Dilute Acid Pretreated Corn Stover Research Articles

Bioconversion of Dilute Acid Pretreated Corn Stover to L-Lactic Acid Using Co-Culture of Furfural Tolerant Enterococcus mundtii WX1 and Lactobacillus rhamnosus SCJ9

This study demonstrated the bioconversion of lignocellulosic by-product corn stover (CS) to the value-added fermentative product L-lactic acid using the furfural tolerant Enterococcus mundtii WX1 and Lactobacillus rhamnosus SCJ9. The efficacy of dilute acid pretreatment by sulfuric and formic acids varying from 1% to 4% (v/v) concentration was compared. CS pretreated with 1% (v/v) sulfuric acid was selected for L-LA fermentation regarding the highest efficacy of fermentable sugar release when combined with the enzymatic hydrolysis process. Optimal conditions achieved a highest sugar release of 24.5 g/L glucose and 11.2 g/L of xylose from 100 g/L pretreated CS with 1% (v/v) sulfuric acid at 121 °C for 30 min, followed by enzymatic hydrolysis with Cellic CTec2 30 FPU/g pretreated CS at 50 °C for 48 h. The maximum L-LA titer, yield, and average productivity reached 31.4 g/L, 0.90 g/g, and 1.73 g/L/h, respectively. Moreover, addition of a hemicellulose-degrading enzyme complex combined with Cellic CTec2 led to an increase in xylose release, which resulted in a higher L-LA titer of 36.7 g/L at 48 h fermentation. Moreover, the purification of LA from culture broth by a process of electrodialysis with 331 g/L of LA and purity of 99.7% (w/w), was successful, with an optically pure L-LA of 99.9%. This study not only presents a feasible process for L-LA production from lignocellulose hydrolysate derived from abundant corn stover; this study also showed an alternative approach for solving the problem of haze air pollution caused by inappropriate management of corn production residuals.

Effects of potential inhibitors present in dilute acid-pretreated corn stover on fermentative hydrogen production by Escherichia coli

This study reports the inhibitory compounds present in dilute sulfuric acid-pretreated corn stover for fermentative hydrogen production by Escherichia coli. The E. coli W3110-derived strain deficient in lactate and succinate production pathways was used. When dilute acid-treated corn stover liquor containing 175 mM acetate, 21 mM furfural, and 2.6 mM 5-hydroxymethylfurfural was added at fourfold dilution to the xylose-containing culture medium, the microbial growth and hydrogen production were almost completely inhibited. The addition of acetate, furfural, or 5-hydroxymethylfurfural, at up to 200, 80, or 40 mM, respectively, to the xylose- or glucose-containing medium led to dose-dependent inhibition of fermentative hydrogen production. No synergistic inhibition by furfural with acetate or 5-hydroxymethylfurfural at concentrations based on the corn stover hydrolysate was observed. Altogether, these results suggest that the inhibition of the E. coli-based hydrogen production by the corn stover hydrolysate is primarily attributed to acetate under the conditions used.

The advanced performance of microbial consortium for simultaneous utilization of glucose and xylose to produce lactic acid directly from dilute sulfuric acid pretreated corn stover

