A toxicogenomic approach for the risk assessment of the food contaminant acetamide.

Acetamide (CAS 60-35-5) is detected in common foods. Chronic rodent bioassays led to its classification as a group 2B possible human carcinogen due to the induction of liver tumors in rats. We used a toxicogenomics approach in Wistar rats gavaged daily for 7 or 28 days at doses of 300 to 1500 mg/kg/day (mkd) to determine a point of departure (POD) and investigate its mode of action (MoA). Ki67 labeling was increased at doses ≥750 mkd up to 3.3-fold representing the most sensitive apical endpoint. Differential gene expression analysis by RNA-Seq identified 1110 and 1814 differentially expressed genes in male and female rats, respectively, following 28 days of treatment. Down-regulated genes were associated with lipid metabolism while up-regulated genes included cell signaling, immune response, and cell cycle functions. Benchmark dose (BMD) modeling of the Ki67 labeling index determined the BMD10 lower confidence limit (BMDL10) as 190 mkd. Transcriptional BMD modeling revealed excellent concordance between transcriptional POD and apical endpoints. Collectively, these results indicate that acetamide is most likely acting through a mitogenic MoA, though specific key initiating molecular events could not be elucidated. A POD value of 190 mkd determined for cell proliferation is suggested for risk assessment purposes.

Scale-up and operation of a pilot-scale ammonia fiber expansion reactor.

Pretreatment and densification of agricultural residues at regional depots can simplify feedstock supply logistics for the production of biofuels in commercial biorefineries. We have previously reported the performance of a laboratory-scale (5 L) packed-bed ammonia fiber expansion (AFEX) reactor system, which showed significant promise for biomass pretreatment at distributed depots. In this paper, we describe the performance of a 90-fold larger pilot-scale packed-bed AFEX-reactor system, used to produce over 1,500 batches (~36 tons) of pretreated crop residues over a 5-year period. Virtually all unreacted ammonia was successfully removed from the biomass, and 76% of the ammonia was recycled and reused. Pretreatment performance at pilot scale was comparable to laboratory-scale, averaging 74% glucose and 75% xylose yield in a standard test compared with 71% and 73%, respectively. Other operating and maintenance aspects are also discussed.

The food contaminant acetamide is not an in vivo clastogen, aneugen, or mutagen in rodent hematopoietic tissue.

Acetamide (CAS 60-35-5) is classified by IARC as a Group 2B, possible human carcinogen, based on the induction of hepatocellular carcinomas in rats following chronic exposure to high doses. Recently, acetamide was found to be present in a variety of human foods, warranting further investigation. The regulatory body JECFA has previously noted conflicting reports on acetamide's ability to induce micronuclei (MN) in mice in vivo. To better understand the potential in vivo genotoxicity of acetamide, we performed acute MN studies in rats and mice, and a subchronic study in rats, the target species for liver cancer. In the acute exposure, animals were gavaged with water vehicle control, 250, 1000, or 2000 mg/kg acetamide, or the positive control (1 mg/kg mitomycin C). In the subchronic assay, bone marrow of rats gavaged at 1000 mg/kg/day (limit dose) for 28 days was evaluated. Both acute and subchronic exposures showed no change in the ratio of polychromatic to total erythrocytes (P/E) at any dose, nor was there any increase in the incidence of micronucleated polychromatic erythrocytes (MN-PCE). Potential mutagenicity of acetamide was evaluated in male rats gavaged with vehicle control or 1500 mg/kg/day acetamide using the in vivoPig-a gene mutation assay. There was no increase in mutant red blood cells or reticulocytes in acetamide-treated animals. In both acute and sub-chronic studies, elevated blood plasma acetamide in treated animals provided evidence of systemic exposure. We conclude based on this study that acetamide is not clastogenic, aneugenic, or mutagenic in vivo in rodent hematopoietic tissue warranting a formal regulatory re-evaluation.

Effect of ammonia fiber expansion on the available energy content of wheat straw fed to lactating cattle and buffalo in India.

