A novel mechanism and kinetic model to explain enhanced xylose yields from dilute sulfuric acid compared to hydrothermal pretreatment of corn stover

Pretreatment of corn stover in 0.5% sulfuric acid at 160 °C for 40 min realized a maximum monomeric plus oligomeric xylose yield of 93.1% compared to a maximum of only 71.5% for hydrothermal (no added mineral acid) pretreatment at 180 °C for 30 min. To explain differences in dilute acid and hydrothermal yields, a fast reacting xylan fraction (0.0889) was assumed to be able to directly form monomeric xylose while a slow reacting portion (0.9111) must first form oligomers during hydrothermal pretreatment. Two reactions to oligomers were proposed: reversible from fast reacting xylan and irreversible from slow reacting xylan. A kinetic model and its analytical solution simulated xylan removal data well for dilute acid and hydrothermal pretreatment of corn stover. These results suggested that autocatalytic reactions from xylan to furfural in hydrothermal pretreatment were controlled by oligomeric xylose decomposition, while acid-catalytic reactions in dilute acid pretreatment were controlled by monomeric xylose decomposition.     

Dilute sulfuric acid cycle spray flow-through pretreatment of corn stover for enhancement of sugar recovery

A cycle spray flow-through reactor was designed and used to pretreat corn stover in dilute sulfuric acid medium. The dilute sulfuric acid cycle spray flow-through (DCF) process enhanced xylose sugar yields and cellulose digestibility while increasing the removal of lignin. Within the DCF system, the xylose sugar yields of 90–93% could be achieved for corn stover pretreated with 2% (w/v) dilute sulfuric acid at 95 °C during the optimal reaction time (90 min). The remaining solid residue exhibited enzymatic digestibility of 90–95% with cellulase loading of 60 FPU/g glucan that was due to the effective lignin removal (70–75%) in this process. Compared with flow-through and compress-hot water pretreatment process, the DCF method produces a higher sugar concentration and higher xylose monomer yield. The novel DCF process provides a feasible approach for lignocellulosic material pretreatment.     

Combined sugar yields for dilute sulfuric acid pretreatment of corn stover followed by enzymatic hydrolysis of the remaining solids

A number of previous studies determined dilute acid pretreatment conditions that maximize xylose yields from pretreatment or glucose yields from subsequent digestion of the pretreated cellulose, but our emphasis was on identifying conditions to realize the highest yields of both sugars from both stages. Thus, individual xylose and glucose yields are reported as a percentage of the total potential yield of both sugars over a range of sulfuric acid concentrations of 0.22%, 0.49% and 0.98% w/w at 140, 160, 180 and 200 degrees C. Up to 15% of the total potential sugar in the substrate could be released as glucose during pretreatment and between 15% and 90+% of the xylose remaining in the solid residue could be recovered in subsequent enzymatic hydrolysis, depending on the enzyme loading. Glucose yields increased from as high as 56% of total maximum potential glucose plus xylose for just enzymatic digestion to 60% when glucose released in pretreatment was included. Xylose yields similarly increased from as high as 34% of total potential sugars for pretreatment alone to between 35% and 37% when credit was taken for xylose released in digestion. Yields were shown to be much lower if no acid was used. Conditions that maximized individual sugar yields were often not the same as those that maximized total sugar yields, demonstrating the importance of clearly defining pretreatment goals when optimizing the process. Overall, up to about 92.5% of the total sugars originally available in the corn stover used could be recovered for coupled dilute acid pretreatment and enzymatic hydrolysis. These results also suggest that enhanced hemicellulase activity could further improve xylose yields, particularly for low cellulase loadings.     

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.     

Structural changes of corn stover lignin during acid pretreatment

In this study, raw corn stover was subjected to dilute acid pretreatments over a range of severities under conditions similar to those identified by the National Renewable Energy Laboratory (NREL) in their techno-economic analysis of biochemical conversion of corn stover to ethanol. The pretreated corn stover then underwent enzymatic hydrolysis with yields above 70?% at moderate enzyme loading conditions. The enzyme exhausted lignin residues were characterized by (31)P NMR spectroscopy and functional moieties quantified and correlated to enzymatic hydrolysis yields. Results from this study indicated that both xylan solubilization and lignin degradation are important for improving the enzyme accessibility and digestibility of dilute acid pretreated corn stover. At lower pretreatment temperatures, there is a good correlation between xylan solubilization and cellulose accessibility. At higher pretreatment temperatures, lignin degradation correlated better with cellulose accessibility, represented by the increase in phenolic groups. During acid pretreatment, the ratio of syringyl/guaiacyl functional groups also gradually changed from less than 1 to greater than 1 with the increase in pretreatment temperature. This implies that more syringyl units are released from lignin depolymerization of aryl ether linkages than guaiacyl units. The condensed phenolic units are also correlated with the increase in pretreatment temperature up to 180?°C, beyond which point condensation reactions may overtake the hydrolysis of aryl ether linkages as the dominant reactions of lignin, thus leading to decreased cellulose accessibility.     