BackgroundLignocellulosic feedstocks have attracted much attention as a potential carbon source for lactic acid (LA) production because of their ready availability, sustainability, and renewability. However, there are at least two major technical challenges to producing LA from lignocellulose. Inhibitors derived from lignocellulose pretreatment have a negative impact on the growth of cells producing LA. Furthermore, pentose sugars produced from the pretreatment are difficultly utilized by most LA producers, which is known as the carbon catabolite repression (CCR) effect. This complex feedstock can be utilized by a robust microbial consortium with high bioconversion efficiency.ResultsIn this study, a thermophilic consortium DUT50 producing LA was enriched and employed to improve corn stover (CS) utilization. Enterococcus was the dominant family in the consortium DUT50, accounting for 93.66% of the total abundance, with Lactobacillus, Bacillus, Lactococcus, and Trichococcus accounted for the remaining 2.68%. This consortium could be resistant to inhibitors concentration up to 9.74 g/L (2.88 g/L acetic acid, 2.46 g/L furfural, 2.20 g/L 5-HMF, and 2.20 g/L vanillin derived from pretreatment of CS), and simultaneously metabolizes hexose and pentose without CCR effect. Based on the promising consortium features, an efficient process of simultaneous saccharification and co-fermentation (SSCF) was developed to produce LA from acid pretreated corn stover, in which solid–liquid separation and detoxification were avoided. The key influencing factors were investigated and optimized, including dry biomass and cellulase loading, corn steep liquor powder concentration, and the pre-hydrolysis time. The highest LA titer of 71.04 g/L with a yield of 0.49 g/g-CS was achieved at a dry biomass loading of 20% (w/v), which is the highest LA production from non-detoxified acid pretreated corn stover via the SSCF process without wastewater generation reported to date. The simultaneous metabolism of hexose and pentose revealed collaboration between Enterococcus in the consortium, whereas xylose may be efficiently metabolized by Lactobacillus and Bacillus with low abundance via the pentose phosphate pathway.ConclusionsThe experimental results demonstrated the potential advantage of symbiosis in microbial consortia used for LA production from lignocellulosic biomass.

Impact of dilute acid pretreatment conditions on p-coumarate removal in diverse maize lines

Prior work has identified that lignins recovered from dilute acid-pretreated corn stover exhibit superior performance in phenol–formaldehyde resins used in wood adhesive applications when compared to diverse process-modified lignins derived from other sources. This improved performance is hypothesized to be due to the higher content of unsubstituted phenolic groups specifically p-coumarate lignin esters. In this work, a diverse set of corn stover samples are employed that exhibit diversity in p-coumarate content and total lignin content to explore the relationship between dilute acid pretreatment conditions, p-coumarate ester hydrolysis, xylan solubilization, and the resulting glucose enzymatic hydrolysis yields. The goal of this study is to identify pretreatment conditions that preserve a significant fraction of the p-coumarate esters while simultaneously achieving high enzymatic hydrolysis yields. Kinetic parameters for p-coumarate ester hydrolysis were quantified and pretreatment-biomass combinations were identified that result in glucose hydrolysis yields of more than 90% while retaining nearly 50 mg p-coumarate/g lignin.

Physico-chemical properties of lignin fractions from acid pretreated corn stover and their effects on enzymatic hydrolysis of microcrystalline cellulose

Lignin has been shown to be a recalcitrance factor in many biomass conversion studies. To better understand the effects of lignin on cellulose conversion, different lignin fractions were extracted from the same dilute acid pretreated corn stover by three sequential isolation methods, namely ethanol extraction, dioxane extraction, and enzyme purification. The physicochemical properties of each lignin fraction including molecular weight distribution, surface area, surface charge, and other structural features varied, depending on the isolation methods. All three lignin fractions had negative surface charges, and ethanol-extracted lignin carried the highest surface charges, followed by dioxane-extracted lignin and cellulase-purified residual lignin. These physicochemical properties of lignin fractions also resulted in different extent of inhibitory effects on enzymatic hydrolysis of microcrystalline cellulose (MCC). Dioxane-extracted lignin exhibited the highest inhibitory effect on glucose release from MCC, followed by the cellulase-purified residual lignin fraction and ethanol-extracted lignin. Furthermore, lignin fractions with higher contents of syringyl (S) substructure and β-O-4 aryl ether interunit linkages showed a stronger negative effect on cellulase hydrolysis of MCC.

Recycle of fermentation process water through mitigation of inhibitors in dilute-acid corn stover hydrolysate

Water consumption is an important consideration for production of advanced biofuels and chemicals. Recycling process water to reduce water consumption concentrates inhibitors formed during biomass pretreatment processes. To lessen their impact, Coniochaeta ligniaria NRRL 30616 was used to biologically abate inhibitors in dilute-acid pretreated corn stover hydrolysate. Biological abatement of inhibitors enabled recombinant ethanologenic E. coli FBR5 to complete fermentation before cultures using untreated hydrolysate emerged from the lag phase. Biological abatement allowed 50% reuse of the liquor in subsequent fermentations, and hydrolysate could be recycled five times. Although growth of E. coli FBR5 was variable in fermentations using recycled hydrolysate, ethanol productivity was essentially the same as in single-use hydrolysate. In contrast to biological abatement, overliming did not adequately eliminate inhibitors and led to unsatisfactory fermentation results. Bioabatement also enabled recovery of failed fermentations and allowed recycling of spent liquor for use as process water in a corn stover pretreatment process.

