Evaluation of a kinetic model for computer simulation of growth and fermentation by Scheffersomyces (Pichia) stipitis fed D-xylose.

Scheffersomyces (formerly Pichia) stipitis is a potential biocatalyst for converting lignocelluloses to ethanol because the yeast natively ferments xylose. An unstructured kinetic model based upon a system of linear differential equations has been formulated that describes growth and ethanol production as functions of ethanol, oxygen, and xylose concentrations for both growth and fermentation stages. The model was validated for various growth conditions including batch, cell recycle, batch with in situ ethanol removal and fed-batch. The model provides a summary of basic physiological yeast properties and is an important tool for simulating and optimizing various culture conditions and evaluating various bioreactor designs for ethanol production.

Dehydration of ethanol: new approach gives positive energy balance.

Water was removed from aqueous ethanol by using cellulosic materials, starch, corn, and other agents. The combustion energy of the ethanol product can exceed the energy needed to carry out the dehydration by a factor of 10.

Cellulose to sugars: new path gives quantitative yield.

Cellulosic residues that had been treated with a small amount of chemical solvent under room conditions were quantitatively saccharified on enzyme hydrolysis. This treatment can be used to obtain simple sugars for the production of alcohol and other chemicals.

Hydrophobic interaction chromatography.

Hydrophobic interaction chromatography (HIC) is emerging as a useful technique for the separation of biological compounds. Advances in the past two years in HIC applications, stationary phases, eluents, and theory are reviewed. Recent applications of HIC processes include analytical and semi-preparative separations of a variety of proteins, such as isolectins, hemoglobins, calmodulin, and cardiotoxins. Additionally, HIC is being employed as a tool to investigate protein properties and mechanisms. Advances in HIC stationary phases include development of non-porous, microparticulate supports as well as supports with pore sizes up to 1000 Angstroms. Studies of HIC eluents have further shown the effects of mobile phase pH, water-structuring characterization, and surface tension increments on retention. Various retention mechanisms which have been presented are reviewed; and a correlation relating resolution to column and solute parameters is presented. Protein conformational effects at specific sites have been shown to have a significant impact on retention and specific examples illustrating such effects are discussed.

Electrochromatographic separation of proteins.

We have developed a modified electrochromatography system which minimizes Joule heating at electric field strengths up to 125 V/cm. A non-linear equilibrium model is described which incorporates electrophoretic mobility, hydrodynamic flow velocity, and an electrically induced concentration polarization at the surface of the stationary phase. This model is able to provide useful estimates of protein retention time and velocity in a column packed with Sephadex gel and subjected to an electric field. A correlation of electrophoretic mobility of peptide and proteins with respect to their charge, molecular mass, and asymmetry enables the selection of solute target molecules for electrochromatographic separations. Good separation of protein mixtures have been obtained.

Recombinant human insulin.

Insulin is a well-characterized peptide that can be produced by recombinant DNA technology for human therapeutic use. A brief overview of insulin production from both traditional mammalian pancreatic extraction and recombinant bacterial and yeast systems is presented, and detection techniques, including electrophoresis, are reviewed. Analytical systems for insulin separation are principally based on reversed-phase chromatography, which resolves the deamidation product(s) (desamido insulin) of insulin, proinsulin, and insulin. Process-scale separation is a multistep process and includes ion exchange, reversed-phase, and size exclusion chromatography. Advantages and/or disadvantages of various separation approaches, as described by the numerous literature references on insulin purification, are presented.

Simulation of diauxic production of cephalosporin C by Cephalosporium acremonium: lag model for fed-batch fermentation.

