Loren B. Iten

Dilute acid pretreatment, enzymatic saccharification, and fermentation of rice hulls to ethanol.

Rice hulls, a complex lignocellulosic material with high lignin (15.38 ± 0.2%) and ash (18.71 ± 0.01%) content, contain 35.62 ± 0.12% cellulose and 11.96 ± 0.73% hemicellulose and has the potential to serve as a low‐cost feedstock for production of ethanol. Dilute H2SO4 pretreatments at varied temperature (120–190 °C) and enzymatic saccharification (45 °C, pH 5.0) were evaluated for conversion of rice hull cellulose and hemicellulose to monomeric sugars. The maximum yield of monomeric sugars from rice hulls (15%, w/v) by dilute H2SO4 (1.0%, v/v) pretreatment and enzymatic saccharification (45 °C, pH 5.0, 72 h) using cellulase, β‐glucosidase, xylanase, esterase, and Tween 20 was 287 ± 3 mg/g (60% yield based on total carbohydrate content). Under this condition, no furfural and hydroxymethyl furfural were produced. The yield of ethanol per L by the mixed sugar utilizing recombinant Escherichia coli strain FBR 5 from rice hull hydrolyzate containing 43.6 ± 3.0 g fermentable sugars (glucose, 18.2 ± 1.4 g; xylose, 21.4 ± 1.1 g; arabinose, 2.4 ± 0.3 g; galactose, 1.6 ± 0.2 g) was 18.7 ± 0.6 g (0.43 ± 0.02 g/g sugars obtained; 0.13 ± 0.01 g/g rice hulls) at pH 6.5 and 35 °C. Detoxification of the acid‐ and enzyme‐treated rice hull hydrolyzate by overliming (pH 10.5, 90 °C, 30 min) reduced the time required for maximum ethanol production (17 ± 0.2 g from 42.0 ± 0.7 g sugars per L) by the E. coli strain from 64 to 39 h in the case of separate hydrolysis and fermentation and increased the maximum ethanol yield (per L) from 7.1 ± 2.3 g in 140 h to 9.1 ± 0.7 g in 112 h in the case of simultaneous saccharification and fermentation.

Media and Fermentation Processes for the Rapid Production of High Concentrations of Stable Blastospores of the Bioinsecticidal Fungus Paecilomyces fumosoroseus

In shake flask and fermentor studies, various media components and culture inocula were tested to improve P. fumosoroseus spore production rates, yield and stability. To evaluate inoculum potential and inoculum scale-up for fermentor studies, conidia and liquid culture-produced spores of various strains of P. fumosoroseus were compared as inoculum. Inoculation of liquid cultures with blastospores at concentrations of at least 1×106 spores mL−1 resulted in the rapid production of high concentrations of blastospores (∼1×109 spores mL−1, 48 h fermentation time) for all strains tested. The rapid germination rate of blastospores (90% after 6 h incubation) compared to conidia (>90% after 16 h incubation) and the use of higher inoculum rates reduced the fermentation time from 96 to 48 h for maximal spore yields. A comparison of various complex nitrogen sources showed that liquid media supplemented with acid hydrolyzed casein or yeast extract supported the production of high concentrations of blastospores that were significantly more desiccation-tolerant (79-82% survival after drying) when compared to blastospores produced in media supplemented with other nitrogen sources (12-50% survival after drying). For rapid spore production, requirements for trace metals and vitamin supplementation were dependent on the type of hydrolyzed casein used in the medium. Fermentor studies with two strains of P. fumosoroseus showed that high concentrations (1.3-1.8×109 spores mL−1) of desiccation-tolerant blastospores could be produced in 48-h fermentations. These studies have demonstrated that the infective spores of various strains of the fungal bioinsecticide Paecilomyces fumosoroseus can be rapidly produced using deep-tank, liquid culture fermentation techniques.

Pilot scale conversion of wheat straw to ethanol via simultaneous saccharification and fermentation

The production of ethanol from wheat straw (WS) by dilute acid pretreatment, bioabatement of fermentation inhibitors by a fungal strain, and simultaneous saccharification and fermentation (SSF) of the bio-abated WS to ethanol using an ethanologenic recombinant bacterium was studied at a pilot scale without sterilization. WS (124.2g/L) was pretreated with dilute H2SO4 in two parallel tube reactors at 160°C. The inhibitors were bio-abated by growing the fungus aerobically. The maximum ethanol produced by SSF of the bio-abated WS by the recombinant Escherichia coli FBR5 at pH 6.0 and 35°C was 36.0g/L in 83h with a productivity of 0.43gL(-1)h(-1). This value corresponds to an ethanol yield of 0.29g/g of WS which is 86% of the theoretical ethanol yield from WS. This is the first report on the production of ethanol by the recombinant bacterium from a lignocellulosic biomass at a pilot scale.

