EFFICIENT PRODUCTION OF BIOETHANOL FROM CORN STOVER BY PRETREATMENT WITH A COMBINATION OF SULFURIC ACID AND SODIUM HYDROXIDE

Corn stover is the most abundant agricultural residue in China and a valuable reservoir for bioethanol production. In this study, we proposed a process for producing bioethanol from corn stover; the pretreatment prior to presaccharification, followed by simultaneous saccharification and fermentation (SSF) by using a flocculating Saccharomyces cerevisiae strain, was optimized. Pretreatment with acid–alkali combination (1% H₂SO₄, 150°C, 10 min, followed by 1% NaOH, 80°C, 60 min) resulted in efficient lignin removal and excellent recovery of xylose and glucose. A glucose recovery efficiency of 92.3% was obtained by enzymatic saccharification, when the pretreated solid load was 15%. SSF was carried out at 35°C for 36 hr after presaccharification at 50°C for 24 hr, and an ethanol yield of 88.2% was achieved at a solid load of 15% and an enzyme dosage of 15 FPU/g pretreated corn stover.     

Simultaneous Saccharification and Ethanol Fermentation of Corn Stover at High Temperature and High Solids Loading by a Thermotolerant Strain Saccharomyces cerevisiae DQ1

The thermotolerant strain Saccharomyces cerevisiae DQ1 was applied to the simultaneous saccharification and fermentation (SSF) at high temperature and high solids loading of the dilute acid-pretreated corn stover in the present study. The SSF using S. cerevisiae DQ1 was operated at 30?% solids loading of the pretreated corn stover with three-step SSF mode and achieved up to ethanol titer of 48?g/L and yield of 65.6?%. S. cerevisiae DQ1 showed strong thermotolerance in both the regular one-step SSF and the three-step SSF with changing temperature in each step. The three-step SSF at 40°C using S. cerevisiae DQ1 tolerated the greater cellulase dosage and solids loading of the pretreated corn stover and resulted in increased ethanol production. The present study provided a practical potential for the future SSF of lignocellulose feedstock at high temperature to reach high ethanol titer.     

Simultaneous saccharification and fermentation of lime-treated biomass

Simultaneous saccharification and fermentation (SSF) was performed on lime-treated switchgrass and corn stover, and oxidatively lime-treated poplar wood to determine their compatibility with Saccharomyces cerevisiae. Cellulose-derived glucose was extensively utilized by the yeast during SSF. The ethanol yields from pretreated switchgrass, pretreated corn stover, and pretreated-and-washed poplar wood were 72%, 62% and 73% of theoretical, respectively, whereas those from -cellulose were 67 to 91% of theoretical. The lower ethanol yields from treated biomass resulted from lower cellulose digestibilities rather than inhibitors produced by the pretreatment. Oxidative lime pretreatment of poplar wood increased the ethanol yield by a factor of 5.6, from 13% (untreated) to 73% (pretreated-and-washed).     

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.     

Acetic acid-catalyzed hydrothermal pretreatment of corn stover for the production of bioethanol at high-solids content

Corn stover (CS) was hydrothermally pretreated using CH₃COOH (0.3 %, v/v), and subsequently its ability to be utilized for conversion to ethanol at high-solids content was investigated. Pretreatment conditions were optimized employing a response surface methodology (RSM) with temperature and duration as independent variables. Pretreated CS underwent a liquefaction/saccharification step at a custom designed free-fall mixer at 50 °C for either 12 or 24 h using an enzyme loading of 9 mg/g dry matter (DM) at 24 % (w/w) DM. Simultaneous enzymatic saccharification and fermentation (SSF) of liquefacted corn stover resulted in high ethanol concentration (up to 36.8 g/L), with liquefaction duration having a negligible effect. The threshold of ethanol concentration of 4 % (w/w), which is required to reduce the cost of ethanol distillation, was surpassed by the addition of extra enzymes at the start up of SSF achieving this way ethanol titer of 41.5 g/L.     