We extend a previously reported model (Chu, W.B.; Constantinides, A. Biotechnol. Bioeng. 1988, 32, 277-288) for the batch fermentation of cephalosporin C under the diauxic growth of Cephalosporium acremonium on glucose and sucrose to a fed-batch system. For this purpose, a novel lag model is proposed for diauxie, which has two functional forms, each embodying the dependence of lag on total cell mass and secondary substrate concentration. This lag model is applicable for batch simulations for arbitrary initial glucose and sucrose concentrations. We used the previously reported batch data to perform locally optimized fed-batch simulations. When applied to fed-batch fermentations, multiple lag times were accounted for. These studies showed that fed-batch fermentations (under the restriction that cell mass concentration did not exceed 25 g/L) could be more productive than simple batch runs. A representative result for a glucose-pulse fed-batch run at optimal cephalosporin production is a productivity of 4.22 mg of cephalosporin C/(L.h) and a yield of 9.25 mg of cephalosporin C/g of total sugar used.

Characterization of the swelling of a size-exclusion gel.

The swelling of a dextran gel, Sephadex G-75, was observed in an aqueous environment at room temperature by a noninvasive technique that uses light microscopy coupled to an image analysis system via a video camera. The rate of swelling was found to follow the Tanaka and Fillmore theory, from which the overall gel diffusion coefficient was estimated as 6.3 x 10(-7) cm2/s. In addition to giving a quantitative measure of gel swelling that could be useful in the mechanical design of liquid chromatography columns, this approach provides data on wet particle size and particle size range, which is needed for the modeling of diffusional and mass transfer effects in size-exclusion chromatography. In this context, key observations are that the gel particles are nearly spherical with an elliptical shape factor of 0.98 (perfect sphere = 1) and that there is little difference between sizes of particles obtained in water, 50 mM Tris-glycine buffer (pH 10.2), and buffer containing 1 mg/mL protein. The diameter of the dry material ranged from 20 to 100 microns, while the hydrated particles had diameters of 40-350 microns. The rate of swelling is rapid, with 50% swelling occurring in about 10 s and swelling to 99% of the final wet particle size being obtained in less than 90 s.

Characterization of dicarboxylic acids for cellulose hydrolysis.

In this paper, we show that dilute maleic acid, a dicarboxylic acid, hydrolyzes cellobiose, the repeat unit of cellulose, and the microcrystalline cellulose Avicel as effectively as dilute sulfuric acid but with minimal glucose degradation. Maleic acid, superior to other carboxylic acids reported in this paper, gives higher yields of glucose that is more easily fermented as a result of lower concentrations of degradation products. These results are especially significant because maleic acid, in the form of maleic anhydride, is widely available and produced in large quantities annually.

Chromatography for rapid buffer exchange and refolding of secretory leukocyte protease inhibitor.

A DEAE-cellulose stationary phase in a rolled configuration was used to separate recombinant secretory leukocyte protease inhibitor (rSLPI) from denaturants and reducing agents (3 M guanidine-HCl and 5 mM DTT) in less than 5 min to promote refolding of the protein to an active form. The mobile phase consisted of buffer and 500 mM NaCl, where NaCl suppressed binding of protein to this stationary phase. Separation of an initial concentration of 2 mg/mL protein from the other constituents resulted in 96% recovery of the rSLPI at an average concentration of 1.28 mg/mL. When incubated for 4 h at 20 degrees C, the fractionated rSLPI gave a 46% yield of properly refolded protein. The protein concentration was 6.4 times higher than that reported in a previously published method, where refolding was carried out by diluting the mixture of protein, denaturants, and reducing agents by a factor of 10. The results show that a combination of rapid chromatographic separation over a cellulosic stationary phase followed by protein refolding will significantly enhance process throughput by minimizing tankage, water requirements, and process time.

Analysis of plant harvest indices for bioregenerative life support systems.

Harvest indices, which are measures of the ratio of edible to total plant weight, are redefined to include edible sugars derived from enzymatic hydrolysis of the cellulose content of inedible plant components. Compositional analysis and carbohydrate contents of rapeseed, rice, soybeans, cowpea, wheat, sweet potato, white potato, and lettuce were analyzed to develop such generalized harvest indices. Cellulose conversion is shown to extend considerably the food available from plants otherwise grown for their oil and protein content in a bioregenerative life support system.