Conversion of corn fiber to ethanol by recombinant E. coli strain FBR3

Escherichia coli strain FBR3 that is an efficient biocatalyst for converting mixed sugar streams (eg, arabinose, glucose, and xylose) into ethanol. In this report, the strain was tested for conversion of corn fiber hydrolysates into ethanol. Corn fiber hydrolysates with total sugar concentrations of 7.5% (w/v) were prepared by reacting corn fiber with dilute sulfuric acid at 145°C. Initial fermentations of the hydrolysate by strain FBR3 had lag times of approximately 30 h judged by ethanol production. Further experiments indicated that the acetate present in the hydrolysate could not solely account for the long lag. The lag phase was greatly reduced by growing the pre-seed and seed cultures on corn fiber hydrolysate. Ethanol yields for the optimized fermentations were 90% of theoretical. Maximum ethanol concentrations were 2.80% w/v, and the fermentations were completed in approximately 50 h. The optimal pH for the fermentation was 6.5. Below this pH, sugar consumption was incomplete and above it, excess base addition was required throughout the fermentation. Two alternative neutralization methods (overliming and overliming with sulfite addition) have been reported for improving the fermentability of lignocellulosic hydrolysates. These methods further reduced the lag phase of the fermentation, albeit by a minor amount.

Fate of Bt Protein and Influence of Corn Hybrid on Ethanol Production

ABSTRACT Corn hybrids were compared to determine the fate of recombinant Bt protein (CRY1Ab from Bacillus thuringiensis) in coproducts from dry grind and wet-milled corn during production of fuel ethanol. Two pairs of Bt and non-Bt hybrids were wet milled, and each fraction was examined for the presence of the Bt protein. Bt protein was found in the germ, gluten, and fiber fractions of Bt hybrids. In addition, one set of Bt and non-Bt hybrids were treated by the dry-grind ethanol process and Bt protein was monitored during each step of the process. The Bt protein was not detected after liquefaction. Subsequent experiments determined that the Bt protein is rapidly denatured at liquefaction temperatures. Finally, five hybrids were compared for ethanol yield after dry grinding. Analysis of fermentation data with an F-test revealed the percent of total starch available for conversion into ethanol varied significantly among the hybrids (P < 0.002), indicating ethanol yield is not exclusively dependent on starc...

Shaping Reactor Microbiomes to Produce the Fuel Precursor n-Butyrate from Pretreated Cellulosic Hydrolysates

To maximize the production of carboxylic acids with open cultures of microbial consortia (reactor microbiomes), we performed experiments to understand which factors affect the community dynamics and performance parameters. We operated six thermophilic (55 °C) bioreactors to test how the factors: (i) biomass pretreatment; (ii) bioreactor operating conditions; and (iii) bioreactor history (after perturbations during the operating period) affected total fermentation product and n-butyrate performance parameters with corn fiber as the cellulosic biomass waste. We observed a maximum total fermentation product yield of 39%, a n-butyrate yield of 23% (both on a COD basis), a maximum total fermentation production rate of 0.74 g COD l(-1) d(-1) and n-butyrate production rate of 0.47 g COD l(-1) d(-1) in bioreactors that were fed with dilute-acid pretreated corn fiber at a pH of 5.5. Pyrosequencing of 16S rRNA genes with constrained ordination and other statistical methods showed that changes in operating conditions to enable dilution of toxic carboxylic acid products, which lead to these maximum performance parameters, also altered the composition of the microbiome, and that the microbiome, in turn, affected the performance. Operating conditions are an important factor (tool for operators) to shape reactor microbiomes, but other factors, such as substrate composition after biomass pretreatment and bioreactor history are also important. Further optimization of operating conditions must relieve the toxicity of carboxylic acids at acidic bioreactor pH levels even more, and this can, for example, be accomplished by extracting the product from the bioreactor solutions.

Conversion of switchgrass to ethanol using dilute ammonium hydroxide pretreatment: influence of ecotype and harvest maturity

Switchgrass (Panicum virgatum L.) is a perennial C4 grass that is being developed as a bioenergy crop because it has high production yields and suitable agronomic traits. Five switchgrass biomass samples from upland and lowland switchgrass ecotypes harvested at different stages or maturity were used in this study. Switchgrass samples contained 317.0–385.0 g glucans/kg switchgrass dry basis (db) and 579.3–660.2 g total structural carbohydrates/kg switchgrass, db. Carbohydrate contents were greater for the upland ecotype versus lowland ecotype and increased with harvest maturity. Pretreatment of switchgrass with dilute ammonium hydroxide (8% w/w ammonium loading) at 170°C for 20 min was determined to be effective for preparing switchgrass for enzymatic conversion to monosaccharides; glucose recoveries were 66.9–90.5% and xylose recoveries 60.1–84.2% of maximum and decreased with increased maturity at harvest. Subsequently, pretreated switchgrass samples were converted to ethanol by simultaneous saccharification and fermentation using engineered xylose-fermenting Saccharomyces cerevisiae strain YRH400. Ethanol yields were 176.2–202.0 l/Mg of switchgrass (db) and followed a similar trend as observed for enzymatic sugar yields.

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.

Utilizing pretreatment and fungal incubation to enhance the nutritional value of canola meal

The objective of this study was to determine the optimal pretreatment and fungal strain to reduce glucosinolates (GLS), fibre and residual sugars while increasing the nutritional value of canola meal.