Conversion of biomass hydrolysates and other substrates to ethanol and other chemicals by Lactobacillus buchneri*

Aims: A Lactobacillus buchneri strain NRRL B‐30929 can convert xylose and glucose into ethanol and chemicals. The aims of the study were to survey three strains (NRRL B‐30929, NRRL 1837 and DSM 5987) for fermenting 17 single substrates and to exam NRRL B‐30929 for fermenting mixed substrates from biomass hydrolysates. Methods and Results: Mixed acid fermentation was observed for all three L. buchneri strains using various carbohydrates; the only exception was uridine which yielded lactate, acetate and uracil. Only B‐30929 is capable of utilizing cellobiose, a desired trait in a potential biocatalyst for biomass conversion. Flask fermentation indicated that the B‐30929 strain can use all the sugars released from pretreated hydrolysates, and producing 1·98–2·35 g l^?1 ethanol from corn stover hydrolysates and 2·92–3·01 g l^?1 ethanol from wheat straw hydrolysates when supplemented with either 0·25× MRS plus 1% corn steep liquor or 0·5× MRS. Conclusions: The L. buchneri NRRL B‐30929 can utilize mixed sugars in corn stover and wheat straw hydrolysates for ethanol and other chemical production. Significance and Impact of the Study: These results are valuable for future research in engineering L. buchneri NRRL B‐30929 for fermentative production of ethanol and chemicals from biomass.     

Simultaneous saccharification and fermentation of steam-exploded corn stover at high glucan loading and high temperature

Background

Simultaneous saccharification and fermentation (SSF) is a promising process for bioconversion of lignocellulosic biomass. High glucan loading for hydrolysis and fermentation is an efficient approach to reduce the capital costs for bio-based products production. The SSF of steam-exploded corn stover (SECS) for ethanol production at high glucan loading and high temperature was investigated in this study.

Results

Glucan conversion of corn stover biomass pretreated by steam explosion was maintained at approximately 71 to 79% at an enzyme loading of 30 filter paper units (FPU)/g glucan, and 74 to 82% at an enzyme loading of 60 FPU/g glucan, with glucan loading varying from 3 to 12%. Glucan conversion decreased obviously with glucan loading beyond 15%. The results indicated that the mixture was most efficient in enzymatic hydrolysis of SECS at 3 to 12% glucan loading. The optimal SSF conditions of SECS using a novel Saccharomyces cerevisiae were inoculation optical density (OD)₆₀₀?=?4.0, initial pH 4.8, 50% nutrients added, 36 hours pre-hydrolysis time, 39°C, and 12% glucan loading (20% solid loading). With the addition of 2% Tween 20, glucan conversion, ethanol yield, final ethanol concentration reached 78.6%, 77.2%, and 59.8 g/L, respectively, under the optimal conditions. The results suggested that the solid and degradation products’ inhibitory effect on the hydrolysis and fermentation of SECS were also not obvious at high glucan loading. Additionally, glucan conversion and final ethanol concentration in SSF of SECS increased by 13.6% and 18.7%, respectively, compared with separate hydrolysis and fermentation (SHF).

Conclusions

Our research suggested that high glucan loading (6 to 12% glucan loading) and high temperature (39°C) significantly improved the SSF performance of SECS using a thermal- and ethanol-tolerant strain of S. cerevisiae due to the removal of degradation products, sugar feedback, and solid’s inhibitory effects. Furthermore, the surfactant addition obviously increased ethanol yield in SSF process of SECS.

     

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.     

