Process design and techno-economic analysis of 2'-fucosyllactose enriched distiller's dried grains with solubles production in dry grind ethanol process using genetically engineered Saccharomyces cerevisiae.

2'-fucosyllactose (2'-FL) has been linked positively with piglet gut health. Genetically engineered Saccharomyces cerevisiae strains producing 2'-FL can be used in the dry grind process to enrich Distiller's dried grains with solubles (DDGS) with 2'-FL and supplement swine diets with 2'-FL. The objectives of our study were to modify dry grind ethanol process for 2'-FL enriched DDGS production and evaluate the techno-economic feasibility of the process. Concentrations of 19.8 g 2'-FL/kg dry DDGS were achieved in the dry grind process using engineered strain without negatively affecting the ethanol yield. Process models for conventional and modified dry grind processes producing 2'-FL enriched DDGS (1150 MT corn/day capacity) were developed using SuperPro Designer. Capital and ethanol production costs for modified dry grind processes were higher than the conventional process. The internal rate of return for the modified processes was higher than the conventional process for $300/MT 2'-FL enriched DDGS selling price.

Improving ethanol yields with deacetylated and two-stage pretreated corn stover and sugarcane bagasse by blending commercial xylose-fermenting and wild type Saccharomyces yeast.

Corn stover and sugarcane bagasse are the most widely available agriculture processing biomass and could serve as feedstocks for production of biofuel. In this study, three different technologies are combined to develop a more efficient conversion process for each of these feedstocks. The three technologies are diluted alkaline deacetylation process, combined thermochemical and mechanical shear pretreatment, and fermentation using a combined inoculum of two commercial Saccharomyces yeast strains. The two yeast strains used were a non-GMO and GMO strain engineered for xylose fermentation. The final ethanol concentrations obtained were 35.7 g/L from deacetylated corn stover and 32.9 g/L from sugarcane bagasse. Blending the two yeast reduced residual xylose content from 1.24 g/L to 0.48 g/L and increased ethanol production by 6.5% compared to solely using the C5/C6 yeast. The optimized yeast blend also lowered the amount of C5/C6 yeast required for inoculation by 80%.

Increasing ethanol yield through fiber conversion in corn dry grind process.

Conversion of corn fiber to ethanol in the dry grind process could increase ethanol yields, reduce downstream processing costs and improve overall process profitability. This work investigates the in-situ conversion of corn fiber into ethanol (cellulase addition during simultaneous saccharification and fermentation) during dry grind process. Addition of 30 FPU/g fiber cellulase resulted in 4.6% increase in ethanol yield compared to the conventional process. Use of excess cellulase (120 FPU/g fiber) resulted in incomplete fermentation and lower ethanol yield compared to the conventional process. Multiple factors including high concentrations of ethanol and phenolic compounds were responsible for yeast stress and incomplete fermentation in excess cellulase experiments.

Fermentation of undetoxified sugarcane bagasse hydrolyzates using a two stage hydrothermal and mechanical refining pretreatment.

In this study, liquid hot water pretreatment was combined with disk milling for pretreatment of sugarcane bagasse. Sugarcane bagasse was pretreated using liquid hot water (LHW) at 140-180 °C for 10 min (20% w/w solids content) and then disk milled. Disk milling improved glucose release 41-177% and ethanol production from glucose/xylose cofermentation by 80% compared to only using LHW pretreatment. The highest ethanol conversion efficiency achieved was 94%, which was observed when bagasse was treated at 180 °C with LHW and disk milled. However, a small amount of residual xylose (3 g/L) was indicative that further improvement could be achieved to increase ethanol production.

Impact of disk milling on corn stover pretreated at commercial scale.

In cellulosic biofuel production, chemical pretreatment performed at laboratory or pilot scale, followed by mechanical refining, has been demonstrated to be effective to increase feedstock enzyme digestibility. To take the combined pretreatment process one step closer to commercialization, disk milling was performed with commercially pretreated corn stover. Dilute acid pretreated samples with combined severity factors (cSF) of 0.09 (DA09) and 0.43 (DA43) were obtained from a commercial plant. Effects of pretreatment conditions (DA09 and DA43), milling cycles (0, 3, 9, and 15) and enzyme dosages (7.8, 15.6 and 31.2mgcellulase/g dry biomass) were evaluated. Milling improved glucose yields by 0.7 to 1.2-fold. Higher enzyme dosages enhanced sugar yields. Milling was more effective to improve glucose yields, while enzyme dosage was more effective to improve xylose yields. However, dilute acid pretreatment condition was the most important factor to increase final sugar yields compared to milling cycles and enzyme dosages.