The seasonal lack of availability of lush green forages can force dairy farmers in developing nations to rely on crop residues such as wheat and rice straw as the major feed source. We tested whether ammonia fiber expansion (AFEX) treatment of wheat straw would increase the energy available to Murrah buffalo and Karan-Fries cattle consuming 70% of their diet as wheat straw in India. Forty lactating animals of each species were blocked by parity and days in milk and randomly assigned to 1 of 4 treatment diets (n = 10). Treatments were a nutrient-rich diet with 0 to 20% straw (positive control; PC) and 3 high-straw diets with various levels of AFEX-treatment: (1) 70% untreated straw (no AFEX), (2) 40 to 45% untreated straw with 25 to 30% AFEX-treated straw (low AFEX), and (3) 20% untreated straw with 50% AFEX-treated straw (high AFEX). The AFEX-treated straw was pelleted. Urea was added to the no and low AFEX diets so they were isonitrogenous with the high AFEX diet. Animals were individually fed the PC diet for 14 d followed by 7 d of adaptation to treatments, full treatments for 28 to 35 d, and finally PC diets for 21 d. Compared with buffalo fed the PC diet, those fed high-straw diets consumed 29% less feed dry matter, put out 16% less milk energy, and lost 0.8 kg/d more body weight; the AFEX treatment of straw did not alter intake or milk production but greatly ameliorated the body weight loss (-1.0 kg/d for no AFEX and -0.07 kg/d for high AFEX). In Karan-Fries cattle, high-straw diets decreased dry matter intake by 39% and milk energy by 24%, and the high AFEX diet increased intake by 42% and milk energy by 18%. The AFEX treatment increased digestibilities of organic matter, dry matter, neutral detergent fiber, acid detergent fiber, and crude protein by 6 to 13 percentage points in buffalo and 5 to 10 points in cattle. In conclusion, AFEX treatment increased the digestibility and energy availability of wheat straw for lactating buffalo and cattle and has commercial potential to improve milk production and feed efficiency when high-quality forages or grains are not available.

Toward high solids loading process for lignocellulosic biofuel production at a low cost.

High solids loadings (>18 wt%) in enzymatic hydrolysis and fermentation are desired for lignocellulosic biofuel production at a high titer and low cost. However, sugar conversion and ethanol yield decrease with increasing solids loading. The factor(s) limiting sugar conversion at high solids loading is not clearly understood. In the present study, we investigated the effect of solids loading on simultaneous saccharification and co-fermentation (SSCF) of AFEX™ (ammonia fiber expansion) pretreated corn stover for ethanol production using a xylose fermenting strain Saccharomyces cerevisiae 424A(LNH-ST). Decreased sugar conversion and ethanol yield with increasing solids loading were also observed. End-product (ethanol) was proven to be the major cause of this issue and increased degradation products with increasing solids loading was also a cause. For the first time, we show that with in situ removal of end-product by performing SSCF aerobically, sugar conversion stopped decreasing with increasing solids loading and monomeric sugar conversion reached as high as 93% at a high solids loading of 24.9 wt%. Techno-economic analysis was employed to explore the economic possibilities of cellulosic ethanol production at high solids loadings. The results suggest that low-cost in situ removal of ethanol during SSCF would significantly improve the economics of high solids loading processes. Biotechnol. Bioeng. 2017;114: 980-989. © 2016 Wiley Periodicals, Inc.

Enzymatic hydrolysis of pelletized AFEX™-treated corn stover at high solid loadings.

Ammonia fiber expansion (AFEX™) pretreatment can be performed at small depots, and the pretreated biomass can then be pelletized and shipped to a centralized refinery. To determine the feasibility of this approach, pelletized AFEX-treated corn stover was hydrolyzed at high (18-36%) solid loadings. Water absorption and retention by the pellets was low compared to unpelletized stover, which allowed enzymatic hydrolysis slurries to remain well mixed without the need for fed-batch addition. Glucose yields of 68% and xylose yields of 65% were obtained with 20 mg enzyme/g glucan and 18% solid loading after 72 h, compared to 61% and 59% for unpelletized corn stover. Pelletization also slightly increased the initial rate of hydrolysis compared to unpelletized biomass. The ease of mixing and high yields obtained suggests that pelletization after AFEX pretreatment could have additional advantages beyond improved logistical handling of biomass.

