Burkholderia sacchari DSM 17165: A source of compositionally-tunable block-copolymeric short-chain poly(hydroxyalkanoates) from xylose and levulinic acid.

Burkholderia sacchari was used to produce poly-3-hydroxybutyrate-co-3-hydroxyvalerate block copolymers from xylose and levulinic acid. Levulinic acid was the preferred substrate resulting in 3-hydroxyvalerate (3HV) contents as high as 95 mol% at 24 h. The 3HB:3HV ratios were controlled by the initial levulinic acid media concentration and fermentation length. Higher levulinic acid concentrations and longer durations, resulted in polymers with two glass transition temperatures, each approximating those associated with poly-3HB and poly-3HV. 13C NMR confirmed the presence of high concentrations of 3HB-3HB and 3HV-3HV homopolymeric dyads, while mass spectrometry of the partial hydrolysis products did not conform to Bernoullian statistics for randomness, confirming block sequences. MS/MS analysis of specific oligomers showed the mass-loss of 86 amu (a 3HB unit) and 100 amu (a 3HV unit) attesting to some randomness within the polymers. This study verifies the potential for producing Poly-3HB-block-3HV copolymers from inexpensive biorenewable feedstocks without sequential addition of carbon sources.

Protease increases fermentation rate and ethanol yield in dry-grind ethanol production.

The effects of acid protease and urea addition during the fermentation step were evaluated. The fermentations were also tested with and without the addition of urea to determine if protease altered the nitrogen requirements of the yeast. Results show that the addition of the protease had a statistically significant effect on the fermentation rate and yield. Fermentation rates and yields were improved with the addition of the protease over the corresponding controls without protease. Protease addition either with or with added urea resulted in a higher final ethanol yield than without the protease addition. Urea addition levels >1200 ppm of supplemental nitrogen inhibited ethanol production. The economic effects of the protease addition were evaluated by using process engineering and economic models developed at the Eastern Regional Research Center. The decrease in overall processing costs from protease addition was as high as $0.01/L (4 ¢/gal) of denatured ethanol produced.

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.

Scale-up of ethanol production from winter barley by the EDGE (enhanced dry grind enzymatic) process in fermentors up to 300 l.

A fermentation process, which was designated the enhanced dry grind enzymatic (EDGE) process, has recently been developed for barley ethanol production. In the EDGE process, in addition to the enzymes normally required for starch hydrolysis, commercial ß-glucanases were used to hydrolyze (1,3)(1,4)-ß-D: -glucans to smaller molecules, thus reducing the viscosity of the mash to levels sufficiently low to allow transport and mixing in commercial equipment. Another enzyme, a developmental ß-glucosidase, then was used to hydrolyze the resulting oligomers to glucose, which subsequently was fermented to produce additional ethanol. The EDGE process was developed with Thoroughbred, a winter hulled barley, using a shake flask model. To move toward commercialization, it is necessary to prove that the developed process would be applicable to other barley varieties and also to demonstrate its scalability. Experiments were performed in 7.5, 70, and 300-l fermentors using Thoroughbred and Eve, a winter hull-less barley. It was shown that the process was scalable for both barley varieties. Low levels of glucose throughout the course of the fermentations demonstrated the high efficiency of the simultaneous saccharification and fermentation process. Final ethanol concentrations of 14% (v/v) were achieved for initial total solids of 28.5-30% (w/w), which gave an ethanol yield of 83-87% of the theoretical values. The distillers dried grains with solubles co-products contained very low levels of ß-glucans and thus were suitable for use in feed formulations for all animal species.

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.

Economic analysis of fuel ethanol production from winter hulled barley by the EDGE (Enhanced Dry Grind Enzymatic) process.

