R. Mark Worden

Reactor Design Issues for Synthesis‐Gas Fermentations

Synthesis gas is readily obtained by gasifying coal, oil, biomass, or waste organics and represents an abundant, potentially inexpensive, feedstock for bioprocessing. The primary components of synthesis gas, carbon monoxide and hydrogen, can be converted into methane, organic acids, and alcohols via anaerobic fermentations. Bioconversion of synthesis gas is an attractive alternative to catalytic processing because the biological catalysts are highly specific and often more tolerant of sulfur contaminants than inorganic catalysts. However, because the aqueous solubilities of carbon monoxide and hydrogen are low, synthesis‐gas fermentations are typically limited by the rate of gas‐to‐liquid mass transfer. Consequently, a major engineering challenge in commercial development of synthesis‐gas fermentations is to provide sufficient gas mass transfer in an energy‐efficient manner. This paper reviews recent progress in the development of synthesis‐gas fermentations, with emphasis on efforts to increase the efficiency of gas mass transfer. Metabolic properties of several microbes able to ferment synthesis gas are described. Results of synthesis‐gas fermentations conducted in various bioreactor configurations are summarized. Recent results showing enhancement of synthesis‐gas fermentations using microbubble dispersions are presented, and studies of the mass‐transfer and coalescence properties of microbubbles are described.

Mass-Transfer Properties of Microbubbles. 1. Experimental Studies

Synthesis‐gas fermentations have typically been gas‐to‐liquid mass‐transfer‐limited due to low solubilities of the gaseous substrates. A potential method to enhance mass‐transfer rates is to sparge with microbubble dispersions. Mass‐transfer coefficients for microbubble dispersions were measured in a bubble column. Oxygen microbubbles were formed in a dilute Tween 20 solution using a spinning disk apparatus. Axial dispersion coefficients measured for the bubble column ranged from 1.5 to 7.2 cm2/s and were essentially independent of flow rate. A laser‐diffraction technique was used to determine the interfacial area per unit gas volume, a. The mass‐transfer coefficient, KL, was determined by fitting a plug‐flow model to the experimental, steady‐state, liquid‐phase oxygen‐concentration profile. The KL values ranged from 2.9 × 10−5 to 2.2 × 10−4 m/s. Volumetric mass‐transfer coefficients, KLa, for microbubbles with an average initial diameter of 60 μm ranged from 200 to 1800 h−1. Enhancement of mass transfer using microbubbles was demonstrated for a synthesis‐gas fermentation. Butyribacterium methylotrophicum was grown in a continuous, stirred‐tank reactor using a tangential filter for total cell recycle. The fermentation KLa values were 14 h−1 for conventional gas sparging through a stainless steel frit and 91 h−1 for microbubble sparging. The Power number of the microbubble generator was determined to be 0.036. Using this value, an incremental power‐to‐volume ratio to produce microbubbles for a B. methylotrophicum fermentation was estimated to be 0.01 kW/m3 of fermentation capacity.

Evidence for Production of n-Butanol from Carbon Monoxide by Butyribacterium methylotrophicum

Abstract Biological conversion of synthesis gas, primarily a mixture of carbon monoxide (CO) and hydrogen gases, is a potential alternative to chemical processing for production of fuels and chemicals. In addition to acetate and butyrate as metabolic end products, Butyribacterium methylotrophicum has now exhibited n-butanol production directly from CO gas. A butanol concentration as high as 2.7 g/l has been achieved using the CO strain of this organism. These findings suggest the existence of a unique metabolic pathway for butanol production from CO in this strain.

A Clark-type oxidase enzyme-based amperometric microbiosensor for sensing glucose, galactose, or choline

Abstract Microbiosensors for glucose, galactose, or choline were constructed by attaching the respective oxidase enzyme to the tip of a Clark-type oxygen microelectrode. The enzyme is immobilized on the electrode tip in a polyacrylamide matrix and then coated with a polyurethane membrane. The analyte concentration in the sample controls the amount of oxygen consumed by the electrode and, hence, the biosensors output. These microbiosensors had tip diameters of 15–40 μm, response times of 0.5–5s, and could detect as little as 2 μM of analyte. The linear range of response was dependent on the thickness of the polyurethane coating, and extended up to 10 mM for glucose and galactose biosensors. The glucose sensors were the most stable and could remain operational for up to 6 months. Galactose sensors remained operational for at least 1 month. Choline sensors remained operational for about 2 weeks and were generally less sensitive. The specific activity of the enzyme was a key determinant in the longevity and linearity of the biosensor response. In continuous operation tests, the glucose sensors were relatively drift-free and showed little deterioration of response over 72 h. These sensors exhibited little stirring dependence. As a result a glucose sensor accurately measured the glucose gradient in an unmixed, semisolid gel. These microsensors should prove to be versatile tools for measuring specific analytes in unstirred environments with a spatial resolution of 100 μm or less, and with extremely rapid response times.

Ethanol production by immobilized cells of Zymomonas mobilis

SummaryColumnar reactors containing immobilized cells of Zymomonas mobilis were utilized for the continuous production of ethanol from glucose. Two different immobilization strategies were investigated. In one case, cells were entrapped in borosilicate glass fiber pads, while in the other, cells were immobilized via flocculation. The reactors were operated in both the fixed-bed and expanded-bed manner. Ethanol productivities as high as 132 g/l·h were achieved. Data obtained from studies employing 5.0 and 10.0% glucose concentrations are presented. Problems encountered during the operation of the continuous, immobilized cell reactors are discussed.

