high-throughput-microscale-protocol-for-analysis-processing

High-throughput, Microscale Protocol for the Analysis of Processing Parameters and Nutritional Qualities in Maize (<em>Zea mays</em> L.)

Maize is an important grain crop in the United States and worldwide. However, maize grain must be processed prior to human consumption. Furthermore, whole ...

University of Illinois at Urbana-Champaign

Increased demand for ethanol as a fuel additive has resulted in dramatic growth in ethanol production. Ethanol is produced from corn by either wet milling or dry-grind processing. In wet milling, the corn kernel is fractionated into different components, resulting in several coproducts. Wet-milling plants are capital intensive because of equipment requirements; they produce large volumes of ethanol and are corporate owned. In dry-grind processing, the corn kernel is not fractionated and only one coproduct, distillers' dried grains with solubles (DDGS), is generated. Dry-grind plants require less equipment and capital than wet mills. They generate smaller volumes of ethanol, are producer owned, and add direct benefits to rural economies. Most of the increase in ethanol production during the past decade is attributed to growth in the dry-grind industry. The marketing of coproducts provides income to offset processing costs. For dry-grind plants, this is especially important, because only one coproduct is available. Several issues could affect DDGS marketing. The increasing volume of DDGS accompanying ethanol production could reduce market value; high phosphorous content could limit the use of DDGS, because of animal waste disposal issues. Water removal is a costly processing step and affects the economics of ethanol processing. Technologies to remove germ and fiber from DDGS could produce a new coproduct suitable for feeding to nonruminants; this would expand the markets for DDGS. Reducing phosphorus in DDGS would sustain markets for conventional DDGS. The development of more efficient methods of water removal would increase the efficiency of ethanol processing and reduce the costs of processing. New technologies could contribute to greater stability of dry-grind plants.

The future of coproducts from corn processing.

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.

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

We investigated bacterial and archaeal community structures and population dynamics in two anaerobic bioreactors processing a carbohydrate- and sulfate-rich synthetic wastewater. A five-compartment anaerobic migrating blanket reactor (AMBR) was designed to promote biomass and substrate staging, which partially separates the processes of methanogenesis and sulfidogenesis in the middle and outer compartment(s) respectively. The second reactor was a conventional, single-compartment upflow anaerobic sludge blanket (UASB) reactor. Both reactors, which were seeded with the same inoculum, performed well when the influent chemical oxygen demand (COD)/SO(4) (2-) mass ratio was 24.4. The AMBR performed worse than the UASB reactor when the influent COD/SO(4) (2-) mass ratio was decreased to 5.0 by raising the sulfate load. Terminal restriction fragment length polymorphism analyses of bacterial 16S rRNA genes showed that the increase in sulfate load had a greater impact on bacterial diversity and community structure for the five AMBR compartments than for the UASB reactor. Moreover, bacterial community profiles across AMBR compartments became more similar through time, indicating a converging, rather than a staged community. While similar populations were abundant in both reactors at the beginning of the experiment, fermenting bacteria (clostridia, streptococci), and sulfate-reducing bacteria became more abundant in the AMBR, after shifting to a higher sulfate load, while a novel Thermotogales-like population eventually became predominant in the UASB reactor. A similar shift in the community structure of the hydrogenotrophic methanogens in the AMBR occurred: representatives of the Methanobacteriaceae out-competed the Methanospirillaceae after increasing the sulfate load in the AMBR, while the archaeal community structure was maintained in the UASB.

Microbial diversity and dynamics in multi- and single-compartment anaerobic bioreactors processing sulfate-rich waste streams.

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).

Thin stillage fractionation using ultrafiltration: resistance in series model.

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.

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

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.

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

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.

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

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.

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

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.

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

Although the system for producing yellow corn grain is well established in the US, its role among other biofeedstock alternatives to petroleum-based energy sources has to be balanced with its predominant purpose for food and feed as well as economics, land use, and environmental stewardship. We model land usage attributed to corn ethanol production in the US to evaluate the effects of anticipated technological change in corn grain production, ethanol processing, and livestock feeding through a multi-disciplinary approach. Seven scenarios are evaluated: four considering the impact of technological advances on corn grain production, two focused on improved efficiencies in ethanol processing, and one reflecting greater use of ethanol co-products (that is, distillers dried grains with solubles) in diets for dairy cattle, pigs, and poultry. For each scenario, land area attributed to corn ethanol production is estimated for three time horizons: 2011 (current), the time period at which the 15 billion gallon cap for corn ethanol as per the Renewable Fuel Standard is achieved, and 2026 (15 years out).

Land usage attributed to corn ethanol production in the United States: sensitivity to technological advances in corn grain yield, ethanol conversion, and co-product utilization.

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.

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

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.

Miscanthus×giganteus xylooligosaccharides: Purification and fermentation.

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%).

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

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.

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

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.

Article	Year
The future of coproducts from corn processing. Applied biochemistry and biotechnology\| PubMed ID: 16415480	2006
Improvement in fermentation characteristics of degermed ground corn by lipid supplementation. Journal of industrial microbiology & biotechnology\| PubMed ID: 16550435	2006
Microbial diversity and dynamics in multi- and single-compartment anaerobic bioreactors processing sulfate-rich waste streams. Environmental microbiology\| PubMed ID: 17227415	2007
Thin stillage fractionation using ultrafiltration: resistance in series model. Bioprocess and biosystems engineering\| PubMed ID: 18594866	2009
Effect of resistant starch on hydrolysis and fermentation of corn starch for ethanol. Applied biochemistry and biotechnology\| PubMed ID: 19415528	2010
Nutrient recovery from the dry grind process using sequential micro and ultrafiltration of thin stillage. Bioresource technology\| PubMed ID: 20138754	2010
Heat transfer fouling characteristics of microfiltered thin stillage from the dry grind process. Bioresource technology\| PubMed ID: 20395132	2010
Ultrafiltration of thin stillage from conventional and e-mill dry grind processes. Applied biochemistry and biotechnology\| PubMed ID: 21104340	2011
Autohydrolysis of Miscanthus x giganteus for the production of xylooligosaccharides (XOS): kinetics, characterization and recovery. Bioresource technology\| PubMed ID: 24463409	2014
Land usage attributed to corn ethanol production in the United States: sensitivity to technological advances in corn grain yield, ethanol conversion, and co-product utilization. Biotechnology for biofuels\| PubMed ID: 24725504	2014
In Vitro Fermentation of Xylooligosaccharides Produced from Miscanthus × giganteus by Human Fecal Microbiota. Journal of agricultural and food chemistry\| PubMed ID: 26648520	2016
Miscanthus×giganteus xylooligosaccharides: Purification and fermentation. Carbohydrate polymers\| PubMed ID: 26876832	2016
Improvement of sugar yields from corn stover using sequential hot water pretreatment and disk milling. Bioresource technology\| PubMed ID: 27289063	2016
Impact of disk milling on corn stover pretreated at commercial scale. Bioresource technology\| PubMed ID: 28242386	2017
Fermentation of undetoxified sugarcane bagasse hydrolyzates using a two stage hydrothermal and mechanical refining pretreatment. Bioresource technology\| PubMed ID: 29677659	2018

Kent D. Rausch

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