Gas holdup in a three-phase fluidized-bed bioreactor

The effects of system parameters on the gas holdup in a three-phase fluidized-bed bioreactor were examined. A valve technique was used to measure the average gas holdup in the reactor. Gas holdup was found to be strongly affected by fluid superficial velocities, bead size and density, electrolyte concentration, type of gas sparger, and temperature. Quantitative and qualitative observations were made regarding the differences between conventional glass-particle systems and low-density gel-particle systems. Most notably, the degree of bubble coalescence and mode of fluidization were highly dependent on each system.     

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.     

Dispersion and holdup in a three-phase fluidized-bed bioreactor

Axial dispersion and phase holdup measurements were made using electroconductivity in a fermenting fluidized-bed bioreactor (FBR) and in a model nonfermenting three-phase FBR. Multiple axial conductivity probes were used to nonintrusively monitor the bed conductivity. The gas phase holdup was estimated from a ratio of the average bed conductivity and bulk conductivity. The solid fraction in the three-phase FBR can be estimated from the two-phase liquid-solid FBR. The response to a salt pulse was used to estimate the liquid axial dispersion coefficient. Particle Peclet numbers on the order of 10^-2 were estimated as a function of flowrates and compared to literature correlations.     

Technical and economic evaluation of different reactors for methanotrophic cultures for propylene oxide production

A two-stage process for the manufacture of propylene oxide is described. The preliminary economics based on use of methanol as a regeneration factor has resulted in a production cost of $12.10/lb of propylene oxide based on propylene oxide production rate of 40 mg/g-cell/h in conventional reactor. Increasing the propylene oxide production from 40 to 500 mg/g-cell/h resulted in a cost reduction from $12.10 to 5.8/lb of propylene oxide. The granular-activated, carbon-fluidized bed reactor (GAC-FBR) absorbs the propylene oxide and when saturated is eluted with ethyl acetate, and the bed is regenerated by steam to drive off the residual solvents. The estimated manufacturing costs are approx 59% lower (from $12.10/lb in conventional reactors to $5.00/lb for GAC-FBRs) for products that are highly inhibitory such as epoxides. In the GAC-FBR reactor, enhancing the propylene oxide production rate from 120 to 1500 mg/g-cell/h has resulted in the cost reduction to $2.00/lb. Enhancing the production capacity from 1 million lb to 10 million lb/yr has further reduced the cost of production to $1.00/lb.     

Novel hollow fiber immobilization techniques for whole cells and advanced bioreactors

A novel microporous hollow fiber membrane-based immobilization technique for whole cells has been developed. Yeast cells (Saccharomyces cerevisiae) were grown on chopped hydrophobic microporous hollow fibers as well as on hydrophilic hollow fibers. This immobilization support was used to carry out fermentation in a tubular bioreactor. Air was passed from time to time to facilitate cell growth. The microbial culture reached a very high cell density level of around 10¹⁰/mL of fiber lumen volume. An ethanol concentration of 45 g/L and productivity of 41 g/L-h were obtained with an initial glucose concentration of 100 g/L. The present technique does not have the shortcomings of conventional immobilization methods.     

Heaps as bioreactors for coal bioprocessing

The long time required for the microbial removal of pyritic sulfur from coal does not become an economic issue if the coal is treated in heaps over which bacteria and nutrients are trickled. Correct heap design is dictated by the variation of the inherent process rate with coal particle size, the ability of water to trickle through heaps of fine coal without waterlogging owing to capillary forces, and the ability of oxygen and carbon dioxide to diffuse into the heap. A theoretical analysis of oxygen penetration combined with experimental measurements of water holdup suggests an optimum coal particle size in the range 6×30 mesh. This is confirmed by experiments on 4 in×3 ft heaps of Illinois #6 coal ground to 30×100 and 12×30 mesh. The former plugs with biomass and ferric precipitates, whereas the latter shows good depyritization rate with minimal gradients through the heap.     

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.     

Gas Hydrate Formation in Reversed Micelles

We describe a technique to modify protein solubility and optimize enzyme activity in reversed micellar solutions. The technique is based on the ability of hydrates of natural gas to form in the micro-aqueous phase. Clathrate hydrates are crystalline inclusions of water and gas, and their formation in bulk water has traditionally been studied with relevance to natural gas recovery. We have found that hydrates can form in the environment of the microaqueous pools of reversed micelles, and that their extent of formation can be well controlled through the thermodynamic variables of temperature and pressure. Additionally, formation of hydrates affects the size and aggregation number of the micelles, and thus influences the solubility and conformation of encapsulated proteins. We demonstrate how the concept can be used in two applications: (i) protein extraction into reversed micelles and subsequent recovery, and (ii) optimization of enzyme activity in reversed micelles.     

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.     