Can dispersed biomass processing protect the environment and cover the bottom line for biofuel?

This paper compares environmental and profitability outcomes for a centralized biorefinery for cellulosic ethanol that does all processing versus a biorefinery linked to a decentralized array of local depots that pretreat biomass into concentrated briquettes. The analysis uses a spatial bioeconomic model that maximizes profit from crop and energy products, subject to the requirement that the biorefinery must be operated at full capacity. The model draws upon biophysical crop input-output coefficients simulated with the Environmental Policy Integrated Climate (EPIC) model as well as market input and output prices, spatial transportation costs, ethanol yields from biomass, and biorefinery capital and operational costs. The model was applied to 82 cropping systems simulated across 37 subwatersheds in a 9-county region of southern Michigan in response to ethanol prices simulated to rise from $1.78 to $3.36 per gallon. Results show that the decentralized local biomass processing depots lead to lower profitability but better environmental performance, due to more reliance on perennial grasses than the centralized biorefinery. Simulated technological improvement that reduces the processing cost and increases the ethanol yield of switchgrass by 17% could cause a shift to more processing of switchgrass, with increased profitability and environmental benefits.

Technoeconomic evaluation of recent process improvements in production of sugar and high-value lignin co-products via two-stage Cu-catalyzed alkaline-oxidative pretreatment.

BACKGROUND: A lignocellulose-to-biofuel biorefinery process that enables multiple product streams is recognized as a promising strategy to improve the economics of this biorefinery and to accelerate technology commercialization. We recently identified an innovative pretreatment technology that enables of the production of sugars at high yields while simultaneously generating a high-quality lignin stream that has been demonstrated as both a promising renewable polyol replacement for polyurethane applications and is highly susceptible to depolymerization into monomers. This technology comprises a two-stage pretreatment approach that includes an alkaline pre-extraction followed by a metal-catalyzed alkaline-oxidative pretreatment. Our recent work demonstrated that H2O2 and O2 act synergistically as co-oxidants during the alkaline-oxidative pretreatment and could significantly reduce the pretreatment chemical input while maintaining high sugar yields (~ 95% glucose and ~ 100% xylose of initial sugar composition), high lignin yields (~ 75% of initial lignin), and improvements in lignin usage. RESULTS: This study considers the economic impact of these advances and provides strategies that could lead to additional economic improvements for future commercialization. The results of the technoeconomic analysis (TEA) demonstrated that adding O2 as a co-oxidant at 50 psig for the alkaline-oxidative pretreatment and reducing the raw material input reduced the minimum fuel selling price from $1.08/L to $0.85/L, assuming recoverable lignin is used as a polyol replacement. If additional lignin can be recovered and sold as more valuable monomers, the minimum fuel selling price (MFSP) can be further reduced to $0.73/L. CONCLUSIONS: The present work demonstrated that high sugar and lignin yields combined with low raw material inputs and increasing the value of lignin could greatly increase the economic viability of a poplar-based biorefinery. Continued research on integrating sugar production with lignin valorization is thus warranted to confirm this economic potential as the technology matures.

Economic comparison of multiple techniques for recovering leaf protein in biomass processing.

Leaf protein concentrates (LPC) can be used as a valuable co-product to cellulosic biofuel production and can also mitigate the food versus fuel controversy. Two major approaches have been considered for LPC production: a well-characterized mechanical pressing method and a less studied method involving aqueous extraction with recovery using ultrafiltration. Experimental results with switchgrass extracts show low protein recovery after filtration, particularly if protein is recovered after cellulose hydrolysis. Economic modeling suggests that aqueous extraction costs less than mechanical pressing, but due to lower protein yields and lower quality, overall profit is higher for mechanical pressing versus aqueous extraction ($26/Mg feedstock vs. $14/Mg). If modest improvements can be made in extraction yields, filtration recovery, and protein quality, then the profitability of the aqueous extraction approach can be increased to $37/Mg feedstock. This study suggests that aqueous extraction is a viable alternative for LPC co-production in a biorefinery if key improvements can be made in the process.