Improvement of sugar yields from corn stover using sequential hot water pretreatment and disk milling.

Efficient pretreatment is essential for economic conversion of lignocellulosic feedstocks into monosaccharides for biofuel production. To realize high sugar yields with low inhibitor concentrations, hot water or dilute acid pretreatment followed by disk milling is proposed. Corn stover at 20% solids was pretreated with hot water at 160-200°C for 4-8min with and without subsequent milling. Hot water pretreatment and disk milling acted synergistically to improve glucose and xylose yields by 89% and 134%, respectively, compared to hot water pretreatment alone. Hot water pretreated (180°C for 4min) and milled samples had the highest glucose and xylose yields among all hot water pretreated and milled samples, which were comparable to samples pretreated with 0.55% dilute acid at 160°C for 4min. However, samples pretreated with 1% dilute acid at 150°C for 4min and disk milled had the highest observed glucose (87.3%) and xylose yields (83.4%).

Miscanthus×giganteus xylooligosaccharides: Purification and fermentation.

A procedure was developed to recover xylooligosaccharides (XOS) from Miscanthus×giganteus (M×G) hydrolyzate. M×G hydrolyzate was prepared using autohydrolysis, and XOS rich fractions were acquired using activated carbon adsorption and stepwise ethanol elution. The combined XOS fractions were purified using a series of ion exchange resin treatments. The end product, M×G XOS, had 89.1% (w/w) total substituted oligosaccharides (TSOS) composed of arabinose, glucose, xylose and acetyl group. Bifidobacterium adolescentis and Bifidobacterium catenulatum (health promoting bacteria) were cultured in vitro on M×G XOS and a commercial XOS source, which was used as a comparison. B. adolescentis grew to a higher cell density than B. catenulatum in both XOS cultures. Total xylose consumption for B. adolescentis was 84.1 and 84.8%, respectively for M×G and commercial XOS cultures; and for B. catenulatum was 76.6 and 73.6%, respectively. The xylobiose (X2), xylotriose (X3) and xylotetraose (X4) were almost utilized for both strains. Acetic and lactic acids were the major fermentation products of the XOS cultures.

In Vitro Fermentation of Xylooligosaccharides Produced from Miscanthus × giganteus by Human Fecal Microbiota.

Purified xylooligosaccharides from Miscanthus × giganteus (M×G XOS) were used in an in vitro fermentation experiment inoculated with human fecal microbiota. A commercial XOS product and pectin were used as controls. Decreases in pH by 2.3, 2.4, and 2.0 units and production of short-chain fatty acids (SCFA; acetic acid, 7764.2, 6664.1, and 6387.9 µmol/g; propionic acid, 1006.7, 1089.5, and 661.5 µmol/g; and butyric acid, 955.5, 1252.9, and 917.7 µmol/g) were observed in M×G XOS, commercial XOS, and pectin medium after 12 h of fermentation, respectively. Titers of Bifidobacterium spp., Lactobacillus spp., and Escherichia coli increased when fed all three substrates as monitored by qPCR. There was no significant trend for Clostridium perfringens. During fermentation, M×G XOS was statistically equivalent in performance to the commercial XOS sample as measured by culture acidification and growth of health-promoting bacteria and resulted in the highest SCFA production among the three substrates.

Autohydrolysis of Miscanthus x giganteus for the production of xylooligosaccharides (XOS): kinetics, characterization and recovery.

The optima conditions of production and purification of xylooligosaccharides (XOS) from Miscanthus x giganteus (MxG) were investigated. Using autohydrolysis, XOS were produced at 160, 180 and 200°C at 60, 20 and 5min, respectively. XOS yield up to 13.5% (w/w) of initial biomass and 69.2% (w/w) of xylan were achieved. Results from HPAEC-PAD analysis revealed that X1-X9 sugar oligomers were produced. Higher temperature and longer reaction time resulted in lower product molecular weight. The three optimum conditions had similar degrees of polymerization XOS. Using 10% activated carbon (w/v) with ethanol/water elution recovered 47.9% (w/w) of XOS from pretreated liquid phase. The XOS could be fractionated by degree of polymerization according to ethanol concentration in the ethanol/water elution. Most of the XOS were washed out in 30% and 50% ethanol/water (v/v) fractions. Recoveries of 91.8% xylobiose, 86.9% xylotriose, 66.3% xylotetrose, 56.2% xylopentose and 48.9% xylohexaose were observed in XOS.

Contamination issues in a continuous ethanol production corn wet milling facility.