Cellulose pretreatments of lignocellulosic substrates.

Cellulose in inedible plant materials, forestry residues, and municipal wastes must be pretreated to disrupt its physical structure, thereby making its hydrolysis to glucose practical. Developments since 1991 are summarized.

Bioprocessing in space

NASA: Two approaches for biomass processing in Controlled Ecological Life Support Systems are compared in a literature survey. The approaches are based on (1) total oxidation of plant matter and (2) the potential of bioregenerative recovery.^ieng

Effect of pH on subunit association and heat protection of soybean alpha-galactosidase.

Soybeans contain the enzyme alpha-galactosidase, which hydrolyzes alpha-1, 6 linkages in stachyose and raffinose to give sucrose and galactose. We have found that galactose, a competitive product inhibitor of alpha-galactosidase, strongly promotes the heat stability of the tetrameric form of the enzyme at pH 4.0 and at temperatures of up to 70 degrees C for 60 min. Stachyose and raffinose also protect alpha-galactosidase from denaturation at pH 4.0 although to a lesser extent. Glucose and mannose have little effect. At pH 7.0 the enzyme is a monomer, and galactose has no effect on the heat stability of the enzyme. In the absence of heat protection of the enzyme by added sugars, a series deactivation mechanism was found to describe the deactivation data. In comparison, a unimolecular, non-first order deactivation model applies at pH 4.0, where heat protection effects were observed. At a temperature above 60 degrees C, simple deactivation is a suitable model. The results suggest that alpha-galactosidase conformation and heat stability are directly related.

Mechanism and potential applications of bio-ligninolytic systems in a CELSS.

A large amount of inedible plant material, generated as a result of plant growth in a Controlled Ecological Life Support System (CELSS), should be pretreated and converted into forms that can be recycled on earth as well as in space. The main portion of the inedible biomass is lignocellulosic material. Enzymatic hydrolysis of this cellulose would provide sugars for many other uses by recycling carbon, hydrogen, oxygen, and nitrogen through formation of carbon dioxide, heat, and sugars, which are potential foodstuffs. To obtain monosaccharides from cellulose, the protective effect of lignin should be removed. White-rot fungi degrade lignin more extensively and rapidly than other microorganisms. Pleurotus ostreatus degrades lignin effectively, and produces edible and flavorful mushrooms that increase the quality and nutritional value of the diet. This mushroom is also capable of metabolizing hemicellulose, thereby providing a food use of this pentose containing polysaccharide. This study presents the current knowledge of physiology and biochemistry of primary and secondary metabolisms of basidiomycetes, and degradation mechanism of lignin. A better understanding of the ligninolytic activity of white-rot fungi will impact the CELSS Program by providing insights on how edible fungi might be used to recycle the inedible portions of the crops.

Solid-state fermentation of lignocellulosic plant residues from Brassica napus by Pleurotus ostreatus.

Solid-state fermentation (SSF) of inedible parts of rapeseed was carried out using a white-rot fungus, Pleurotus ostreatus, to degrade lignocellulosic material for mycelial-single cell protein (SCP) production. This SSF system has the potential to be adapted to a controlled ecological life support system in space travel owing to the lack of storage space. The system for converting lignocellulosic material to SCP by P. ostreatus is simple; it can be carried out in a compact reactor. The fungal vegetative growth was better with a particle size of plant material ranging from 0.42 to 10 mm, whereas lignin degradation of the lignocellulose was the highest with particle sizes ranging from 0.42 to 0.84 mm. The addition of veratryl alcohol (3,4-dimethoxybenzyl alcohol), hydrogen peroxide, and glycerol promotes lignocellulose degradation by P. ostreatus. The enhancement of bioconversion was also observed when a gas-flow bioreactor was used to supply oxygen and to maintain the constant moisture of the reactor. With this reactor, approx 85% of the material was converted to fungal and other types of biomass after 60 d of incubation.

Enzyme conversion of lignocellulosic plant materials for resource recovery in a Controlled Ecological Life Support System.