Comparison of separate hydrolysis and fermentation and simultaneous saccharification and fermentation processes for ethanol production from wheat straw by recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> strain FBR5

Ethanol production by recombinant Escherichia coli strain FBR5 from dilute acid pretreated wheat straw (WS) by separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) was studied. The yield of total sugars from dilute acid (0.5% H₂SO₄) pretreated (160 °C, 10 min) and enzymatically saccharified (pH 5.0, 45 °C, 72 h) WS (86 g/l) was 50.0 ± 1.4 g/l. The hydrolyzate contained 1,184 ± 19 mg furfural and 161 ± 1 mg hydroxymethyl furfural per liter. The recombinant E. coli FBR5 could not grow at all at pH controlled at 4.5 to 6.5 in the non-abated wheat straw hydrolyzate (WSH) at 35 °C. However, it produced 21.9 ± 0.3 g ethanol from non-abated WSH (total sugars, 44.1 ± 0.4 g/l) in 90 h including the lag time of 24 h at controlled pH 7.0 and 35 °C. The bioabatement of WS was performed by growing Coniochaeta ligniaria NRRL 30616 in the liquid portion of the pretreated WS aerobically at pH 6.5 and 30 °C for 15 h. The bacterium produced 21.6 ± 0.5 g ethanol per liter in 40 h from the bioabated enzymatically saccharified WSH (total sugars, 44.1 ± 0.4 g) at pH 6.0. It produced 24.9 ± 0.3 g ethanol in 96 h and 26.7 ± 0.0 g ethanol in 72 h per liter from bioabated WSH by batch SSF and fed-batch SSF, respectively. SSF offered a distinct advantage over SHF with respect to reducing total time required to produce ethanol from the bioabated WS. Also, fed-batch SSF performed better than the batch SSF with respect to shortening the time requirement and increase in ethanol yield.     

Effect of surfactants on separate hydrolysis fermentation and simultaneous saccharification fermentation of pretreated lodgepole pine

The effects of surfactants addition on enzymatic hydrolysis and subsequent fermentation of steam exploded lodgepole pine (SELP) and ethanol pretreated lodgepole pine (EPLP) were investigated in this study. Supplementing Tween 80 during cellulase hydrolysis of SELP resulted in a 32% increase in the cellulose‐to‐glucose yield. However, little improvement was obtained from hydrolyzing EPLP in the presence of the same amount of surfactant. The positive effect of surfactants on SELP hydrolysis led to an increase in final ethanol yield after the fermentation. It was found that the addition of surfactant led to a substantial increase in the amount of free enzymes in the 48 h hydrolysates derived from both substrates. The effect of surfactant addition on final ethanol yield of simultaneous saccharification and fermentation (SSF) was also investigated by using SELP in the presence of additional furfural and hydroxymethylfurfural (HMF). The results showed that the surfactants slightly increased the conversion rates of furfural and HMF during SSF process by Saccharomyces cerevisiae. The presence of furfural and HMF at the experimental concentrations did not affect the final ethanol concentration either. The strategy of applying surfactants in cellulase recycling to reduce enzyme cost is presented. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Hydrothermal pretreatment of sugarcane bagasse using response surface methodology improves digestibility and ethanol production by SSF

Sugarcane bagasse was characterized as a feedstock for the production of ethanol using hydrothermal pretreatment. Reaction temperature and time were varied between 160 and 200°C and 5–20 min, respectively, using a response surface experimental design. The liquid fraction was analyzed for soluble carbohydrates and furan aldehydes. The solid fraction was analyzed for structural carbohydrates and Klason lignin. Pretreatment conditions were evaluated based on enzymatic extraction of glucose and xylose and conversion to ethanol using a simultaneous saccharification and fermentation scheme. SSF experiments were conducted with the washed pretreated biomass. The severity of the pretreatment should be sufficient to drive enzymatic digestion and ethanol yields, however, sugars losses and especially sugar conversion into furans needs to be minimized. As expected, furfural production increased with pretreatment severity and specifically xylose release. However, provided that the severity was kept below a general severity factor of 4.0, production of furfural was below an inhibitory concentration and carbohydrate contents were preserved in the pretreated whole hydrolysate. There were significant interactions between time and temperature for all the responses except cellulose digestion. The models were highly predictive for cellulose digestibility (R ² = 0.8861) and for ethanol production (R ² = 0.9581), but less so for xylose extraction. Both cellulose digestion and ethanol production increased with severity, however, high levels of furfural generated under more severe pretreatment conditions favor lower severity pretreatments. The optimal pretreatment condition that gave the highest conversion yield of ethanol, while minimizing furfural production, was judged to be 190°C and 17.2 min. The whole hydrolysate was also converted to ethanol using SSF. To reduce the concentration of inhibitors, the liquid fraction was conditioned prior to fermentation by removing inhibitory chemicals using the fungus Coniochaeta ligniaria.     