Low ethanol yields and poor yeast viability were investigated at a continuous ethanol production corn wet milling facility. Using starch slurries and recycle streams from a commercial ethanol facility, laboratory hydrolysates were prepared by reproducing starch liquefaction and saccharification steps in the laboratory. Fermentations with hydrolysates prepared in the laboratory were compared with plant hydrolysates for final ethanol concentrations and total yeast counts. Fermentation controls were prepared using hydrolysates (plant and laboratory) that were not inoculated with yeast. Hydrolysates prepared in the laboratory resulted in higher final ethanol concentrations (15.8 % v/v) than plant hydrolysate (13.4 % v/v). Uninoculated controls resulted in ethanol production from both laboratory (12.2 % v/v) and plant hydrolysates (13.7 % v/v), indicating the presence of a contaminating microorganism. Yeast colony counts on cycloheximide and virginiamycin plates confirmed the presence of a contaminant. DNA sequencing and fingerprinting studies also indicated a number of dissimilar communities in samples obtained from fermentors, coolers, saccharification tanks, and thin stillage.

A simultaneous saccharification and fermentation model for dynamic growth environments.

Many mathematical models by researchers have been formulated for Saccharomyces cerevisiae which is the common yeast strain used in modern distilleries. A cybernetic model that can account for varying concentrations of glucose, ethanol and organic acids on yeast cell growth dynamics does not exist. A cybernetic model, consisting of 4 reactions and 11 metabolites simulating yeast metabolism, was developed. The effects of variables such as temperature, pH, organic acids, initial inoculum levels and initial glucose concentration were incorporated into the model. Further, substrate and product inhibitions were included. The model simulations over a range of variables agreed with hypothesized trends and to observations from other researchers. Simulations converged to expected results and exhibited continuity in predictions for all ranges of variables simulated. The cybernetic model did not exhibit instability under any conditions simulated.

Design and evaluation of an optimal controller for simultaneous saccharification and fermentation process.

Ethanol from corn is produced using dry grind corn process in which simultaneous saccharification and fermentation (SSF) is one of the most critical unit operations. In this work an optimal controller based on a previously validated SSF model was developed by formulating the SSF process as a Bolza problem and using gradient descent methods. Validation experiments were performed to evaluate the performance of optimal controller under different process disturbances that are likely to occur in practice. Use of optimal control algorithm for the SSF process resulted in lower peak glucose concentration, similar ethanol yields (13.38±0.36% v/v and 13.50±0.15% v/v for optimally controlled and baseline experiments, respectively). Optimal controller improved final ethanol concentrations as compared to process without optimal controller under conditions of temperature (13.35±1.28 and 12.52±1.19% v/v for optimal and no optimal control, respectively) and pH disturbances (12.65±0.74 and 11.86±0.49% v/v for optimal and no optimal control, respectively). Cost savings due to lower enzyme usage and reduced cooling requirement were estimated to be up to $1 million for a 151 million L/yr (40 million gal/yr) dry grind plant.

Starch hydrolysis modeling: application to fuel ethanol production.

Efficiency of the starch hydrolysis in the dry grind corn process is a determining factor for overall conversion of starch to ethanol. A model, based on a molecular approach, was developed to simulate structure and hydrolysis of starch. Starch structure was modeled based on a cluster model of amylopectin. Enzymatic hydrolysis of amylose and amylopectin was modeled using a Monte Carlo simulation method. The model included the effects of process variables such as temperature, pH, enzyme activity and enzyme dose. Pure starches from wet milled waxy and high-amylose corn hybrids and ground yellow dent corn were hydrolyzed to validate the model. Standard deviations in the model predictions for glucose concentration and DE values after saccharification were less than ± 0.15% (w/v) and ± 0.35%, respectively. Correlation coefficients for model predictions and experimental values were 0.60 and 0.91 for liquefaction and 0.84 and 0.71 for saccharification of amylose and amylopectin, respectively. Model predictions for glucose (R2 = 0.69-0.79) and DP4+ (R2 = 0.8-0.68) were more accurate than the maltotriose and maltose for hydrolysis of high-amylose and waxy corn starch. For yellow dent corn, simulation predictions for glucose were accurate (R2 > 0.73) indicating that the model can be used to predict the glucose concentrations during starch hydrolysis.

Ultrafiltration of thin stillage from conventional and e-mill dry grind processes.