Biofuels done right: land efficient animal feeds enable large environmental and energy benefits.

There is an intense ongoing debate regarding the potential scale of biofuel production without creating adverse effects on food supply. We explore the possibility of three land-efficient technologies for producing food (actually animal feed), including leaf protein concentrates, pretreated forages, and double crops to increase the total amount of plant biomass available for biofuels. Using less than 30% of total U.S. cropland, pasture, and range, 400 billion liters of ethanol can be produced annually without decreasing domestic food production or agricultural exports. This approach also reduces U.S. greenhouse gas emissions by 670 Tg CO2-equivalent per year, or over 10% of total U.S. annual emissions, while increasing soil fertility and promoting biodiversity. Thus we can replace a large fraction of U.S. petroleum consumption without indirect land use change.

Presence of Acetamide in Milk and Beef from Cattle Consuming AFEX-Treated Crop Residues.

AFEX treatment of crop residues can greatly increase their nutrient availability for ruminants. This study investigated the concentration of acetamide, an ammoniation byproduct, in AFEX-treated crop residues and in milk and meat from ruminants fed these residues. Acetamide concentrations in four AFEX-treated cereal crop residues were comparable and reproducible (4-7 mg/g dry matter). A transient acetamide peak in milk was detected following introduction of AFEX-treated residues to the diet, but an alternative regimen showed the peak can be effectively mitigated. Milk acetamide concentration following this transition was 6 and 10 ppm for cattle and buffalo, respectively, but also decreased over time for cattle while tending to decrease (p = 0.08) for buffalo. There was no difference in acetamide concentration in the meat of cattle consuming AFEX-treated residues for 160 days compared to controls. Further investigation is necessary to determine the metabolism of acetamide in ruminants and a maximum acceptable daily intake for humans.

Improving the corn-ethanol industry: studying protein separation techniques to obtain higher value-added product options for distillers grains.

Currently in America the biofuel ethanol is primarily being produced by the dry grind technique to obtain the starch contained in the corn grains and subsequently subjected to fermentation. This so-called 1st generation technology has two setbacks; first the lingering debate whether its life cycle contributes to a reduction of fossil fuels and the animal feed sectors future supply/demand imbalance caused by the co-product dry distillers grains (DDGS). Additional utilization of the cellulosic components and separation of the proteins for use as chemical precursors have the potential to alleviate both setbacks. Several different corn feedstock layouts were treated with 2nd generation ammonia fiber expansion (AFEX) pre-treatment technology and tested for protein separation options (protease solubilization). The resulting system has the potential to greatly improve ethanol yields with lower bioprocessing energy costs and satisfy a significant portion of the organic chemical industry.

Enzyme hydrolysis and ethanol fermentation of liquid hot water and AFEX pretreated distillers' grains at high-solids loadings.