Microbial lipid production from dilute acid and dilute alkali pretreated corn stover via Trichosporon dermatis

Microbial lipid production from lignocellulosic biomass has attracted much attention recently. In this study, T. dermatis 32903 was selected from eleven promising oleaginous yeast strains. Carbon to nitrogen ratio (C/N) was investigated and optimized to maximize lipid production. Dilute acid (DA) pretreated corn stover (CS) and dilute alkali (AL) pretreated CS were then used for microbial lipid production, resulting in lipid concentrations of 7.46 g/L and 6.81 g/L, with sugar to lipid yields reached 0.104 g/g and 0.101 g/g, respectively. Washing of DA-CS and AL-CS enhanced lipid production to 11.43 g/L and 20.36 g/L with sugar to lipid yields improved to 0.156 g/g and 0.186 g/g, respectively. As degradation products in pretreated biomass showed severe inhibition on lipid fermentation, eight typical degradation products were further investigated for their effects on lipid fermentation. T. dermatis 32903 exhibited high tolerance to furan derivatives and week acids, but lower tolerance to phenolic compounds.

Boosting Ethanol Productivity of Zymomonas mobilis 8b in Enzymatic Hydrolysate of Dilute Acid and Ammonia Pretreated Corn Stover Through Medium Optimization, High Cell Density Fermentation and Cell Recycling.

The presence of toxic degradation products in lignocellulosic hydrolysate typically reduced fermentation rates and xylose consumption rate, resulting in a decreased ethanol productivity. In the present study, Zymomonas mobilis 8b was investigated for high cell density fermentation with cell recycling to improve the ethanol productivity in lignocellulosic hydrolysate. The fermentation performances of Z. mobilis 8b at various conditions were first studied in yeast extract-tryptone medium. It was found that nutrient level was essential for glucose and xylose co-fermentation by Z. mobilis 8b and high cell density fermentation with cell recycling worked well in yeast extract-tryptone medium for 6 rounds fermentation. Z. mobilis 8b was then studied in enzymatic hydrolysates derived from dilute acid (DA) pretreated corn stover (CS) and ammonia pretreated CS for high cell density fermentation with cell recycling. Ethanol productivity obtained was around three times higher compared to traditional fermentation. Ethanol titer and metabolic yield were also enhanced with high cell density fermentation. Z. mobilis 8b cells showed high recyclability in ammonia pretreated CS hydrolysate.

Process integration for ethanol production from corn and corn stover as mixed substrates

This work investigated all possible process integration strategies for ethanol production from corn and dilute acid pretreated corn stover (CS) as mixed substrates. Three corn to pretreated CS ratios (20%:10%, 10%:20% and 5%:25%) were examined. When the ratio of corn to pretreated CS was 20%:10%, the process integration strategy that mixed corn with CS hydrolysate for liquefaction followed by SSF resulted in the highest ethanol titer of 99.3 g/L. Mixing liquefied corn with pretreated CS for hydrolysis/saccharification followed by fermentation was the best strategy for the other two ratios. The strategy of mixing liquefied corn with pretreated CS for 6 h hydrolysis followed by fermentation showed the highest productivity for all the tested ratios.

Improving xylose utilization and ethanol production from dry dilute acid pretreated corn stover by two-step and fed-batch fermentation

It is a great challenge to efficiently utilize xylose in fermentation with high solid loadings for the economically feasible production of lignocellulosic ethanol. However, 20% solid loading of dry dilute acid pretreated corn stover (DDAPCS) decreased 66.3% of the xylose consumption compared to yeast extract peptone dextrose and xylose (YPDX) medium, which was significantly larger than 16% solid loading (9.4%). To overcome those problems, several two-step processes for simultaneous saccharification and co-fermentation (SSCF) were developed by inoculating a water extract of 16% solid loading DDAPCS with microorganisms in the first fermentation step. During the first step, the relatively lower inhibitor concentrations (compared to 20% solid loading), decreased glucose concentration, and the absence of enzymatic hydrolysis residues (EHR) were beneficial for the use of xylose and enabled up to 75.6% of the xylose to be converted to ethanol. With an optimized fed-batch strategy of two-step process, the residual xylose concentration reduced by 55.6% with 20% solid loading, and the maximum ethanol concentration and yield increased by 30% and 16.6%, respectively.