A large amount of inedible plant material composed primarily of the carbohydrate materials cellulose, hemicellulose, and lignin is generated as a result of plant growth in a Controlled Ecological Life-Support System (CELSS). Cellulose is a linear homopolymer of glucose, which when properly processed will yield glucose, a valuable sugar because it can be added directly to human diets. Hemicellulose is a heteropolymer of hexoses and pentoses that can be treated to give a sugar mixture that is potentially a valuable fermentable carbon source. Such fermentations yield desirable supplements to the edible products from hydroponically-grown plants such as rapeseed, soybean, cowpea, or rice. Lignin is a three-dimensionally branched aromatic polymer, composed of phenyl propane units, which is susceptible to bioconversion through the growth of the white rot fungus, Pluerotus ostreatus. Processing conditions, that include both a hot water pretreatment and fungal growth and that lead to the facile conversion of plant polysaccharides to glucose, are presented.

Liquefaction of sugarcane bagasse for enzyme production.

The objective of this paper is to report liquefaction of pretreated and sterilized sugarcane bagasse for enhancing endoglucanase production through submerged fermentation by Aspergillus niger. After initial solid state fermentation of steam pretreated bagasse solids by A. niger, fed-batch addition of the substrate to cellulase in buffer over a 12h period, followed by 36h reaction, resulted in a liquid slurry with a viscosity of 0.30±0.07Pas at 30% (w/v) solids. Addition of A. niger for submerged fermentation of sterile liquefied bagasse at 23% w/v solids resulted in an enzyme titer of 2.5IUmL(-1) or about 15× higher productivity than solid-state fermentation of non-liquefied bagasse (final activity of 0.17IUmL(-1)). Bagasse not treated by initial solid-state fermentation but liquefied with enzyme gave 2IUmL(-1). These results show the utility of liquefied bagasse as a culture medium for enzyme production in submerged fermentations.

Effect of modulator sorption on gradient shape in ion-exchange chromatography.

Mobile phase additives, or modulators, are used in gradient elution chromatography to facilitate separation and reduce separation time. The modulators are usually assumed to be linearly adsorbed or unadsorbed. Here, the consequences of nonlinear modulator adsorption are examined for ion-exchange gradient elution through a series of simulations. Even when the buffer salt is identical to the modulator salt, gradient deformation is observed; the extent of deformation increases as the volume of the feed is increased. When the modulator salt is different from the buffer salt, unusual effects are observed, and the chromatograms are quite different from those predicted by classical gradient elution theory. In particular, local increases in the buffer concentration are found between feed bands, and serve to improve the separation. These effects become more pronounced as the feed volume increases, and could therefore prove valuable in preparative applications.

Correlation of electrophoretic mobilities of proteins and peptides with their physicochemical properties.

Electrophoretic mobilities, mu, of nine proteins (M(r) 14,200 to 70,000) in 28 mM Tris/47 mM glycine buffer at pH 8.77 and 5 mM ionic strength were measured by laser Doppler velocimetry and correlated to ratios of charge (q) to molecular weight (M(r)) and shape factor (f/f0) by the equation mu(f/f0) = (Aq/Mpr-B). This correlation was previously reported for peptides and proteins for mu measured at 100 mM ionic strength. When A = 6.048 x 10(-3), B = 1.13 x 10(-5), and p = 2/3, the correlation fitted 51 measured and literature values over the molecular weight range of 178 to 140,000 for components whose electrophoretic mobilities ranged from +13.35 x 10(-5) to -19.7 x 10(-5) cm2/(V.s). The experimental measurements confirm the general suitability of p = 2/3 and show that the familiar charge/mass relation for electrophoresis is applicable to proteins in low-ionic-strength buffers which are typical of electrochromatography systems. Extrapolation of the correlation to different ionic strengths indicates that a low-ionic-strength buffer amplifies differences of electrophoretic mobility as a function of charge/mass, while high ionic strength diminishes such differences.