Comparison of microwaves to fluidized sand baths for heating tubular reactors for hydrothermal and dilute acid batch pretreatment of corn stover

Heating of batch tubular reactors with fluidized sand baths and with microwaves resulted in distinctive sugar yield profiles from pretreatment and subsequent enzymatic hydrolysis of corn stover at the same time, temperature, and dilute sulfuric acid concentration combinations and hydrothermal pretreatment conditions. Microwave heated pretreatment led to faster xylan, lignin, and acetyl removal as well as earlier xylan degradation than sand baths, but maximum sugar recoveries were similar. Solid state CP/MAS NMR revealed that microwave heating was more effective in altering cellulose structural features especially in breakdown of amorphous regions of corn stover than sand bath heating. Enzymatic hydrolysis of pretreated corn stover was improved by microwave heating compared to sand bath heating. Mechanisms were proposed to explain the differences in results for the two systems and provide new insights into pretreatment that can help advance this technology.     

Supercritical carbon dioxide pretreatment of corn stover and switchgrass for lignocellulosic ethanol production

Supercritical CO₂ (SC-CO₂), a green solvent suitable for a mobile lignocellulosic biomass processor, was used to pretreat corn stover and switchgrass at various temperatures and pressures. The CO₂ pressure was released as quickly as possible by opening a quick release valve during the pretreatment. The biomass was hydrolyzed after pretreatment using cellulase combined with β-glucosidase. The hydrolysate was analyzed for the amount of glucose released. Glucose yields from corn stover samples pretreated with SC-CO₂ were higher than the untreated sample’s 12% glucose yield (12 g/100 g dry biomass) and the highest glucose yield of 30% was achieved with SC-CO₂ pretreatment at 3500 psi and 150 °C for 60 min. The pretreatment method showed very limited improvement (14% vs. 12%) in glucose yield for switchgrass. X-ray diffraction results indicated no change in crystallinity of the SC-CO₂ treated corn stover when compared to the untreated, while SEM images showed an increase in surface area.     

Feasibility of filamentous fungi for biofuel production using hydrolysate from dilute sulfuric acid pretreatment of wheat straw

ABSTRACT: BACKGROUND: Lipids produced from filamentous fungi show great promise for biofuel production, but a major limiting factor is the high production cost attributed to feedstock. Lignocellulosic biomass is a suitable feedstock for biofuel production due to its abundance and low value. However, very limited study has been performed on lipid production by culturing oleaginous fungi with lignocellulosic materials. Thus, identification of filamentous fungal strains capable of utilizing lignocellulosic hydrolysates for lipid accumulation is critical to improve the process and reduce the production cost. RESULTS: The growth performances of eleven filamentous fungi were investigated when cultured on glucose and xylose. Their dry cell weights, lipid contents and fatty acid profiles were determined. Six fungal strains with high lipid contents were selected to culture with the hydrolysate from dilute sulfuric acid pretreatment of wheat straw. The results showed that all the selected fungal strains were able to grow on both detoxified liquid hydrolysate (DLH) and non-detoxified liquid hydrolysate (NDLH). The highest lipid content of 39.4% was obtained by Mortierella isabellina on NDLH. In addition, NDLH with some precipitate could help M. isabellina form pellets with an average diameter of 0.11 mm. CONCLUSION: This study demonstrated the possibility of fungal lipid production from lignocellulosic biomass. M. isabellina was the best lipid producer grown on lignocellulosic hydrolysates among the tested filamentous fungi, because it could not only accumulate oils with a high content by directly utilizing NDLH to simplify the fermentation process, but also form proper pellets to benefit the downstream harvesting. Considering the yield and cost, fungal lipids from lignocellulosic biomass are promising alternative sources for biodiesel production.     