A process and cost model was developed for fuel ethanol production from winter barley based on the EDGE (Enhanced Dry Grind Enzymatic) process. In this process, in addition to ß-glucanases, which are added to reduce the viscosity of the mash, ß-glucosidase is also added to completely hydrolyze the oligomers obtained during the hydrolysis of ß-glucans to glucose. The model allows determination of capital costs, operating costs, and ethanol production cost for a plant producing 40 million gallons of denatured fuel ethanol annually. A sensitivity study was also performed to examine the effects of ß-glucosidase and barley costs on the final ethanol production cost. The results of this study clearly demonstrate the economic benefit of adding ß-glucosidase. Lower ethanol production cost was obtained compared to that obtained without ß-glucosidase addition in all cases except one where highest ß-glucosidase cost allowance and lowest barley cost were used.

Fractionation of corn fiber treated by soaking in aqueous ammonia (SAA) for isolation of hemicellulose B and production of C5 sugars by enzyme hydrolysis.

A process was developed to fractionate and isolate the hemicellulose B component of corn fiber generated by corn wet milling. The process consisted of pretreatment by soaking in aqueous ammonia followed by enzymatic cellulose hydrolysis, during which the hemicellulose B was solubilized by cleavage into xylo-oligosaccharides and subsequently recovered by precipitation with ethanol. The pretreatment step resulted in high retention of major sugars and improvement of subsequent enzymatic hydrolysis. The recovered hemicellulose B was hydrolyzed by a cocktail of enzymes that consisted of ß-glucosidase, pectinase, xylanase, and ferulic acid esterase (FAE). Xylanase alone was ineffective, demonstrating yields of less than 2% of xylose and arabinose. The greatest xylose and arabinose yields, 44% and 53%, respectively, were obtained by the combination of pectinase and FAE. A mass balance accounted for 87% of the initially present glucan, 91% of the xylan, and 90% of the arabinan. The developed process offered a means for production of corn fiber gum as a value-added co-product and C5 sugars, which could be converted to other valuable co-products through fermentation in a corn wet-milling biorefinery.

Production of astaxanthin from cellulosic biomass sugars by mutants of the yeast Phaffia rhodozyma.

Astaxanthin is a potential high-value coproduct in an ethanol biorefinery. Three mutant strains of the astaxanthin-producing yeast Phaffia rhodozyma, which were derived from the parent strain ATCC 24202 (UCD 67-210) and designated JTM166, JTM185, and SSM19, were tested for their capability of utilizing the major sugars that can be generated from cellulosic biomass, including glucose, xylose, and arabinose, for astaxanthin production. While all three strains were capable of metabolizing these sugars, individually and in mixtures, JTM185 demonstrated the greatest sugar utilization and astaxanthin production. Astaxanthin yield by this strain (milligrams astaxanthin per gram of sugar consumed) was highest for xylose, followed by arabinose and then glucose. The kinetics of sugar utilization by strain JTM185 was studied in fermenters using mixtures of glucose, xylose, and arabinose at varied concentrations. It was found that glucose was utilized preferentially, followed by xylose, and lastly, arabinose. Astaxanthin yield was significantly affected by sugar concentrations. Highest yields were observed with sugar mixtures containing the highest concentrations of xylose and arabinose. Hydrolysates produced from sugarcane bagasse and barley straw pretreated by the soaking in aqueous ammonia method and hydrolyzed with the commercial cellulase preparation, Accellerase™ 1000, were used for astaxanthin production by the mutant strain JTM185. The organism was capable of metabolizing all of the sugars present in the hydrolysates from both biomass sources and produced similar amounts of astaxanthin from both hydrolysates, although these amounts were lower when compared to yields obtained with reagent grade sugars.

Integration of succinic acid and ethanol production with potential application in a corn or barley biorefinery.