High efficiency bioethanol production from barley straw using a continuous pretreatment reactor

We developed a new pretreatment process for producing high-efficiency bioethanol from a lignocellulosic biomass. Barley straw was pretreated with sodium hydroxide in a twin-screw extruder for continuous pretreatment. The biomass to ethanol ratio (BTER) for optimal pretreatment conditions was evaluated by response surface methodology. Simultaneous saccharification and fermentation (SSF) was conducted to investigate the BTER with 30 FPU/g cellulose of enzyme and 7% (v/v) yeast (Saccharomyces cerevisiae CHY 1011) using 10% (w/v) pretreated biomass under various pretreatment conditions. The maximum BTER was 73.00% under optimal pretreatment conditions (86.61 °C, 0.58 M, and 84.79 mL/min for temperature, sodium hydroxide concentration, and solution flow rate, respectively) and the experimental BTER was 70.01 ± 0.59%. SSF was performed to investigate the optimal enzyme and biomass dosage. As a result, maximum ethanol concentration and ethanol yield were 46.00 g/L and 77.36% at a loading pretreated biomass of 20% with 30 FPU/g cellulose of the enzyme dosage for barley straw to bioethanol. These results are a significant contribution to the production of bioethanol from barley straw.     

BSA treatment to enhance enzymatic hydrolysis of cellulose in lignin containing substrates

Cellulase and bovine serum albumin (BSA) were added to Avicel cellulose and solids containing 56% cellulose and 28% lignin from dilute sulfuric acid pretreatment of corn stover. Little BSA was adsorbed on Avicel cellulose, while pretreated corn stover solids adsorbed considerable amounts of this protein. On the other hand, cellulase was highly adsorbed on both substrates. Adding a 1% concentration of BSA to dilute acid pretreated corn stover prior to enzyme addition at 15 FPU/g cellulose enhanced filter paper activity in solution by about a factor of 2 and beta-glucosidase activity in solution by about a factor of 14. Overall, these results suggested that BSA treatment reduced adsorption of cellulase and particularly beta-glucosidase on lignin. Of particular note, BSA treatment of pretreated corn stover solids prior to enzymatic hydrolysis increased 72 h glucose yields from about 82% to about 92% at a cellulase loading of 15 FPU/g cellulose or achieved about the same yield at a loading of 7.5 FPU/g cellulose. Similar improvements were also observed for enzymatic hydrolysis of ammonia fiber explosion (AFEX) pretreated corn stover and Douglas fir treated by SO(2) steam explosion and for simultaneous saccharification and fermentation (SSF) of BSA pretreated corn stover. In addition, BSA treatment prior to hydrolysis reduced the need for beta-glucosidase supplementation of SSF. The results are consistent with non-specific competitive, irreversible adsorption of BSA on lignin and identify promising strategies to reduce enzyme requirements for cellulose hydrolysis.     