We used ultrafiltration (UF) to evaluate membrane filtration characteristics of thin stillage and determine solids and nutrient compositions of filtered streams. To obtain thin stillage, corn was fermented using laboratory methods. UF experiments were conducted in batch mode under constant temperature and flow rate conditions. Two regenerated cellulose membranes (10 and 100 kDa molecular weight cutoffs) were evaluated with the objective of retaining solids as well as maximizing permeate flux. Optimum pressures for 10 and 100 kDa membranes were 207 and 69 kPa, respectively. Total solids, ash, and neutral detergent fiber contents of input TS streams of dry grind and E-Mill processes were similar; however, fat and protein contents were different (p < 0.05). Retentate obtained from conventional thin stillage fractionation had higher mean total solids contents (27.6% to 27.8%) compared to E-Mill (22.2% to 23.4%). Total solids in retentate streams were found similar to those from commercial evaporators used in industry (25% to 35% total solids). Fat contents of retentate streams ranged from 16.3% to 17.5% for the conventional process. A 2% increment in fat concentration was observed in the E-Mill retentate stream. Thin stillage ash content was reduced 60% in retentate streams.

Heat transfer fouling characteristics of microfiltered thin stillage from the dry grind process.

We investigated effects of microfiltration (MF) on heat transfer fouling tendencies of thin stillage. A stainless steel MF membrane (0.1 micron pore size) was used to remove solids from thin stillage. At filtration conditions of 690kPa, the MF process effectively recovered total solids from thin stillage. Thin stillage was concentrated from 7.0% to 22.4% solids with average permeate flux rates of 180+/-30 L/m(2)/h at 75 degrees C. In retentate streams, protein and fat contents were increased from 23.5 and 16.7% db to 27.6 and 31.1% db, respectively, and ash content was reduced from 10.5% to 3.8% db. Removal of solids, protein and fat generated a microfiltration permeate (MFP) that was used as an input stream to the fouling probe system; MFP fouling tendencies were measured. An annular fouling probe was used to measure fouling tendencies of thin stillage from a commercial dry grind facility. When comparing diluted thin stillage (DTS) stream and MFP, a reduction in solids concentration was not the only reason of fouling decrement. Selective removal of protein and fat played an important role in mitigating the fouling. At t=10h, mean fouling rates of MFP were an order of magnitude lower when compared to thin stillage and diluted streams. When maximum probe temperature (200 degrees C) was reached, mean fouling rates for thin stillage, DTS and MFP were 7.1x10(-4), 4.2x10(-4) and 2.6x10(-4) m(2) degrees C/kW/min, respectively. In DTS and MFP, the induction period was prolonged by factors of 4.3 and 9.5, respectively, compared to the induction period for thin stillage fouling. Mean fouling rates were decreased by factors of 2.3 and 23.4 for DTS and MFP, respectively. Fouling of MFP took twice the time to reach a probe temperature of 200 degrees C than did thin stillage (22 h vs 10 h, respectively). A reduction in heat transfer fouling could be achieved by altering process stream composition using microfiltration.

Nutrient recovery from the dry grind process using sequential micro and ultrafiltration of thin stillage.

The effectiveness of microfiltration (MF) and ultrafiltration (UF) for nutrient recovery from a thin stillage stream was determined. When a stainless steel MF membrane (0.1microm pore size) was used, the content of solids increased from 7.0% to 22.8% with a mean permeate flux rate of 45L/m(2)/h (LMH), fat increased and ash content decreased. UF experiments were conducted in batch mode under constant temperature and flow rate conditions. Permeate flux profiles were evaluated for regenerated cellulose membranes (YM1, YM10 and YM100) with molecular weight cut offs of 1, 10 and 100kDa. UF increased total solids, protein and fat and decreased ash in retentate stream. When permeate streams from MF were subjected to UF, retentate total solids concentrations similar to those of commercial syrup (23-28.8%) were obtained. YM100 had the highest percent permeate flux decline (70% of initial flux) followed by YM10 and YM1 membranes. Sequential filtration improved permeate flux rates of the YM100 membrane (32.6-73.4LMH) but the percent decline was also highest in a sequential MF+YM100 system. Protein recovery was the highest in YM1 retentate. Removal of solids, protein and fat from thin stillage may generate a permeate stream that may improve water removal efficiency and increase water recycling.

Effect of resistant starch on hydrolysis and fermentation of corn starch for ethanol.

Starch samples with 0% or 30% amylose were subjected to four different liquefaction enzyme treatments (at various temperature and pH conditions) followed by simultaneous saccharification and fermentation (SSF). Resistant starch (RS) measurements were conducted for the initial starch sample, after liquefaction and after SSF. Initial RS was higher for 30% amylose starch samples (16.53 g/100 g sample) compared with 0% amylose (0.76 g/100 g sample). Higher initial RS resulted in lower conversion of starch into sugars and lower final ethanol yields. The four enzymes hydrolyzed RS, but in varying amounts. Higher temperature liquefaction hydrolyzed a larger portion of RS, resulting in higher ethanol concentrations and lower final residual solids (non-fermentables), whereas lower temperature liquefaction hydrolyzed a smaller portion of RS and resulted in lower ethanol concentrations and higher final residual solids. Decreases in resistant starch after high temperature liquefaction were 55% to 74%, whereas low temperature liquefaction decreases were 11% to 43%. For all enzyme treatments, RS content of starch samples decreased further after SSF.