The dry milling ethanol industry produces distiller's grains as major co-products, which are composed of unhydrolyzed and unfermented polymeric sugars. Utilization of the distiller's grains as an additional source of fermentable sugars has the potential to increase overall ethanol yields in current dry grind processes. In this study, controlled pH liquid hot water pretreatment (LHW) and ammonia fiber expansion (AFEX) treatment have been applied to enhance enzymatic digestibility of the distiller's grains. Both pretreatment methods significantly increased the hydrolysis rate of distiller's dried grains with solubles (DDGS) over unpretreated material, resulting in 90% cellulose conversion to glucose within 24h of hydrolysis at an enzyme loading of 15FPU cellulase and 40 IU beta-glucosidase per gram of glucan and a solids loading of 5% DDGS. Hydrolysis of the pretreated wet distiller's grains at 13-15% (wt of dry distiller's grains per wt of total mixture) solids loading at the same enzyme reduced cellulose conversion to 70% and increased conversion time to 72h for both LHW and AFEX pretreatments. However, when the cellulase was supplemented with xylanase and feruloyl esterase, the pretreated wet distiller's grains at 15% or 20% solids (w/w) gave 80% glucose and 50% xylose yields. The rationale for supplementation of cellulases with non-cellulolytic enzymes is given by Dien et al., later in this journal volume. Fermentation of the hydrolyzed wet distiller's grains by glucose fermenting Saccharomyces cerevisiae ATCC 4124 strain resulted in 100% theoretical ethanol yields for both LHW and AFEX pretreated wet distiller's grains. The solids remaining after fermentation had significantly higher protein content and are representative of a protein-enhanced wet DG that would result in enhanced DDGS. Enhanced DDGS refers to the solid product of a modified dry grind process in which the distiller's grains are recycled and processed further to extract the unutilized polymeric sugars. Compositional changes of the laboratory generated enhanced DDGS are also presented and discussed.

Exposure Assessment of Acetamide in Milk, Beef, and Coffee Using Xanthydrol Derivatization and Gas Chromatography/Mass Spectrometry.

Acetamide has been classified as a possible human carcinogen, but uncertainties exist about its levels in foods. This report presents evidence that thermal decomposition of N-acetylated sugars and amino acids in heated gas chromatograph injectors contributes to artifactual acetamide in milk and beef. An alternative gas chromatography/mass spectrometry protocol based on derivatization of acetamide with 9-xanthydrol was optimized and shown to be free of artifactual acetamide formation. The protocol was validated using a surrogate analyte approach based on d3-acetamide and applied to analyze 23 pasteurized whole milk, 44 raw sirloin beef, and raw milk samples from 14 different cows, and yielded levels about 10-fold lower than those obtained by direct injection without derivatization. The xanthydrol derivatization procedure detected acetamide in every food sample tested at 390 ± 60 ppb in milk, 400 ± 80 ppb in beef, and 39 000 ± 9000 ppb in roasted coffee beans.

Optimization of ammonia fiber expansion (AFEX) pretreatment and enzymatic hydrolysis of Miscanthus x giganteus to fermentable sugars.

Miscanthus x giganteus is a tall perennial grass whose suitability as an energy crop is presently being appraised. There is very little information on the effect of pretreatment and enzymatic saccharification of Miscanthus to produce fermentable sugars. This paper reports sugar yields during enzymatic hydrolysis from ammonia fiber expansion (AFEX) pretreated Miscanthus. Pretreatment conditions including temperature, moisture, ammonia loading, residence time, and enzyme loadings are varied to maximize hydrolysis yields. In addition, further treatments such as soaking the biomass prior to AFEX as well as washing the pretreated material were also attempted to improve sugar yields. The optimal AFEX conditions determined were 160 degrees C, 2:1 (w/w) ammonia to biomass loading, 233% moisture (dry weight basis), and 5 min reaction time for water-soaked Miscanthus. Approximately 96% glucan and 81% xylan conversions were achieved after 168 h enzymatic hydrolysis at 1% glucan loading using 15 FPU/(g of glucan) of cellulase and 64 p-NPGU/(g of glucan) of beta-glucosidase along with xylanase and tween-80 supplementation. A mass balance for the AFEX pretreatment and enzymatic hydrolysis process is presented.

Extraction of proteins from switchgrass using aqueous ammonia within an integrated biorefinery.

Switchgrass (Panicum vergatum) is a potential feedstock for future cellulosic biorefineries. Such a feedstock may also provide protein, most likely for use as an animal feed. In this paper, we present a potential scheme for integrating fiber processing with extractions to obtain both sugar and protein products from switchgrass pretreated using Ammonia Fiber Expansion (AFEX). Solutions of 3% aqueous ammonia at pH 10.5 provided optimal extraction of proteins. Addition of the nonionic surfactant Tween-80 improved protein recovery for AFEX-treated materials. It was determined that an extraction following AFEX solubilized approximately 40% of the protein, while a subsequent hydrolysis solubilized much of the remaining protein while producing 325 g sugar per kg biomass. The remaining insoluble residue contained very little protein or ash, making it ideal for heat and power production. In contrast, an extraction following hydrolysis solubilized only 68% of the original protein in the biomass, while obtaining slightly higher sugar yields.