Production of succinic acid from glucose by Escherichia coli strain AFP184 was studied in a batch fermentor. The bases used for pH control included NaOH, KOH, NH(4)OH, and Na(2)CO(3). The yield of succinic acid without and with carbon dioxide supplied by an adjacent ethanol fermentor using either corn or barley as feedstock was examined. The carbon dioxide gas from the ethanol fermentor was sparged directly into the liquid media in the succinic acid fermentor without any pretreatment. Without the CO(2) supplement, the highest succinic acid yield was observed with Na(2)CO(3), followed by NH(4)OH, and lowest with the other two bases. When the CO(2) produced in the ethanol fermentation was sparged into the media in the succinic acid fermentor, no improvement of succinic acid yield was observed with Na(2)CO(3). However, several-fold increases in succinic acid yield were observed with the other bases, with NH(4)OH giving the highest yield increase. The yield of succinic acid with CO(2) supplement from the ethanol fermentor when NH(4)OH was used for pH control was equal to that obtained when Na(2)CO(3) was used, with or without CO(2) supplementation. The benefit of sparging CO(2) from ethanol fermentation on the yield of succinic acid demonstrated the feasibility of integration of succinic acid fermentation with ethanol fermentation in a biorefinery for production of fuels and industrial chemicals.

Continuous high-solids corn liquefaction and fermentation with stripping of ethanol.

Removal of ethanol from the fermentor during fermentation can increase productivity and reduce the costs for dewatering the product and coproduct. One approach is to recycle the fermentor contents through a stripping column, where a non-condensable gas removes ethanol to a condenser. Previous research showed that this approach is feasible. Savings of $0.03 per gallon were predicted at 34% corn dry solids. Greater savings were predicted at higher concentration. Now the feasibility has been demonstrated at over 40% corn dry solids, using a continuous corn liquefaction system. A pilot plant, that continuously fed corn meal at more than one bushel (25 kg) per day, was operated for 60 consecutive days, continuously converting 95% of starch and producing 88% of the maximum theoretical yield of ethanol. A computer simulation was used to analyze the results. The fermentation and stripping systems were not significantly affected when the CO(2) stripping gas was partially replaced by nitrogen or air, potentially lowering costs associated with the gas recycle loop. It was concluded that previous estimates of potential cost savings are still valid.

Enzymatic corn wet milling: engineering process and cost model.

BACKGROUND: Enzymatic corn wet milling (E-milling) is a process derived from conventional wet milling for the recovery and purification of starch and co-products using proteases to eliminate the need for sulfites and decrease the steeping time. In 2006, the total starch production in USA by conventional wet milling equaled 23 billion kilograms, including modified starches and starches used for sweeteners and ethanol production 1. Process engineering and cost models for an E-milling process have been developed for a processing plant with a capacity of 2.54 million kg of corn per day (100,000 bu/day). These models are based on the previously published models for a traditional wet milling plant with the same capacity. The E-milling process includes grain cleaning, pretreatment, enzymatic treatment, germ separation and recovery, fiber separation and recovery, gluten separation and recovery and starch separation. Information for the development of the conventional models was obtained from a variety of technical sources including commercial wet milling companies, industry experts and equipment suppliers. Additional information for the present models was obtained from our own experience with the development of the E-milling process and trials in the laboratory and at the pilot plant scale. The models were developed using process and cost simulation software (SuperPro Designer) and include processing information such as composition and flow rates of the various process streams, descriptions of the various unit operations and detailed breakdowns of the operating and capital cost of the facility. RESULTS: Based on the information from the model, we can estimate the cost of production per kilogram of starch using the input prices for corn, enzyme and other wet milling co-products. The work presented here describes the E-milling process and compares the process, the operation and costs with the conventional process. CONCLUSION: The E-milling process was found to be cost competitive with the conventional process during periods of high corn feedstock costs since the enzymatic process enhances the yields of the products in a corn wet milling process. This model is available upon request from the authors for educational, research and non-commercial uses.

Fractionation, characterization, and study of the emulsifying properties of corn fiber gum.

Corn fiber gum (CFG) has been fractionated by hydrophobic interaction chromatography on Amberlite XAD-1180 resin using ionic, acidic, basic, and hydrophobic solvents of different polarities. Characterization, including determination of total carbohydrate, acidic sugar, and protein content, has been done for each fraction together with measurements of molar mass, polydispersity, radius of gyration, Mark-Houwink exponent, and intrinsic viscosity using multiangle laser light scattering and online viscosity measurements. Emulsification properties of all fractions in an oil-in-water emulsion system with 20:1 oil to gum ratio were studied by measuring turbidity over 14 days. The results indicate that CFG consists of different components differing in their molecular weights and carbohydrate and protein contents. The main fraction eluted with NaCl, although low in protein content, has the highest average molecular weight and was determined to be a better emulsifier than the other fractions. The unfractionated CFG, which contains different molecular species, is the best emulsifier.

