06. Ethanol yields close to 81% of theoretical were achieved in the two-step process (SHF) at hydrolysis and fermentation temperatures of 45°C and 37°C, respectively. The broth required addition of nutrients. Sterilisation of the biomass hydrolysate in SHF and of reaction medium in SSF can be avoided as can the use of different buffers in the two stages. The optimum temperature for the single-step process (SSF) was found to be 37°C and ethanol yields close to 68% of theoretical were achieved. The SSF process required a much shorter overall process time (≈30 h) than the SHF process (96 h) and resulted in a large increase in ethanol productivity (0.837 g l⁻¹ h⁻¹ for SSF compared to 0.313 g l⁻¹ h⁻¹ for SHF). Journal of Industrial Microbiology & Biotechnology (2000) 25, 184–192. Received 02 December 1999/ Accepted in revised form 20 July 2000     

On-line identification of fermentation processes for ethanol production

Bioprocess and Biosystems Engineering - A strategy for monitoring fermentation processes, specifically, simultaneous saccharification and fermentation (SSF) of corn mash, was developed. The...     

Caffeic acid production by simultaneous saccharification and fermentation of kraft pulp using recombinant Escherichia coli

Caffeic acid (3,4-dihydroxycinnamic acid) serves as a building block for thermoplastics and a precursor for biologically active compounds and was recently produced from glucose by microbial fermentation. To produce caffeic acid from inedible cellulose, separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) reactions were compared using kraft pulp as lignocellulosic feedstock. Here, a tyrosine-overproducing Escherichia coli strain was metabolically engineered to produce caffeic acid from glucose by introducing the genes encoding a 4-hydroxyphenyllactate 3-hydroxylase (hpaBC) from Pseudomonas aeruginosa and tyrosine ammonia lyase (fevV) from Streptomyces sp. WK-5344. Using the resulting recombinant strain, the maximum yield of caffeic acid in SSF (233 mg/L) far exceeded that by SHF (37.9 mg/L). In the SSF with low cellulase loads (≤2.5 filter paper unit/g glucan), caffeic acid production was markedly increased, while almost no glucose accumulation was detected, indicating that the E. coli cells experienced glucose limitation in this culture condition. Caffeic acid yield was also negatively correlated with the glucose concentration in the fermentation medium. In SHF, the formation of by-product acetate and the accumulation of potential fermentation inhibitors increased significantly with kraft pulp hydrolysate than filter paper hydrolysate. The combination of these inhibitors had synergistic effects on caffeic acid fermentation at low concentrations. With lower loads of cellulase in SSF, less potential fermentation inhibitors (furfural, 5-hydroxymethyfurfural, and 4-hydroxylbenzoic acid) accumulated in the medium. These observations suggest that glucose limitation in SSF is crucial for improving caffeic acid yield, owing to reduced by-product formation and fermentation inhibitor accumulation.

     

Modelling ethanol production from cellulose: separate hydrolysis and fermentation versus simultaneous saccharification and fermentation

In ethanol production from cellulose, enzymatic hydrolysis, and fermentative conversion may be performed sequentially (separate hydrolysis and fermentation, SHF) or in a single reaction vessel (simultaneous saccharification and fermentation, SSF). Opting for either is essentially a trade-off between optimal temperatures and inhibitory glucose concentrations on the one hand (SHF) vs. sub-optimal temperatures and ethanol-inhibited cellulolysis on the other (SSF). Although the impact of ethanol on cellobiose hydrolysis was found to be negligible, formation of glucose and cellobiose from cellulose were found to be significantly inhibited by ethanol. A previous model for the kinetics of enzymatic cellulose hydrolysis was, therefore, extended with enzyme inhibition by ethanol, thus allowing a rational evaluation of SSF and SHF. The model predicted SSF processing to be superior. The superiority of SSF over SHF (separate hydrolysis and fermentation) was confirmed experimentally, both with respect to ethanol yield on glucose (0.41 g g^?1 for SSF vs. 0.35 g g^?1 for SHF) and ethanol production rate, being 30% higher for an SSF type process. High conversion rates were found to be difficult to achieve since at a conversion rate of 52% in a SSF process the reaction rate dropped to 5% of its initial value. The model, extended with the impact of ethanol on the cellulase complex proved to predict reaction progress accurately.