Ethanol and furfural production from corn stover using a hybrid fractionation process with zinc chloride and simultaneous saccharification and fermentation (SSF)

A two-stage hybrid fractionation process was investigated to produce cellulosic ethanol and furfural from corn stover. In the first stage, zinc chloride (ZnCl₂) was used to selectively solubilize hemicellulose. During the second stage, the remaining treated solids were converted into ethanol using commercial cellulase and Saccharomyces cerevisiae or recombinant Escherichia coli, KO11. This hybrid fractionation process recovered 93.8% of glucan, 89.7% of xylan, 71.1% of arabinan, and 74.9% of lignin under optimal reaction conditions (1st stage: 5% acidified ZnCl₂, 7.5 ml/min, 150 °C (10 min) and 170 °C (10 min); 2nd stage: simultaneous saccharification and fermentation (SSF) using S. cerevisiae). The furfural yield from the hemicellulose hydrolysates was 58%. The SSF of the treated solids resulted in 69–98% of the theoretical maximum ethanol yields based on the glucan content in the treated solids. After fermentation, the solid residues contained primarily lignin. Based on the total lignin in untreated corn stover, the lignin recovery yield was 74.9%.     

Simultaneous saccharification and co-fermentation of glucose and xylose in steam-pretreated corn stover at high fiber content with Saccharomyces cerevisiae TMB3400

The two main sugars in the agricultural by-product corn stover are glucose and xylose. Co-fermentation of glucose and xylose at high content of water-insoluble solids (WIS) without detoxification is a prerequisite to obtain high ethanol concentration and to reduce production costs. A recombinant strain of Saccharomyces cerevisiae, TMB3400, was used in simultaneous saccharification and fermentation (SSF) of whole pretreated slurry of corn stover at high WIS. TMB3400 co-fermented glucose and xylose with relatively high ethanol yields giving high final ethanol concentration. The ethanol productivity increased with increasing concentration of pretreatment hydrolysate in the yeast production medium and when SSF was performed in a fed-batch mode.     

Production of ethanol from corn stover hemicellulose hydrolyzate using <Emphasis Type="Italic">Pichia stipitis</Emphasis>

Hemicellulose liquid hydrolyzate from dilute acid pretreated corn stover was fermented to ethanol using Pichia stipitis CBS 6054. The fermentation rate increased with aeration but the pH also increased due to consumption of acetic acid by Pichia stipitis. Hemicellulose hydrolyzate containing 34 g/L xylose, 8 g/L glucose, 8 g/L Acetic acid, 0.73 g/L furfural, and 1 g/L hydroxymethyl furfural was fermented to 15 g/L ethanol in 72 h. The yield in all the hemicellulose hydrolyzates was 0.37–0.44 g ethanol/g (glucose + xylose). Nondetoxified hemicellulose hydrolyzate from dilute acid pretreated corn stover was fermented to ethanol with high yields, and this has the potential to improve the economics of the biomass to ethanol process.     

Industrial yeast strain engineered to ferment ethanol from lignocellulosic biomass

In this study an industrial Saccharomyces cerevisiae yeast strain capable of fermenting ethanol from pretreated lignocellulosic material was engineered. Genes encoding cellulases (endoglucanase, exoglucanase and β-glucosidase) were integrated into the chromosomal ribosomal DNA and delta regions of a derivative of the K1-V1116 wine yeast strain. The engineered cellulolytic yeast produces ethanol in one step through simultaneous saccharification and fermentation of pretreated biomass without the addition of exogenously produced enzymes. When ethanol fermentation was performed with 10% dry weight of pretreated corn stover, the recombinant strain fermented 63% of the cellulose in 96 h and the ethanol titer reached 2.6% v/v. These results demonstrate that cellulolytic S. cerevisiae strains can be used as a platform for developing an economical advanced biofuel process.     

Fungal pretreatment of lignocellulose by Phanerochaete chrysosporium to produce ethanol from rice straw