Axial dispersion in a liquid fluidized bed of particles akin to immobilized enzymes

The axial dispersion of a liquid fluidized bed of controlled pore silica (CPS) particles has been determined by the pulse tracer method. The CPS used was the same as for enzyme immobilization, having an average diameter of 0.436 mm and mean pore size of 37.5 nm. The fluidization liquid is α-amylase liquefied manioc starch, 30% w/v, 45°C pH=4.5. Nominal bed porosities tested were 0.7 and 0.8. The results show that the axial dispersion coefficient increases with greater superficial liquid velocities. Various available correlations tested disagree with each other to a large extent and are unable to represent collected experimental data.     

Expanded-bed adsorption utilizing ion-exchange resin to purify extracellular β-galactosidase

The application of expanded-bed ion-exchange resins allows the elimination of intermediary particulate separation steps like filtration or centrifugation prior to adsorption steps in enzyme-purification processes from crude fermentation broths. This work is concerned with the experimental evaluation data of a process related to the adsorption of an extracellular p-galactosidase from the fungiScopulariopsis. The protein recovery in the ion-exchange resin Accell Plus QMA™ was accomplished using a continuous-monitoring method. The direct adsorption step was followed by a elution step with concentrated NaCl solutions aiming to improve the enzyme-specific activity. Experimental data for fixed and expanded bed were compared     

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.     

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.     

Mode of action and synergism of cellulases fromPenicillium funiculosum

A 1,4-β-d-glucan cellobiohydrolase (EC 3.2.1.91) and l,4-β-d-glucan glucanohydrolase (EC 3.2.1.4) were purified from the culture filtrates ofPenicillium funiculosum by using preparative isoelectric focusing. Both the enzymes were homogeneous on polyacrylamide gel with and without sodium dodecyl sulphate. The mol wt of the cellobiohydrolase and endoglucanase were 14,400 and 25,000 respectively. The purified enzymes were free of β-glucosidase activity. Acting in isolation, the cellobiohydrolase had little capacity for solubilizing Avicel or Walseth cellulose, but showed increased rates of hydrolysis when combined with endoglucanase. Cellobiose inhibition (50%) was observed in the initial rate of the hydrolysis of Walseth cellulose. It was also observed that cellobiohydrolase initiates the attack on crystalline cellulose. † NCL communication no. 3898.     

Ethanol from lignocellulosic wastes with utilization of recombinant bacteria

This article presents the advanced technology that has been developed by BioEnergy International of Gainesville, Florida, utilizing novel recombinant strains of bacteria developed by Lonnie Ingram of the University of Florida. The first commercial applications of these unique fermenting organisms convert 5-carbon sugars, as well as 6-carbon sugars, and oligomers of cellulose (e.g., cellobiose and cellotriose) directly to ethanol. The proposed systems that will be utilized for conversion of agricultural wastes, mixed waste papers, and pulp and paper mill waste in forthcoming commercial installations are now under design. This involves the extensive experience of Raphael Katzen Associates International, Inc. in acid hydrolysis, enzyme production, enzymatic hydrolysis, large-scale fermentation engineering, and distillation/dehydration. Specific examples of this advanced technology will be presented in different applications, namely:

Growth inhibition in animal cell culture

Eight independent cell lines accumulated ammonia in culture to concentrations between 1.3 and 2.9 mM. The growth inhibition of such concentrations of ammonium chloride when added to culture medium was variable. The cell lines tested could be divided into 3 groups depending on their growth response to 2 mM added NH4Cl. In the first group (293, HDF, Vero, and PQXB1/2) little (less than 14%) or no growth inhibition occurred. In the second group (McCoy and MDCK) a reduction in final cell yield of 50-60% was observed. The third group (HeLa and BHK) was most sensitive to the effects of NH4Cl with growth inhibition (greater than 75%) compared to controls. The growth inhibitory effect of added lactate up to 20 mM was negligible (less than 10%) for 3 cell lines, although one cell line (PQXB1/2) showed greater sensitivity. The interactive effects of ammonia and lactate were determined in a matrix experiment. At lactate (greater than 12 mM) and ammonia (1-4 mM), the growth inhibitory effects of the two components were synergistic. However, at low concentrations of lactate (less than 12 mM) the toxic effect of ammonia was reduced. A proposed mechanism for the sparing effect of lactate on ammonia toxicity is discussed. This may have importance in developing strategies for the optimal growth of ammonia-sensitive cell lines.     

Preparation of Tyr-C-peptide from genetically altered human insulin precursor

C-peptide radioimmunoassay (C-peptide RIA) is widely used in determination of pancreatic B-cell secretion activity.¹²⁵I labeled TyrC-peptide is indispensable in C-peptide RIA kit. Herein we discuss a way of obtaining recombinant Tyr-C-peptide. Arg32Tyr human proinsulin mutant (R32Y-proinsulin) gene was constructed by site-directed mutagenesis and overexpressed inEscherichia coli. Purified R32Y-proinsulin was converted to insulin and Tyr-C-peptide by trypsin and carboxypeptidase B codigestion. Tyr-C-peptide was isolated through reverse-phase HPLC (RP-HPLC) and identified by C-peptide RIA and amino acid analysis.