Thin stillage fractionation using ultrafiltration: resistance in series model.

The corn based dry grind process is the most widely used method in the US for fuel ethanol production. Fermentation of corn to ethanol produces whole stillage after ethanol is removed by distillation. It is centrifuged to separate thin stillage from wet grains. Thin stillage contains 5-10% solids. To concentrate solids of thin stillage, it requires evaporation of large amounts of water and maintenance of evaporators. Evaporator maintenance requires excess evaporator capacity at the facility, increasing capital expenses, requiring plant slowdowns or shut downs and results in revenue losses. Membrane filtration is one method that could lead to improved value of thin stillage and may offer an alternative to evaporation. Fractionation of thin stillage using ultrafiltration was conducted to evaluate membranes as an alternative to evaporators in the ethanol industry. Two regenerated cellulose membranes with molecular weight cut offs of 10 and 100 kDa were evaluated. Total solids (suspended and soluble) contents recovered through membrane separation process were similar to those from commercial evaporators. Permeate flux decline of thin stillage using a resistance in series model was determined. Each of the four components of total resistance was evaluated experimentally. Effects of operating variables such as transmembrane pressure and temperature on permeate flux rate and resistances were determined and optimum conditions for maximum flux rates were evaluated. Model equations were developed to evaluate the resistance components that are responsible for fouling and to predict total flux decline with respect to time. Modeling results were in agreement with experimental results (R(2) > 0.98).

Element concentrations of dry-grind corn-processing streams.

The dry-grind corn process is one of two technologies used to convert corn into ethanol. In this process, all kernel components are processed through several sequential steps, including fermentation. Only one coproduct (distillers' dried grains with solubles [DDGS]) is available for marketing. DDGS provide income to offset costs of processing; issues that affect marketing have implications in the economic viability of dry-grind plants. Two issues relate to elements in DDGS: high concentrations and excessive variation. Data on element concentrations in dry-grind processing streams could be helpful in addressing these concerns. The objective of this study was to determine element concentrations in primary process streams from dry-grind plants. Samples of corn, ground corn, beer, wet grains, syrup, and DDGS were obtained from nine dry-grind plants, and element concentrations were determined. The concentrations of most elements in corn were not different among processing plants and were similar to published data. However, for the processing streams, there were differences in several element concentrations among processing plants. The concentrations of most elements in beer were about three times those of corn, due to the disappearance of starch during fermentation. Syrup had the highest element concentrations. Variations in element contents of DDGS and parent streams were due to processing conditions and not corn. Appropriate processing of thin stillage (the parent stream of syrup) could reduce the element content of DDGS.

Improvement in fermentation characteristics of degermed ground corn by lipid supplementation.

With rapid growth of fuel ethanol industry, and concomitant increase in distillers dried grains with solubles (DDGS), new corn fractionation technologies that reduce DDGS volume and produce higher value coproducts in dry grind ethanol process have been developed. One of the technologies, a dry degerm, defiber (3D) process (similar to conventional corn dry milling) was used to separate germ and pericarp fiber prior to the endosperm fraction fermentation. Recovery of germ and pericarp fiber in the 3D process results in removal of lipids from the fermentation medium. Biosynthesis of lipids, which is important for cell growth and viability, cannot proceed in strictly anaerobic fermentations. The effects of ten different lipid supplements on improving fermentation rates and ethanol yields were studied and compared to the conventional dry grind process. Endosperm fraction (from the 3D process) was mixed with water and liquefied by enzymatic hydrolysis and was fermented using simultaneous saccharification and fermentation. The highest ethanol concentration (13.7% v/v) was achieved with conventional dry grind process. Control treatment (endosperm fraction from 3D process without lipid supplementation) produced the lowest ethanol concentration (11.2% v/v). Three lipid treatments (fatty acid ester, alkylphenol, and ethoxylated sorbitan ester 1836) were most effective in improving final ethanol concentrations. Fatty acid ester treatment produced the highest final ethanol concentration (12.3% v/v) among all lipid supplementation treatments. Mean final ethanol concentrations of alkylphenol and ethoxylated sorbitan ester 1836 supplemented samples were 12.3 and 12.0% v/v, respectively.