Phanerochaete chrysosporium is a wood‐rot fungus that is capable of degrading lignin via its lignolytic system. In this study, an environmentally friendly fungal pretreatment process that produces less inhibitory substances than conventional methods was developed using P. chrysosporium and then evaluated by various analytical methods. To maximize the production of manganese peroxidase, which is the primary lignin‐degrading enzyme, culture medium was optimized using response surface methodologies including the Plackett–Burman design and the Box–Behnken design. Fermentation of 100 g of rice straw feedstock containing 35.7 g of glucan (mainly in the form of cellulose) by cultivation with P. chrysosporium for 15 days in the media optimized by response surface methodology was resulted in a yield of 29.0 g of glucan that had an enzymatic digestibility of 64.9% of the theoretical maximum glucose yield. In addition, scanning electronic microscopy, confocal laser scanning microscopy, and X‐ray diffractometry revealed significant microstructural changes, fungal growth, and a reduction of the crystallinity index in the pretreated rice straw, respectively. When the fungal‐pretreated rice straw was used as a substrate for ethanol production in simultaneous saccharification and fermentation (SSF) for 24 h, the ethanol concentration, production yield and the productivity were 9.49 g/L, 58.2% of the theoretical maximum, and 0.40 g/L/h, respectively. Based on these experimental data, if 100 g of rice straw are subjected to fungal pretreatment and SSF, 9.9 g of ethanol can be produced after 96 h, which is 62.7% of the theoretical maximum ethanol yield. Biotechnol. Bioeng. 2009; 104: 471–482 © 2009 Wiley Periodicals, Inc.     

Impact of recycling stillage on conversion of dilute sulfuric acid pretreated corn stover to ethanol

Both the current corn starch to ethanol industry and the emerging lignocellulosic biofuels industry view recycling of spent fermentation broth or stillage as a method to reduce fresh water use. The objective of this study was to understand the impact of recycling stillage on conversion of corn stover to ethanol. Sugars in a dilute‐acid pretreated corn stover hydrolysate were fermented to ethanol by the glucose–xylose fermenting bacteria Zymomonas mobilis 8b. Three serial fermentations were performed at two different initial sugar concentrations using either 10% or 25% of the stillage as makeup water for the next fermentation in the series. Serial fermentations were performed to achieve near steady state concentration of inhibitors and other compounds in the corn stover hydrolysate. Little impact on ethanol yields was seen at sugar concentrations equivalent to pretreated corn stover slurry at 15% (w/w) with 10% recycle of the stillage. However, ethanol yields became progressively poorer as the sugar concentration increased and fraction of the stillage recycled increased. At an equivalent corn stover slurry concentration of 20% with 25% recycled stillage the ethanol yield was only 5%. For this microorganism with dilute‐acid pretreated corn stover, recycling a large fraction of the stillage had a significant negative impact on fermentation performance. Although this finding is of concern for biochemical‐based lignocellulose conversion processes, other microorganism/pretreatment technology combinations will likely perform differently. Biotechnol. Bioeng. 2010;105: 992–996. © 2009 Wiley Periodicals, Inc.     

Fungal metabolism of fermentation inhibitors present in corn stover dilute acid hydrolysate

Use of agricultural residues for ethanol production requires pretreatment of the material to facilitate release of sugars. Physical–chemical pretreatment of lignocellulosic biomass can, however, give rise to side-products that may be toxic to fermenting microorganisms and hinder utilization of sugars obtained from biomass. Potentially problematic compounds include furan aldehydes formed by degradation of sugars, organic acids released from hemicellulose side-groups, and aldehydes and phenolics released from lignin. A fungal isolate, Coniochaeta ligniaria NRRL30616, metabolizes furfural and 5-hydroxymethylfurfural (HMF) as well as aromatic and aliphatic acids and aldehydes. NRRL30616 grew in corn stover dilute-acid hydrolysate, and converted furfural to both furfuryl alcohol and furoic acid. Hydrolysate was inoculated with NRRL30616, and the fate of pretreatment side-products was followed in a time-course study. A number of aromatic and aliphatic acids, aldehydes, and phenolic compounds were quantitated by analytical extraction of corn stover hydrolysate, followed by HPLC–UV–MS/MS analysis. Compounds representing all of the classes of inhibitory side-products were removed during the course of fungal growth. Biological abatement of hydrolysates using C. ligniaria improved xylose utilization in subsequent ethanol fermentations.