Biological pretreatment of corn stover with Phlebia brevispora NRRL‐13108 for enhanced enzymatic hydrolysis and efficient ethanol production

Biological pretreatment of lignocellulosic biomass by white‐rot fungus can represent a low‐cost and eco‐friendly alternative to harsh physical, chemical, or physico‐chemical pretreatment methods to facilitate enzymatic hydrolysis. In this work, solid‐state cultivation of corn stover with Phlebia brevispora NRRL‐13018 was optimized with respect to duration, moisture content and inoculum size. Changes in composition of pretreated corn stover and its susceptibility to enzymatic hydrolysis were analyzed. About 84% moisture and 42 days incubation at 28°C were found to be optimal for pretreatment with respect to enzymatic saccharification. Inoculum size had little effect compared to moisture level. Ergosterol data shows continued growth of the fungus studied up to 57 days. No furfural and hydroxymethyl furfural were produced. The total sugar yield was 442 ± 5 mg/g of pretreated corn stover. About 36 ± 0.6 g ethanol was produced from 150 g pretreated stover per L by fed‐batch simultaneous saccharification and fermentation (SSF) using mixed sugar utilizing ethanologenic recombinant Eschericia coli FBR5 strain. The ethanol yields were 32.0 ± 0.2 and 38.0 ± 0.2 g from 200 g pretreated corn stover per L by fed‐batch SSF using Saccharomyces cerevisiae D5A and xylose utilizing recombinant S. cerevisiae YRH400 strain, respectively. This research demonstrates that P. brevispora NRRL‐13018 has potential to be used for biological pretreatment of lignocellulosic biomass. This is the first report on the production of ethanol from P. brevispora pretreated corn stover. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:365–374, 2017     

Optimization of pH controlled liquid hot water pretreatment of corn stover

Controlled pH, liquid hot water pretreatment of corn stover has been optimized for enzyme digestibility with respect to processing temperature and time. This processing technology does not require the addition of chemicals such as sulfuric acid, lime, or ammonia that add cost to the process because these chemicals must be neutralized or recovered in addition to the significant expense of the chemicals themselves. Second, an optimized controlled pH, liquid hot water pretreatment process maximizes the solubilization of the hemicellulose fraction as liquid soluble oligosaccharides while minimizing the formation of monomeric sugars. The optimized conditions for controlled pH, liquid hot water pretreatment of a 16% slurry of corn stover in water was found to be 190 degrees C for 15 min. At the optimal conditions, 90% of the cellulose was hydrolyzed to glucose by 15FPU of cellulase per gram of glucan. When the resulting pretreated slurry, in undiluted form, was hydrolyzed by 11FPU of cellulase per gram of glucan, a hydrolyzate containing 32.5 g/L glucose and 18 g/L xylose was formed. Both the xylose and the glucose in this undiluted hydrolyzate were shown to be fermented by recombinant yeast 424A(LNH-ST) to ethanol at 88% of theoretical yield.     

Optimization of thermal-dilute sulfuric acid pretreatment for enhancement of methane production from cassava residues

In this study, the pretreatment of cassava residues by thermal-dilute sulfuric acid (TDSA) hydrolysis was investigated by means of a statistically designed set of experiments. A three-factor central composite design (CCD) was employed to identify the optimum pretreatment condition of cassava residues for methane production. The individual and interactive effects of temperature, H₂SO₄ concentration and reaction time on increase of methane yield (IMY) were evaluated by applying response surface methodology (RSM). After optimization, the resulting optimum pretreatment condition was 157.84 °C, utilizing 2.99% (w/w TS) H₂SO₄ for 20.15 min, where the maximum methane yield (248 mL/g VS) was 56.96% higher than the control (158 mL/g VS), which was very close to the predict value 56.53%. These results indicate the model obtained through RSM analysis is suit to predict the optimum pretreatment condition and there is great potential of using TDSA pretreatment of cassava residues to enhance methane yield.     

Carbonic acid enhancement of hydrolysis in aqueous pretreatment of corn stover

Carbonic acid and liquid hot water pretreatments were applied to corn stover. Temperatures ranged from 180 to 220 degrees C; reaction times varied between 2 and 32 min and prereaction carbon dioxide pressure was either 0 or 800 psig. Over the range of reaction conditions tested, it was found that the presence of carbonic acid had an effect of increasing the concentrations of xylose and furan compounds in the hydrolysate that was significant at above the 99% confidence level. Thus there appears to be an increase in the severity of the pretreatment conditions with the presence of carbonic acid. These results are contrary to previously reported results on aspen wood, where the presence of carbonic acid was not found to have an effect on either the xylose or furan concentrations. Although pretreatment conditions were more severe with the addition of carbonic acid, the presence of carbonic acid resulted in a hydrolysate with a higher final pH. Thus it appears that the higher severity conditions reduce the accumulation of organic acids in the hydrolysate. This result was consistent with previously reported work on carbonic acid pretreatment of aspen wood.     

