Biosolubilization and liquid fuel production from coal

Recently, several microorganisms have been shown to be capable of directly solubilizing low-rank coals. This bioextract has a high molecular weight and is water soluble, but is not useful as a liquid fuel. This paper presents the results of studies to biologically solubilize coal and convert the solubilized coal into more useful compounds. Preliminary experiments have been conducted to isolate cultures for the serial biological conversion of coal into liquid fuels. Coal particles have been solubilized employing an isolate from the surface of Arkansas lignite. Natural inocula, such as sheep rumen and sewage sludge, are then employed in developing cultures for converting the bioextract into fuels. This paper presents preliminary results of experiments in coal solubilization and bioextract conversion.     

Optimization of sucrose ethanol fermentation for K,Na, Ca,and cu metal contents

The effect of the trace metals Cu, K, Na, and Ca, separately or in mixture, on fermentation time, ethanol production rate, and cell growth in the fermentation of synthetic media containing sucrose is discussed. The results are related to the range of contents found in raw materials, molasses and raisins, in order to determine their optimum concentrations for alcohol production.     

An integrated bioconversion process for production of l-lactic acid from starchy potato feedstocks

The potential market for lactic acid as the feedstock for biodegradable polymers, oxygenated chemicals, and specialty chemicals is significant. L-lactic acid is often the desired enantiomer for such applications. However, stereospecific lactobacilli do not metabolize starch efficiently. In this work, Argonne researchers have developed a process to convert starchy feedstocks into L-lactic acid. The processing steps include starch recovery, continuous liquefaction, and simultaneous saccharification and fermentation. Over 100 g/L of lactic acid was produced in less than 48 h. The optical purity of the product was greater than 95%. This process has potential economical advantages over the conventional process.     

Production of methyl ester fuel from microalgae

Microalgae are unique photosynthetic organisms that are known to accumulate storage lipids in large quantitites and thrive in saline waters. Before these storage lipids can be used, they must be extracted from the microalgae and converted into usable fuel. Transesterification of lipids produces fatty acid methyl esters that can be used as a diesel fuel substitute. Three solvents, 1-butanol, ethanol, and hexane/2-propanol, were evaluated for extraction efficiency of microalgal lipids. Type of catalyst, concentration of catalyst, time of reaction, temperature of reaction, and quality of lipid were examined as variables for transesterification. The most efficient solvent of the three for extraction was 1-butanol (90% efficiency), followed by hexane/2-propanol and ethanol. Optimal yield of fatty acid methyl esters was obtained using 0.6N hydrochloric acid in methanol for 0.1 h at 70°C.     

Xylanase recovery by ethanol and Na2SO4 precipitation

Xylans are the major components of the hemicellulosic fraction of lignocellulosic biomass and their hydrolysis can be obtained using xylanases fromPenicillium janthinellum. In this work, sugarcane bagasse hemicellulosic hydrolysate was used as the substrate for producing xylanase. The precipitation of these enzymes was studied using ethanol and Na₂SO₄ as precipitating agents. Ethanol precipitation experiments were performed batchwise in concentrations ranging from 10 to 80%, pH 4.0 to 7.0, at 4áC. The concentrations used in the precipitations with Na₂SO₄ were from 5 to 60% at pH 5.5 and 25áC. Solubility curves as a function of xylanase activity and total protein for both precipitating agents were made. According to the results, Na₂SO₄ is not appropriate for precipitating xylanases in this medium since at salt concentrations higher than 25%, the enzyme was denaturated and at this concentration less than 80% of the enzyme and total protein were precipitated. Because of differences in xylanase and total protein solubility, a fractionated precipitation using ethanol can be performed, since with 40% ethanol, 49% of the total protein was precipitated and more than 95% of the enzyme was kept in solution. On the other hand approx 100% of the xylanases were recovered by precipitation after adding 80% ethanol.     