Integrated production of gluconic acid and xylonic acid using dilute acid pretreated corn stover by two-stage fermentation

A sustainable and efficient two-stage fermentation was developed to produce gluconic acid (GA) and xylonic acid (XA) from dilute acid pretreated corn stover (DA-CS) using Gluconobacter oxydans. Cells (6.2 g/L) were obtained after four cycles of fermentation in DA-CS enzyme hydrolysate at 50 ml scale in a shake flask. With each cycle showing complete utilization of glucose with GA productivity rate at 5.3 g/L/h. The enriched cells were then collected and used to ferment DA-CS liquid stream (generated during pretreatment containing mostly xylose) at 50 ml scale in a shake flask to produce XA at a rate of 1.9 g/L/h. Subsequently, the process was scaled up to 1 L. Both GA and XA was produced at the rate of 8.7 g/L/h and 3.7 g/L/h respectively in 36 h. From 1 kg of corn stover we were able to produce 296.2 g GA and 167.4 g XA by two stage fermentation.

Xylose induces cellulase production in Thermoascus aurantiacus

BackgroundLignocellulosic biomass is an important resource for renewable production of biofuels and bioproducts. Enzymes that deconstruct this biomass are critical for the viability of biomass-based biofuel production processes. Current commercial enzyme mixtures have limited thermotolerance. Thermophilic fungi may provide enzyme mixtures with greater thermal stability leading to more robust processes. Understanding the induction of biomass-deconstructing enzymes in thermophilic fungi will provide the foundation for strategies to construct hyper-production strains.ResultsInduction of cellulases using xylan was demonstrated during cultivation of the thermophilic fungus Thermoascus aurantiacus. Simulated fed-batch conditions with xylose induced comparable levels of cellulases. These fed-batch conditions were adapted to produce enzymes in 2 and 19 L bioreactors using xylose and xylose-rich hydrolysate from dilute acid pretreatment of corn stover. Enzymes from T. aurantiacus that were produced in the xylose-fed bioreactor demonstrated comparable performance in the saccharification of deacetylated, dilute acid-pretreated corn stover when compared to a commercial enzyme mixture at 50 °C. The T. aurantiacus enzymes retained this activity at of 60 °C while the commercial enzyme mixture was largely inactivated.ConclusionsXylose induces both cellulase and xylanase production in T. aurantiacus and was used to produce enzymes at up to the 19 L bioreactor scale. The demonstration of induction by xylose-rich hydrolysate and saccharification of deacetylated, dilute acid-pretreated corn stover suggests a scenario to couple biomass pretreatment with onsite enzyme production in a biorefinery. This work further demonstrates the potential for T. aurantiacus as a thermophilic platform for cellulase development.

How does cellulosome composition influence deconstruction of lignocellulosic substrates in Clostridium (Ruminiclostridium) thermocellum DSM 1313?

