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.

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.

Ammonia Fiber Expansion (AFEX) Pretreatment of Lignocellulosic Biomass.

Lignocellulosic materials are plant-derived feedstocks, such as crop residues (e.g., corn stover, rice straw, and sugar cane bagasse) and purpose-grown energy crops (e.g., miscanthus, and switchgrass) that are available in large quantities to produce biofuels, biochemicals, and animal feed. Plant polysaccharides (i.e., cellulose, hemicellulose, and pectin) embedded within cell walls are highly recalcitrant towards conversion into useful products. Ammonia fiber expansion (AFEX) is a thermochemical pretreatment that increases accessibility of polysaccharides to enzymes for hydrolysis into fermentable sugars. These released sugars can be converted into fuels and chemicals in a biorefinery. Here, we describe a laboratory-scale batch AFEX process to produce pretreated biomass on the gram-scale without any ammonia recycling. The laboratory-scale process can be used to identify optimal pretreatment conditions (e.g., ammonia loading, water loading, biomass loading, temperature, pressure, residence time, etc.) and generates sufficient quantities of pretreated samples for detailed physicochemical characterization and enzymatic/microbial analysis. The yield of fermentable sugars from enzymatic hydrolysis of corn stover pretreated using the laboratory-scale AFEX process is comparable to pilot-scale AFEX process under similar pretreatment conditions. This paper is intended to provide a detailed standard operating procedure for the safe and consistent operation of laboratory-scale reactors for performing AFEX pretreatment of lignocellulosic biomass.

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.

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.

Understanding factors that limit enzymatic hydrolysis of biomass: characterization of pretreated corn stover.

Spectroscopic characterization of both untreated and treated material is being performed in order to determine changes in the biomass and the effects of pretreatment on crystallinity, lignin content, selected chemical bonds, and depolymerization of hemicellulose and lignin. The methods used are X-ray diffraction for determination of cellulose crystallinity (CrI); diffusive reflectance infrared (DRIFT) for changes in C-C and C-O bonds; and fluorescence to determine lignin content. Changes in spectral characteristics and crystallinity are statistically correlated with enzymatic hydrolysis results to identify and better understand the fundamental features of biomass that govern its enzymatic conversion to monomeric sugars. Models of the hydrolysis initial rate and 72 h extent of conversion were developed and evaluated. Results show that the hydrolysis initial rate is most influenced by the cellulose crystallinity, while lignin content most influences the extent of hydrolysis at 72 h. However, it should be noted that in this study only crystallinity, lignin, and selected chemical bonds were used as inputs to the models. The incorporation of additional parameters that affect the hydrolysis, like pore volume and size and surface area accessibility, would improve the predictive capability of the models.

Pretreatment of switchgrass by ammonia fiber explosion (AFEX).

The effects of ammonia fiber explosion (AFEX) pretreatment of switch grass using its major process variables are reported. The optimal pretreatment conditions for switchgrass were found to be near 100 degrees C reactor temperature, and ammonia loading of 1:1 kg of ammonia: kg of dry matter with 80% moisture content (dry weight basis [dwb]) at 5 min residence time. Hydrolysis results of AFEX-treated and untreated samples showed 93% vs 16% glucan conversion, respectively. The ethanol yield of optimized AFEX-treated switchgrass was measured to be about 0.2 g ethanol/g dry biomass, which is 2.5 times more than that of the untreated sample.

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.

Effects of ammonia fiber explosion treatment on activity of endoglucanase from Acidothermus cellulolyticus in transgenic plant.

A critical parameter affecting the economic feasibility of lignocellulosic bioconversion is the production of inexpensive and highly active cellulase enzymes in bulk quantity. A promising approach to reduce enzyme costs is to genetically transform plants with the genes of these enzymes, thereby producing the desired cellulases in the plants themselves. Extraction and recovery of active proteins or release of active cellulase from the plants during bioconversion could have a significant positive impact on overall lignocellulose conversion economics. The effects of ammonia fiber explosion (AFEX) pretreatment variables (treatment temperature, moisture content, and ammonia loading) on the activity of plant-produced heterologous cellulase enzyme were individually investigated via heat treatment or ammonia treatment. Finally, we studied the effects of all these variables in concert through the AFEX process. The plant materials included transgenic tobacco plants expressing E1 (endoglucanase from Acidothermus cellulolyticus). The E1 activity was measured in untreated and AFEX-treated tobacco leaves to investigate the effects of the treatment on the activity of this enzyme. The maximum observed activity retention in AFEX-treated transgenic tobacco samples compared with untreated samples was approx 35% (at 60 degrees C, 0.5:1 ammonia loading, and 40% moisture). Based on these findings, it is our opinion that AFEX pretreatment is not a suitable option for releasing cellulase enzyme from transgenic plants.