A whole cell biocatalyst for cellulosic ethanol production from dilute acid-pretreated corn stover hydrolyzates

In this research, a recombinant whole cell biocatalyst was developed by expressing three cellulases from Clostridium cellulolyticum--endoglucanase (Cel5A), exoglucanase (Cel9E), and β-glucosidase--on the surface of the Escherichia coli LY01. The modified strain is identified as LY01/pRE1H-AEB. The cellulases were displayed on the surface of the cell by fusing with an anchor protein, PgsA. The developed whole cell biocatalyst was used for single-step ethanol fermentation using the phosphoric acid-swollen cellulose (PASC) and the dilute acid-pretreated corn stover. Ethanol production was 3.59 ± 0.15 g/L using 10 g/L of PASC, which corresponds to a theoretical yield of 95.4 ± 0.15%. Ethanol production was 0.30 ± 0.02 g/L when 1 g/L equivalent of glucose in the cellulosic fraction of the dilute sulfuric acid-pretreated corn stover (PCS) was fermented for 84 h. A total of 0.71 ± 0.12 g/L ethanol was produced in 48 h when the PCS was fermented in the simultaneous saccharification and co-fermentation mode using the hemicellulosic (1 g/L of total soluble sugar) and as well as the cellulosic (1 g/L of glucose equivalent) parts of PCS. In a control experiment, 0.48 g/L ethanol was obtained from 1 g/L of hemicellulosic PCS. It was concluded that the whole cell biocatalyst could convert both cellulosic and hemicellulosic substrates into ethanol in a single reactor. The developed C. cellulolyticum-E. coli whole cell biocatalyst also overcame the incompatible temperature problem of the frequently reported fungal-yeast systems.     

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.     

Lime pretreatment and enzymatic hydrolysis of corn stover

Corn stover was pretreated with an excess of calcium hydroxide (0.5 g Ca(OH)2/g raw biomass) in non-oxidative and oxidative conditions at 25, 35, 45, and 55 degrees C. The optimal condition is 55 degrees C for 4 weeks with aeration. Glucan (91.3%) and xylan (51.8%) were converted to glucose and xylose respectively, when the treated corn stover was enzymatically hydrolyzed with 15 FPU/g cellulose. Only 0.073 g Ca(OH)2 was consumed per g of raw corn stover. Of the initial lignin, 87.5% was maximally removed. Almost all acetyl groups were removed. After 4 weeks at 55 degrees C with aeration, some cellulose and hemicellulose were solubilized as monomers and oligomers in the pretreatment liquor. When considering the dissolved fragments of glucan and xylan in the pretreatment liquor, the overall yields of glucose and xylose were 93.2% and 79.5% at 15 FPU/g cellulose. The pretreatment liquor has no inhibitory effect on ethanol fermentation.     

Raman spectroscopy measurements of glucose and xylose in hydrolysate: role of corn stover pretreatment and enzyme composition

The effect of corn stover pretreatment on glucose quantitation in hydrolysate using Raman spectroscopy is evaluated. Dilute sulfuric-acid pretreatment results in a 20 mg mL⁻¹ glucose limit of detection in hydrolysate. Soaking in aqueous ammonia pretreatment produces a 4 mg mL⁻¹ limit of detection. Water, ethanol or hexane extraction of corn stover reduces the spectral background that limits glucose detection in dilute acid hydrolysate. Additionally, a Raman spectroscopy multi-peak fitting method is presented to simultaneously measure glucose and xylose concentration in hydrolysate. This method yields a 6.1% average relative standard error at total saccharide concentrations above 45 mg mL⁻¹. When only cellulase is present, glucose and xylose yield were measured by Raman spectroscopy to be 32 ± 4 and 7.0 ± 0.8 mg mL⁻¹, respectively. When both cellulase and hemicellulase were present, xylose yield increased to 18.0 ± 0.5 mg mL⁻¹. Enzymatic or colorimetric assays confirmed the validity of the Raman spectroscopy results.     