BackgroundBioethanol production processes involve enzymatic hydrolysis of pretreated lignocellulosic biomass into fermentable sugars. Due to the relatively high cost of enzyme production, the development of potent and cost-effective cellulolytic cocktails is critical for increasing the cost-effectiveness of bioethanol production. In this context, the multi-protein cellulolytic complex of Clostridium (Ruminiclostridium) thermocellum, the cellulosome, was studied here. C. thermocellum is known to assemble cellulosomes of various subunit (enzyme) compositions, in response to the available carbon source. In the current study, different carbon sources were used, and their influence on both cellulosomal composition and the resultant activity was investigated.ResultsGlucose, cellobiose, microcrystalline cellulose, alkaline-pretreated switchgrass, alkaline-pretreated corn stover, and dilute acid-pretreated corn stover were used as sole carbon sources in the growth media of C. thermocellum strain DSM 1313. The purified cellulosomes were compared for their activity on selected cellulosic substrates. Interestingly, cellulosomes derived from cells grown on lignocellulosic biomass showed no advantage in hydrolyzing the original carbon source used for their production. Instead, microcrystalline cellulose- and glucose-derived cellulosomes were equal or superior in their capacity to deconstruct lignocellulosic biomass. Mass spectrometry analysis revealed differential composition of catalytic and structural subunits (scaffoldins) in the different cellulosome samples. The most abundant catalytic subunits in all cellulosome types include Cel48S, Cel9K, Cel9Q, Cel9R, and Cel5G. Microcrystalline cellulose- and glucose-derived cellulosome samples showed higher endoglucanase-to-exoglucanase ratios and higher catalytic subunit-per-scaffoldin ratios compared to lignocellulose-derived cellulosome types.ConclusionThe results reported here highlight the finding that cellulosomes derived from cells grown on glucose and microcrystalline cellulose are more efficient in their action on cellulosic substrates than other cellulosome preparations. These results should be considered in the future development of C. thermocellum-based cellulolytic cocktails, designer cellulosomes, or engineering of improved strains for deconstruction of lignocellulosic biomass.

Constructing xylose-assimilating pathways in Pediococcus acidilactici for high titer d-lactic acid fermentation from corn stover feedstock

Xylose-assimilating pathway was constructed in a d-lactic acid producing Pediococcus acidilactici strain and evolutionary adapted to yield a co-fermentation strain P. acidilactici ZY15 with 97.3g/L of d-lactic acid and xylose conversion of 92.6% obtained in the high solids content simultaneous saccharification and co-fermentation (SSCF) of dry dilute acid pretreated and biodetoxified corn stover feedstock. The heterologous genes encoding xylose isomerase (xylA) and xylulokinase (xylB) were screened and integrated into the P. acidilactici chromosome. The metabolic flux to acetic acid in phosphoketolase pathway was re-directed to pentose phosphate pathway by substituting the endogenous phosphoketolase gene (pkt) with the heterologous transketolase (tkt) and transaldolase (tal) genes. The xylose-assimilating ability of the newly constructed P. acidilactici strain was significantly improved by adaptive evolution. This study provided an important strain and process prototype for high titer d-lactic acid production from lignocellulose feedstock with efficient xylose assimilation.

Dual effect of soluble materials in pretreated lignocellulose on simultaneous saccharification and co-fermentation process for the bioethanol production

In this study, wash liquors isolated from ethylenediamine and dry dilute acid pretreated corn stover were used to evaluate the effect of soluble materials in pretreated biomass on simultaneous saccharification and co-fermentation (SSCF) for ethanol production, respectively. Both of the wash liquors had different impacts on enzymatic hydrolysis and fermentation. Enzymatic conversions of glucan and xylan monotonically decreased as wash liquor concentration increased. Whereas, with low wash liquor concentrations, xylose consumption rate, cell viability and ethanol yield were maximally stimulated in fermentation without nutrient supplementary. Soluble lignins were found as the key composition which promoted sugars utilization and cell viability without nutrient supplementary. The dual effects of soluble materials on enzymatic hydrolysis and fermentation resulted in the reduction of ethanol yield as soluble materials increased in SSCF.

Oxidative production of xylonic acid using xylose in distillation stillage of cellulosic ethanol fermentation broth by Gluconobacter oxydans

An oxidative production process of xylonic acid using xylose in distillation stillage of cellulosic ethanol fermentation broth was designed, experimentally investigated, and evaluated. Dry dilute acid pretreated and biodetoxified corn stover was simultaneously saccharified and fermented into 59.80g/L of ethanol (no xylose utilization). 65.39g/L of xylose was obtained in the distillation stillage without any concentrating step after ethanol was distillated. Then the xylose was completely converted into 66.42g/L of xylonic acid by Gluconobacter oxydans. The rigorous Aspen Plus modeling shows that the wastewater generation and energy consumption was significantly reduced comparing to the previous xylonic acid production process using xylose in pretreatment liquid. This study provided a practical process option for xylonic acid production from lignocellulose feedstock with significant reduction of wastewater and energy consumption.