Comparison of yellow poplar pretreatment between NREL digester and sunds hydrolyzer

Single-stage cocurrent dilute acid pretreatments were carried out on yellow poplar (Liriodendron tulipifera) sawdust using an as-installed and short residence time modified pilot-scale Sunds hydrolyzer and a 4-L bench-scale NREL digester (steam explosion reactor). Pretreatment conditions for the Sunds hydrolyzer, installed in the NREL process development unit (PDU), which operates at 1 t/d (bone-dry t) feed rate, spanned the temperature range of 160 – 210°C, 0.1 – 1.0% (w/w) sulfuric acid, and 4-10-min residence times. The batch pretreatments of yellow poplar sawdust in the bench-scale digester were carried out at 210 and 230°C, 0.26% (w/w) sulfuric acid, and 1-, 3-, and 4-min residence times. The dilute acid prehydrolysis solubilized more than 90% of the hemicellulose, and increased the enzymatic digestibility of the cellulose that remained in the solids. Compositional analysis of the pretreated solids and liquors and mass balance data show that the two pretreatment devices had similar pretreatment performance.     

Evaluation of O2 and CO2 transfer coefficients in a locally integrated tubular hollow fiber bioreactor

We have studied the transfer coefficients for O₂ transport to and CO₂ removal from a model cell-free system using microporous hydrophobic hollow fibers axially placed along a tubular bioreactor for ethanol production with immobilized yeast in the shell. In this locally integrated bioreactor, O₂ and CO₂ transfer rates depend strongly on the shell side liquid flow rate; O₂ flow rate in the fiber bore influences O₂ transport only at very low flow rates. Diffusion of CO₂ does not affect O₂ transport. Local and overall O₂ and CO₂ transfer coefficients have been determined for a wide range of Reynolds Numbers. The efficiency of such transfers has been demonstrated for alcohol fermentation.     

The role of ester groups in resistance of plant cell wall polysaccharides to enzymatic hydrolysis

Xylan backbones in native plant cell walls are extensively acety-lated. Previously, no direct investigations as to their role in cellulolytic enzyme resistance have been done, though indirect results point to their importance. An in vitro deesterification of aspen wood and wheat straw has been completed using hydroxylamine solutions. Yields of 90% acetyl ester removal for both materials have been accomplished, with little disruption of other fractions (i.e., lignin). Apparently, as the xylan becomes increasingly deacetylated, it becomes 5–7 times more digestible. This renders the cellulose fraction more accessible, and 2–3 times more digestible. This effect levels off near an acetyl removal of 75%, where other resistances become limiting.     

Simultaneous saccharification and fermentation of lignocellulose

Simultaneous saccharification and fermentation (SSF) processes for producing ethanol from lignocellulose are capable of improved hydrolysis rates, yields, and product concentrations compared to separate hydrolysis and fermentation (SHF) systems, because the continuous removal of the sugars by the yeasts reduces the end-product inhibition of the enzyme complex. Recent experiments using Genencor 150L cellulase and mixed yeast cultures have produced yields and concentrations of ethanol from cellulose of 80% and 4.5%, respectively. The mixed culture was employed because B.clausenii has the ability to ferment cellobiose (further reducing end-product inhibition), while the brewing yeastS. cerevisiae provides a robust ability to ferment the monomeric sugars. These experimental results are combined with a process model to evaluate the economics of the process and to investigate the effect of alternative processes, conditions, and organisms.     

Purification and partial characterization of two lectin isoforms fromCratylia mollis mart. (camaratu bean)

Two additional electrophoretically distinct molecular forms, isoforms (iso) 2 and 3, with lectin properties were isolated fromCratylia mollis Mart, seeds (FABACEAE), by extraction with 0.15M NaCl and ammonium sulfate fractionation, followed by chromatography on Sephadex G-75 and Bio-Gel P-200 (iso 2), as well as CM-Cellulose and Sephadex G-75 (iso 3). Both isoforms were human group nonspecific and showed distinct specificity. Polyacrylamide gel electrophoresis resolved iso 2 and 3 in polypeptides of apparent mol wts 60 and 31 kDa, respectively; a distinct isoelectric focusing pattern was obtained for iso 2 and 3, under denaturing and reducing conditions.     