Techno-economic analysis of decentralized biomass processing depots.

Decentralized biomass processing facilities, known as biomass depots, may be necessary to achieve feedstock cost, quantity, and quality required to grow the future U.S. bioeconomy. In this paper, we assess three distinct depot configurations for technical difference and economic performance. The depot designs were chosen to compare and contrast a suite of capabilities that a depot could perform ranging from conventional pelleting to sophisticated pretreatment technologies. Our economic analyses indicate that depot processing costs are likely to range from ∼US$30 to US$63 per dry metric tonne (Mg), depending upon the specific technology implemented and the energy consumption for processing equipment such as grinders and dryers. We conclude that the benefits of integrating depots into the overall biomass feedstock supply chain will outweigh depot processing costs and that incorporation of this technology should be aggressively pursued.

Developing a model for assessing biomass processing technologies within a local biomass processing depot.

One solution to the supply chain challenges of cellulosic biofuels is a network of local biomass processing depots (LBPDs) that can produce stable, dense, intermediate commodities and valuable co-products prior to shipping to a refinery. A techno-economic model of an LBPD facility that could incorporate multiple technologies and products was developed in Microsoft Excel to be used to economically and environmentally evaluate potential LBPD systems. In this study, three technologies (ammonia fiber expansion or AFEX™ pretreatment, fast pyrolysis, and leaf protein processing) were assessed for profitability. Pyrolysis was slightly profitable under the base conditions, leaf protein processing was highly unprofitable, and AFEX was profitable if biomass drying was not required. This model can be adapted to multiple feedstocks and end uses, including both economic and environmental modeling.

Protease digestion from wheat stillage within a dry grind ethanol facility.

As the current starch based ethanol market increases at its rapid pace, finding new markets for the primary coproduct, distiller's grains, has gained considerable interest. One possibility is to isolate the protein-rich fraction for use as precursors to biochemicals and bioplastics, further decreasing fossil fuel consumption. This research focuses on enzymatic extraction of protein peptides from wheat heavy stillage using commercially available proteases. The energy saved due to this process ranged from ∼ 1.5 to 3.0 GJ/ton wheat stillage compared to fossil fuel-based chemicals. Using Protex 6L (Genencor), ∼ 57% of the protein in the stillage was soluble 24 h after protease addition at 0.1% w/w loading. Of these proteins, ∼ 32% were already soluble, indicating the importance of using wet heavy stillage as the feedstock rather than dried distiller's grains. Peptide size was less than 6 kDa. Further improvements in protein removal may be obtained through a fed batch addition of protease and improved protease cocktails.

Evaluating the impact of ammonia fiber expansion (AFEX) pretreatment conditions on the cost of ethanol production.

Ammonia fiber expansion (AFEX) pretreatment is an ammonia-based process for improving the susceptibility of lignocellulosic biomass to enzymatic attack. Four parameters--ammonia loading, water loading, reaction temperature, and residence time--can be varied in order to optimize AFEX pretreatment. The effect of these parameters on process economics of ethanol production was studied using a leading biorefinery model. Ammonia loading and residence time had the greatest impact on the economics of ethanol production, primarily due to processing costs for the chilled water condenser and the capital cost of the AFEX reactor. Water loading and reaction temperature had only modest impact on process economics. In addition, the impact of pretreatment conditions on makeup ammonia requirements was explored experimentally, which ranged from 15 to 25 g ammonia/kg biomass. Overall, pretreatment conditions can change the costs of ethanol production by up to 35 cents per gallon ethanol in an 850 ton/day refinery. By linking the results obtained from this Aspen model to experimental results for ethanol production and makeup ammonia recovery, this study can be used to create an economic optimum for AFEX pretreatment in contrast with simply maximizing fermentable sugar production.