In situ detoxification of dry dilute acid pretreated corn stover by co-culture of xylose-utilizing and inhibitor-tolerant Saccharomyces cerevisiae increases ethanol production

Co-culture of xylose-utilizing and inhibitor-tolerant Saccharomyces cerevisiae was developed for bioethanol production from undetoxified pretreated biomass in simultaneously saccharification and co-fermentation (SSCF) process. Glucose accumulation during late fermentation phase in SSCF using xylose-utilizing strain can be eliminated by the introduction of inhibitor-tolerant strain. Effect of different ratios of two strains was investigated and xylose-utilizing strain to inhibitor-tolerant strain ratio of 10:1 (w/w) showed the best xylose consumption and the highest ethanol yield. Inoculating of xylose-utilizing strain at the later stage of SSCF (24–48h) exhibited lower ethanol yield than inoculating at early stage (the beginning 0–12h), probably due to the reduced enzymatic efficiency caused by the unconsumed xylose and oligomeric sugars. Co-culture SSCF increased ethanol concentration by 21.2% and 41.0% comparing to SSCF using individual inhibitor-tolerant and xylose-utilizing strain (increased from 48.5 and 41.7g/L to 58.8g/L), respectively, which suggest this co-culture system was very promising.

Improved methods for the determination of drying conditions and fraction insoluble solids (FIS) in biomass pretreatment slurry

Accurate and precise chemical characterization of biomass feedstocks and process intermediates is a requirement for successful technical and economic evaluation of biofuel conversion technologies. The uncertainty in primary measurements of the fraction insoluble solid (FIS) content of dilute acid pretreated corn stover slurry is the major contributor to uncertainty in yield calculations for enzymatic hydrolysis of cellulose to glucose. This uncertainty is propagated through process models and impacts modeled fuel costs. The challenge in measuring FIS is obtaining an accurate measurement of insoluble matter in the pretreated materials, while appropriately accounting for all biomass derived components. Three methods were tested to improve this measurement. One used physical separation of liquid and solid phases, and two utilized direct determination of dry matter content in two fractions. We offer a comparison of drying methods. Our results show utilizing a microwave dryer to directly determine dry matter content is the optimal method for determining FIS, based on the low time requirements and the method optimization done using model slurries.

Evaluation of soluble fraction and enzymatic residual fraction of dilute dry acid, ethylenediamine, and steam explosion pretreated corn stover on the enzymatic hydrolysis of cellulose

This study is aimed to examine the inhibition of soluble fraction (SF) and enzymatic residual fraction (ERF) in dry dilute acid (DDA), ethylenediamine (EDA) and steam explosion (SE) pretreated corn stover (CS) on the enzymatic digestibility of cellulose. SF of DDA, EDA and SE pretreated CS has high xylose, soluble lignin and xylo-oligomer content, respectively. SF of EDA pretreated CS leads to the highest inhibition, followed by SE and DDA pretreated CS. Inhibition of ERF of DDA and SE pretreated CS is higher than that of EDA pretreated CS. The inhibition degree (A0/A) of SF is 1.76 and 1.21 times to that of ERF for EDA and SE pretreated CS, respectively. The inhibition degree of ERF is 1.05 times to that of SF in DDA pretreated CS. The quantitative analysis shows that SF of EDA pretreated CS, SF and ERF of SE pretreated CS cause significant inhibition during enzymatic hydrolysis.

Fuzzy logic feedback control for fed-batch enzymatic hydrolysis of lignocellulosic biomass.

A fuzzy logic feedback control system was developed for process monitoring and feeding control in fed-batch enzymatic hydrolysis of a lignocellulosic biomass, dilute acid-pretreated corn stover. Digested glucose from hydrolysis reaction was assigned as input while doser feeding time and speed of pretreated biomass were responses from fuzzy logic control system. Membership functions for these three variables and rule-base were created based on batch hydrolysis data. The system response was first tested in LabVIEW environment then the performance was evaluated through real-time hydrolysis reaction. The feeding operations were determined timely by fuzzy logic control system and efficient responses were shown to plateau phases during hydrolysis. Feeding of proper amount of cellulose and maintaining solids content was well balanced. Fuzzy logic proved to be a robust and effective online feeding control tool for fed-batch enzymatic hydrolysis.