Cellulose hydrolysis using zinc chloride as a solvent and catalyst

Cellulose gel with < 10% of crystallinity was prepared by treatment of microcrystalline cellulose, Avicel, with zinc chloride solution at a ratio of zinc chloride to cellulose from 1.5 to 18 (w/w). The presence of zinc ions in the cellulose gels enhanced the rate of hydrolysis and glucose yield. The evidence obtained from X-ray diffraction, iodine absorption experiments; and Nuclear Magnetic Resonance spectra analysis suggested the presence of zinc-cellulose complex after Avicel was treated with zinc chloride. Zinc-cellulose complex was more susceptible to hydrolysis than amorphous cellulose. Under the experimental condition, cellulose gels with zinc ions were hyrolyzed to glucose with 95% theoretical yield and a concentration of 14% (w/v) by cellulases within 20 h. The same gel was hydrolyzed by acid to glucose with 91.5% yield and a concentration of 13.4% (w/v).     

Yeast adaptation on softwood prehydrolysate

Several strains and genera of yeast, includingSaccharomyces cerevisiae D₅A,Pachysolen tannophilus, S. cerevisiae K-l,Brettanomyces custersii, Candida shehatae, andCandida acidothermophilum, are screened for growth on dilute acid-pretreated softwood prehydrolysate. Selected softwood species found in forest underbrush of the western United States, which contain predominantly hexosan hemicellulose, were studied. This phase of the work emphasized debarked Douglas fir. The two best initial isolates were gradually selected for improved growth by adaptation to increasing prehydrolysate concentrations in batch culture, with due consideration of nutrient requirements. Microaerophilic conditions were evaluated to encourage tolerance of pretreatment hydrolysate, as well as ethanol product. Adaptation and simultaneous saccharification and fermentation (SSF) results are used to illustrate improved performance with an adapted strain, compared to the wild type.     

Urease purification from the seeds ofCajanus Cajan and its application in a biosensor construction

Urease has been purified from the seeds of Cajanus Cajan. The purification process involves three solvent extraction steps followed by DEAE-cellulose column chromatography. The specific activity of the purified enzyme is found to be 1920 U/mg with the recovery of 8%. The application of the purified enzyme in a biosensor construction is discussed.     

Synthesis of Cyclodextrin Glucosyl Transferase byBacillus cereus for the production of cyclodextrins

A potent indigenous bacillus isolate identified asBacillus cereus (RJ-30) was found to produce Cyclodextrin Glucosyl Transferase (CGTase) extracellularly. Process optimization of various fermentation parameters has been established for optimal growth of bacillus and the maximum enzyme synthesis. The organism had the highest specific growth rate (0.7μ) with a generation time of 1 h in glucose containing medium at the conditions of pH 7.0, 37°C at 300 rpm, 1.5 vvm of agitation, and aeration. At these conditions, it exhibited the maximum activity of 54 U/mL at the synthesis rate of 2.7 U/L/h. CGTase was produced from the early exponential growth and peaked during the midsporulating stage of about 16 h thereafter maintained at the same level of 50 U/mL. Saccharides containing media were better inducers than starch, and the influence of carbohydrate substrates has shown that enzyme synthesis is promoted by xylose (65 U/mL) and, more remarkably, by the supplementation of wheat bran extract in glucose medium (106 U/mL). This organism produced CGTase stably in a chemostat culturing over a period of 400 h with a maximum productivity of 5.4 kU/L/h (threefold higher than obtained in batch culturing [1.75 kU/L/h]). Comparatively, CGTase was produced by immobilized cells in a continuous fluidized bed reactor for over approx 360 h, at a relatively high dilution rate of 0.88 h⁻¹ resulting in the productivity of 23.0 kU/L/h.     