Ammonia fiber explosion treatment of corn stover.

Optimizing process conditions and parameters such as ammonia loading, moisture content of biomass, temperature, and residence time is necessary for maximum effectiveness of the ammonia fiber explosion process. Approximate optimal pretreatment conditions for corn stover were found to be temperature of 90 degrees C, ammonia:dry corn stover mass ratio of 1:1, moisture content of corn stover of 60% (dry weight basis), and residence time (holding at target temperature), of 5 min. Approximately 98% of the theoretical glucose yield was obtained during enzymatic hydrolysis of the optimal treated corn stover using 60 filter paper units (FPU) of cellulase enzyme/g of glucan (equal to 22 FPU/g of dry corn stover). The ethanol yield from this sample was increased up to 2.2 times over that of untreated sample. Lowering enzyme loading to 15 and 7.5 FPU/g of glucan did not significantly affect the glucose yield compared with 60 FPU, and any differences between effects at different enzyme levels decreased as the treatment temperature increased.

Effect of pelleting process variables on physical properties and sugar yields of ammonia fiber expansion pretreated corn stover.

Pelletization process variables, including grind size (4, 6mm), die speed (40, 50, 60 Hz), and preheating (none, 70°C), were evaluated to understand their effect on pellet quality attributes and sugar yields of ammonia fiber expansion (AFEX) pretreated biomass. The bulk density of the pelletized AFEX corn stover was three to six times greater compared to untreated and AFEX-treated corn stover. Also, the durability of the pelletized AFEX corn stover was>97.5% for all pelletization conditions studied except for preheated pellets. Die speed had no effect on enzymatic hydrolysis sugar yields of pellets. Pellets produced with preheating or a larger grind size (6mm) had similar or lower sugar yields. Pellets generated with 4mm AFEX-treated corn stover, a 60Hz die speed, and no preheating resulted in pellets with similar or greater density, durability, and sugar yields compared to other pelletization conditions.

Separation of glucose and pentose sugars by selective enzyme hydrolysis of AFEX-treated corn fiber.

A process was developed to fractionate corn fiber into glucose- and pentose-rich fractions. Corn fiber was ammonia fiber explosion treated at 90 degrees C, using 1 g anhydrous ammonia pergram of drybiomass, 60% moisture, and 30-min residence time. Twenty four hour hydrolysis of ammonia fiber explosion-treated corn fiber with cellulase converted 83% of available glucanto-glucose. In this hydrolysis the hemicellulose was partially broken down with 81% of the xylan and 68% of the arabinan being contained in the hydrolysate after filtration to remove lignin and other insoluble material. Addition of ethanol was used to precipitate and recover the solubilized hemicellulose from the hydrolysate, followed by hydrolysis with 2% (v/v) sulfuric acid to convert the recovered xylan and arabinan to monomeric sugars. Using this method, 57% of xylose and 54% of arabinose available in corn fiber were recovered in a pentose-rich stream. The carbohydrate composition of the pentose-enriched stream was 5% glucose, 57% xylose, 27% arabinose, and 11% galactose. The carbohydrate composition of the glucose-enriched stream was 87% glucose, 5% xylose, 6% arabinose, and 1% galactose, and contained 83% of glucose available from the corn fiber.

Ammonia fiber expansion pretreatment and enzymatic hydrolysis on two different growth stages of reed canarygrass.

Plant materials from the vegetative growth stage of reed canarygrass and the seed stage of reed canarygrass are pretreated by ammonia fiber expansion (AFEX) and enzymatically hydrolyzed using 15 filter paper units (FPU) cellulase/g glucan to evaluate glucose and xylose yields. Percent conversions of glucose and xylose, effects of temperature and ammonia loading, and hydrolysis profiles are analyzed to determine the most effective AFEX treatment condition for each of the selected materials. The controls used in this study were untreated samples of each biomass material. All pretreatment conditions tested enhanced enzyme digestibility and improved sugar conversions for reed canarygrass compared with their untreated counterparts. Based on 168 h hydrolysis results using 15 FPU Spezyme CP cellulase/g glucan the most effective AFEX treatment conditions were determined as: vegetative growth stage of reed canarygrass--100 degrees C, 60% moisture content, 1.2:1 kg ammonia/kg of dry matter (86% glucose and 78% xylose) and seed stage of reed canarygrass--100 degrees C, 60% moisture content, 0.8:1 kg ammonia/kg of dry matter (89% glucose and 81% xylose). Supplementation by commercial Multifect 720 xylanase along with cellulase further increased both glucose and xylose yields by 10-12% at the most effective AFEX conditions.