Angiotensin I converting enzyme-inhibitory peptides from commercial wet- and dry-milled corn germ.

Bioprocesses were developed to enhance the value of proteins from deoiled corn germ. Proteins were hydrolyzed with trypsin, thermolysin, GC 106, or Flavourzyme to generate the bioactive peptide sequences. At an enzyme to substrate ratio of 1:100, protein hydrolysis of wet-milled germ was greatest using thermolysin followed by trypsin, GC 106, and Flavourzyme. For the dry-milled corn germ, protein hydrolysis was greatest for GC 106 and least for Flavourzyme. Electrophoretic patterns indicated that the hydrolysis conditions used were adequate for generating low molecular weight peptides for both germs. Unhydrolyzed dry- and wet-milled corn germ did not appear to contain angiotensin I converting enzyme (ACE)-inhibitory peptides. After hydrolysis with trypsin, thermolysin, and GC 106 but not Flavourzyme, ACE inhibition was observed. ACE inhibition was greatest for the GC 106 hydrolysate for both wet- and dry-milled corn germ. Denaturing the protein with urea before hydrolysis, in general, increased the amount of ACE-inhibitory peptides found in the hydrolysate. Membrane fractionations of both the wet- and dry-milled hydrolysates indicated that most of the ACE-inhibitory peptides were in the <1 kDa fraction. Examination of the control total protein extracts (before treatment with proteases) from wet- and dry-milled germ revealed that neither had ACE-inhibitory properties. However, when both total corn germ control protein extracts were fractionated, the <1 kDa fraction of wet-milled corn germ proteins exhibited ACE inhibition, whereas the comparable low molecular weight fraction from dry-milled corn germ did not.

Structural characterization of corn fiber gums from coarse and fine fiber and a study of their emulsifying properties.

The stabilities of orange oil emulsions stabilized with various concentrations of two different types of corn fiber gum (CFG-1 and 2) isolated from coarse (pericarp) and fine (endosperm) fiber from corn wet milling have been studied. The emulsion stabilities in all these studies increased with increasing gum concentration up to a gum-to-oil ratio of 0.05, and after that it either levels off or changes very slightly. These results indicate that only 0.25% of CFG is required to make stable emulsion containing 5% orange oil under the experimental conditions used in this study. At this CFG concentration, CFG-2 from each fiber source was found to be a superior emulsifier relative to the corresponding CFG-1 from each source in a 10-day emulsion stability study at room temperature. The emulsion stability was also investigated by confocal laser scanning microscopy measurement, and it was found that CFG-1 and 2 from both coarse and fine fiber made stable emulsions with an average particle size of less than 1 mum for 10 days at room temperature. Sugar composition analysis of CFGs from both sources indicated that they were typical galactoglucuronoarabinoxylans containing mainly 55-59% xylose, 29-36% arabinose, and 4-6% galactose as neutral sugars and 3-5% glucuronic acid. Methylation analysis revealed a highly branched structure of all CFGs, in which only 16-25% of the 1--> 4-linked xylose residues were not substituted at O-2 and/or O-3. Arabinose is present both as a terminal residue and at branch points.

Protein distribution in commercial wet- and dry-milled corn germ.