Modeling of an immobilized-cell three-phase fluidized-bed bioreactor

A mathematical model of a three-phase, tapered, fluidized-bed bioreactor has been developed. This model includes the effects of the tapered bed, a variable dispersion coefficient, and the concentration profile inside the biocatalyst bead on the reaction rate within the bed. Parameters in this model were obtained by adjusting them, within a realistic range, such that the square of the difference between the values predicted by the model and those obtained experimentally was minimized. The model was found to predict experimentally obtained concentration profiles quite accurately. It also demonstrates the need to include the effects of variable dispersion in three-phase systems where the gas phase is being generated inside the reactor, as the dispersion coefficient varied by more than an order of magnitude across the bed.     

Dilute acid pretreatment of softwoods

Selective thinning of forests in the western United States will generate a large, sustainable quantity of softwood residues that can be an attractive feedstock for fuel ethanol production. The major species available from thinning of forests in northern California and the eastern Rocky Mountains include white fir (Abies concolor), Douglas fir (Pseudotsuga menziesii), and Ponderosa pine (Pinus ponderosa). Douglas fir chips were soaked in 0.4% sulfuric acid solution, then pretreated with steam at 200 – 230°C for 1 – 5 min. After pretreatment, 90 – 95% of the hemicellulose and as much as 20% of the cellulose was solubilized in water, and 90% of the remaining cellulose can be hydrolyzed to glucose by cellulase enzyme. The prehydrolysates, at as high as 10% total solid concentration, can be readily fermented by the unadapted yeastSaccharomyces cerevisiae D₅A.     

Coordinating California’s efforts to promote waste to alcohol production

Alcohol fuels produced from biomass can improve air quality, enhance energy security, create employment opportunities, and reduce waste disposal problems. Opportunities in California exist to produce alcohols from waste streams from various sectors of the economy. Government agencies have promoted waste-to-alcohol activities, but efforts have been inconsistent and intermittent. Often these efforts have been hindered by contradictory but mandate-driven policies. A prudent approach to coordinate statewide efforts includes the development of an integrated statewide policy to examine barriers that impede private sector business efforts to produce alcohols from biomass. A multi-agency task force to promote research, development, commercialization, and marketing efforts for biomass-produced alcohols is desirable. The views and opinions contained in this document do not necessarily reflect those of the California Energy Commission, its staff, management, or the State of California.     

Xylose fermentation

The economic impact of conversion of xylose to ethanol for a wood-to-ethanol plant was examined, and the maximum potential reduction in the price of ethanol from utilization of xylose is estimated to be 0.42 per gallon from a base case price of0.42 per gallon from a base case price of 1.65. The sensitivity of the price of ethanol to the yield, ethanol concentration and rate of the xylose fermentation was also examined, and the price of ethanol is most affected by changes in yield and ethanol concentration, with rate of lesser importance. Current performances of various xylose conversion biocatalysts were analyzed, andC. shehatae andP. stipitis appear to be the best yeasts.     

Development of a novel,two-step process for treating municipal biosolids for beneficial reuse

Modern municipal sewage waste treatment plants use conventional mechanical and biological processes to reclaim wastewaters. This process has an overall effect of converting a water pollution problem into a solid waste disposal problem (sludges or biosolids). An estimated 10 million tons of biosolids, which require final disposal, are produced annually in the United States. Although numerous disposal options for biosolids are available, including land application, landfilling, and incineration, disposal costs have risen, partly because of increased federal and local environmental restrictions(1). A novel, thermomechanical biosolids pre-treatment process, which allows for a variety of potential value-added uses, was developed. This two-step process first employs thermal explosive decompression to inactivate or kill the microbial cells and viruses. This primary step also results in the rupture of a small amount of the microbial biomass and increases the intrinsic fluidity of the biosolids. The second step uses shear to effect a near-complete rupturing of the microbial biomass, and shears the nondigested organics, which increases the overall surface area. Pretreated biosolids may be subjected to a secondary anaerobic digestion process to produce additional fuel gas, and to provide for a high-quality, easily dewatered compost product. This novel biosolids pretreatment process was recently allowed a United States patent.