Valorization of olive stones for xylitol and ethanol production from dilute acid pretreatment via enzymatic hydrolysis and fermentation by Pachysolen tannophilus

Olive stones are an agro-industrial by-product abundant in the Mediterranean area that is regarded as a potential lignocellulosic feedstock for sugar production. Statistical modeling of dilute-sulphuric acid hydrolysis of olive stones has been performed using a response surface methodology, with treatment temperature and process time as factors, to optimize the hydrolysis conditions aiming to attain maximum d-xylose extraction from hemicelluloses. Thus, solid yield and composition of solid and liquid phases were assessed by empirical modeling. The highest yield of d-xylose was found at a temperature of 195 °C for 5 min. Under these conditions, 89.7% of the total d-xylose was recovered from raw material. The resulting solids from optimal conditions were assayed as substrate for enzymatic hydrolysis, while fermentability of hemicellulosic hydrolysates was tested using the d-xylose-fermenting yeast Pachysolen tannophilus. Both bioprocesses were considerably influenced by enzyme loading and inoculum size. In the enzymatic hydrolysis step, about 56% of cellulose was converted into d-glucose by using an enzyme/solid ratio of 40 FPU g⁻¹, while in the fermentation carried out with a cell concentration of 2 g L⁻¹ a yield of 0.44 g xylitol/g d-xylose and a global volumetric productivity of 0.11 g L⁻¹ h⁻¹ were achieved.     

Oil production by oleaginous yeasts using the hydrolysate from pretreatment of wheat straw with dilute sulfuric acid

This paper explores the use of the hydrolysate from the dilute sulfuric acid pretreatment of wheat straw for microbial oil production. The resulting hydrolysate was composed of pentoses (24.3 g/L) and hexoses (4.9 g/L), along with some other degradation products, such as acetic acid, furfural, and hydroxymethylfurfural (HMF). Five oleaginous yeast strains, Cryptococcus curvatus, Rhodotorula glutinis, Rhodosporidium toruloides, Lipomyces starkeyi, and Yarrowia lipolytica, were evaluated by using this hydrolysate as substrates. The results showed that all of these strains could use the detoxified hydrolysate to produce lipids while except R. toruloides non-detoxified hydrolysate could also be used for the growth of all of the selective yeast strains. C. curvatus showed the highest lipid concentrations in medium on both the detoxified (4.2 g/L) and non-detoxified (5.8 g/L) hydrolysates. And the inhibitory effect studies on C. curvatus indicated HMF had insignificant impacts at a concentration of up to 3 g/L while furfural inhibited cell growth and lipid content by 72.0% and 62.0% at 1 g/L, respectively. Our work demonstrates that lipid production is a promising alternative to utilize hemicellulosic sugars obtained during pretreatment of lignocellulosic materials.     

Characterization of dilute acid pretreatment of silvergrass for ethanol production

Pretreatment with dilute sulfuric acid of silvergrass was compared with the pretreatment's effect on other commonly used lignocellulosic materials, namely rice straw and bagasse, in order to evaluate the potential of this feedstock for ethanol production. The highest yield of xylose from silvergrass was between 70% and 75%, which was similar to bagasse. However, silvergrass gave a higher level of fermentability than bagasse using the hydrolysate because less acetic acid was formed. The release of sugars resulted in an about 2.0-fold increase in specific surface area of the pretreated silvergrass. Increasing the specific surface area did not obviously enhance enzymatic digestibility. The hydrophilicity of the acid pretreated silvergrass was characterized using its Fourier transform infrared (FTIR) spectra. The increase in hydrophilicity may enhance enzymatic adsorption onto lignin and increase the accumulation of cellobiose for enzymatic hydrolysis as pretreatment severity increases.     

Optimization of CO2 laser-based pretreatment of corn stover using response surface methodology

CO2 laser pretreatment to improve the enzymatic hydrolysis of corn stover for production of monosaccharide, was investigated. Response surface methodology (RSM), at a three-variable, three-level experiment Box-Behnken design (BBD) established the following optimum pretreatment parameters: time, 67.53 min; power, 264.33 W; and liquid-to-solid ratio, 21.29:1 (mL/g). Under these conditions, the reducing sugars produced was 4.941 mg/mL for cellulase hydrolysis. This amount matched the predicted value and increased cellulase hydrolysis from 14.47% to 30.84%. Scanning electron microscopy (SEM) showed that CO2 laser pretreatment converted the smooth into a rough and porous surface, which promoted the enzyme access and resulted in a high specific reducing sugars production rate.