To identify high-valued coproducts from commercially processed corn germ, it was necessary to determine the effect of processing conditions on corn germ proteins. We found that significantly less protein was extracted from commercial wet-milled as compared to dry-milled corn germ using Tris, sodium dodecyl sulfate (SDS) buffer containing 14 mM 2-mercaptoethanol at 100 degrees C for 10 min. SDS-polyacrylamide gel electrophoresis (PAGE) revealed a number of proteins with molecular masses ranging from approximately 10 to 66 kDa for the dry-milled corn germ as compared to only a few significant protein bands centered around 23 kDa in the wet-milled corn germ. The protein content of the wet- and dry-milled corn germ was approximately the same; however, nonprotein nitrogen values were significantly greater for the wet-milled than for the dry-milled germ. The distribution of fractionated germ protein freshly excised from the embryo of yellow dent corn kernels was more similar to that of dry-milled than wet-milled corn. SDS-PAGE of laboratory preparations of wet-milled corn germ more closely resembled commercial dry- than wet-milled corn germ, which could be attributed to limited microbial growth during steeping in the laboratory preparations.

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.

Membrane separation of solids from corn processing streams.

Corn processing streams are characterized by high water content. Removal of water and recovery of solids are major economic and logistical challenges. New technologies are needed to modify processing streams and to reduce variability and improve quality of coproducts. The objective was to determine the effectiveness of microfiltration and ultrafiltration systems in altering water, solids (protein) and ash contents of corn processing streams. Corn was either steeped with SO(2) (STW) or soaked (SKW) in water; STW contained more solids than SKW. Ultrafiltration of STW and SKW had little effect on water removal or solids recovery. Corn was processed by a conventional wet milling process and a wet milling process that used enzymes to eliminate use of SO(2) steeping. Protein streams from the conventional process (CG) and the enzymatic process (EG) were processed by microfiltration. Permeate streams from EG and CG had higher total solids and ash concentrations than retentate streams; much of the ash was recovered in permeate (67% and 83%, respectively). For CG, proteins were largely recovered in retentate, whereas for EG, proteins were recovered in permeate. SDS-PAGE data indicated a decrease in size of proteins in the EG process stream. Permeate streams from microfiltration were subject to ultrafiltration; there was little effect on solids and nutrient separations.

Fermentation of "Quick Fiber" produced from a modified corn-milling process into ethanol and recovery of corn fiber.

Approximately 9% of the 9.7 billion bushels of corn harvested in the United States was used for fuel ethanol production in 2002, half of which was prepared for fermentation by dry grinding. The University of Illinois has developed a modified dry grind process that allows recovery of the fiber fractions prior to fermentation. We report here on conversion of this fiber (Quick Fiber [QF]) to ethanol. QF was analyzed and found to contain 32%wt glucans and 65%wt total carbohydrates. QF was pretreated with dilute acid and converted into ethanol using either ethanologenic Escherichia coli strain FBR5 or Saccharomyces cerevisiae. For the bacterial fermentation the liquid fraction was fermented, and for the yeast fermentation both liquid and solids were fermented. For the bacterial fermentation, the final ethanol concentration was 30 g/L, a yield of 0.44 g ethanol/g of sugar(s) initially present in the hydrolysate, which is 85% of the theoretical yield. The ethanol yield with yeast was 0.096 gal/bu of processed corn assuming a QF yield of 3.04 lb/bu. The residuals from the fermentations were also evaluated as a source of corn fiber oil, which has value as a nutraceutical. Corn fiber oil yields were 8.28%wt for solids recovered following pretreatment.

Design and pharmacology of N-[(3R)-1,2,3,4-tetrahydroisoquinolinium- 3-ylcarbonyl]-(1R)-1-(4-chlorobenzyl)- 2-[4-cyclohexyl-4-(1H-1,2,4-triazol- 1-ylmethyl)piperidin-1-yl]-2-oxoethylamine (1), a potent, selective, melanocortin subtype-4 receptor agonist.

Synthetic and natural peptides that act as nonselective melanocortin receptor agonists have been found to be anorexigenic and to stimulate erectile activity. We report the design and development of 1, a potent, selective (1184-fold vs MC3R, 350-fold vs MC5R), small-molecule agonist of the MC4 receptor. Pharmacological testing confirms the food intake lowering effects of MC4R agonism and suggests another role for the receptor in the stimulation of erectile activity.