Enhancement of mass transfer using colloidal liquid aphrons: measurement of mass transfer coefficients in liquid-liquid extraction.

Interphase mass transfer of a sparingly soluble solute is often the rate-limiting step in multiphase biocatalytic processes. Colloidal liquid aphrons (CLA) provide very large interfacial areas, and thus could enhance mass transfer in such processes. The aim of this study was to characterize mass transfer properties of CLA dispersions during transfer of heptanoic acid from water to limonene. The interfacial area per unit volume (a), film mass transfer coefficient (K(L)), and volumetric mass transfer coefficient (K(L)a) values were determined in a stirred-tank reactor. These results were used, along with a literature correlation, to estimate the mass transfer coefficient of the surfactant-stabilized shell surrounding the CLA. The very large increase in a provided by the CLA was only partially offset by a slight increase in the mass transfer resistance of the shell. As a result, the range of K(L)a values obtained using CLA was about an order of magnitude greater than that obtained using a conventional dispersion. The concentration of the aqueous-phase surfactant used to form the CLA strongly affected the Sauter mean diameter of the CLA; however, the concentration of the nonpolar-phase surfactant had little effect. These results suggest that CLA have considerable potential for multiphase biocatalytic applications.

Functional characterization of PorB class II porin from Neisseria meningitidis using a tethered bilayer lipid membrane

PorB class II from Neisseria meningitidis is a pore-forming, outer-membrane protein that can translocate to the host-cell membrane during Neisserial infections. This report describes development of tethered bilayer lipid membrane (tBLM) system to measure PorB conductance properties. The tBLM was fabricated by depositing a self-assembled monolayer of 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol (DPPTE) tethering lipid on a gold electrode and then using 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes to deposit the upper tBLM leaflet. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were used to monitor tBLM formation and subsequent PorB incorporation. The highly insulating tBLM exhibited a membrane resistance and capacitance of 2.5MOmegacm(2) and 0.7 microF/cm(2), respectively. PorB was incorporated into the tBLM in an active conformation, as evidenced by its mediation of ion passage and the decrease in membrane impedance. After PorB incorporation, the membrane resistance decreased to 0.6MOmegacm(2). As expected, the PorB channel was found to be non-selective, allowing the transport of both cations and anions. Cyclic voltammetry indicated that ferricyanide ions can also pass through the pores. The PorB-containing biomimetic interface developed in this study could potentially be used to screen for compounds that modulate PorB activity.

Fed-batch fermentor synthesis of 3-dehydroshikimic acid using recombinant Escherichia coli

3-Dehydroshikimic acid (DHS), in addition to being a potent antioxidant, is the key hydroaromatic intermediate in the biocatalytic conversion of glucose into aromatic bioproducts and a variety of industrial chemicals. Microbial synthesis of DHS, like other intermediates in the common pathway of aromatic amino acid biosynthesis, has previously been examined only under shake flask conditions. In this account, synthesis of DHS using recombinant Escherichia coli constructs is examined in a fed-batch fermentor where glucose availability, oxygenation levels, and solution pH are controlled. DHS yields and titers are also determined by the activity of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) synthase. This enzymes expression levels, sensitivity to feedback inhibition, and the availability of its substrates, phosphoenolpyruvate (PEP) and D-erythrose 4-phosphate (E4P), dictate its in vivo activity. By combining fed-batch fermentor control with amplified expression of a feedback-insensitive isozyme of DAHP synthase and amplified expression of transketolase, DHS titers of 69 g/L were synthesized in 30% yield (mol/mol) from D-glucose. Significant concentrations of 3-dehydroquinic acid (6.8 g/L) and gallic acid (6.6 g/L) were synthesized in addition to DHS. The pronounced impact of transketolase overexpression, which increases E4P availability, on DHS titers and yields indicates that PEP availability is not a limiting factor under the fed-batch fermentor conditions employed.

Fabrication of highly insulating tethered bilayer lipid membrane using yeast cell membrane fractions for measuring ion channel activity.

A tethered bilayer lipid membrane (tBLM) was fabricated on a gold electrode using 1,2-dipalmitoyl-sn-glycero-phosphothioethanol as a tethering lipid and the membrane fractions of Saccharomyces pombe yeast cells to deposit the upper leaflet. The membrane fractions were characterized using transmission electron microscopy and dynamic light scattering and found to be similar in size to small unilamellar vesicles of synthetic lipids. The dynamics of membrane-fraction deposition and rupture on the tethering-lipid layer were measured using quartz crystal microgravimetry. The electrochemical properties of the resulting tBLM were characterized using electrical impedance spectroscopy and cyclic voltammetry. The tBLMs electrical resistance was greater than 1 MOmegacm(2), suggesting a defect-free membrane. The suitability of tBLM produced using membrane fractions for measuring ion-channel activities was shown by a decrease in membrane resistance from 1.6 to 0.43 MOmegacm(2) following addition of gramicidin. The use of membrane fractions to form high-quality tBLM on gold electrodes suggests a new approach to characterize membrane proteins, in which the upper leaflet of the tBLM is deposited, and overexpressed membrane proteins are incorporated, in a single step. This approach would be especially useful for proteins whose activity is lost or altered during extraction, purification, and reconstitution, or whose activities are strongly influenced by the lipid composition of the bilayer.

Modeling microbial chemotaxis in a diffusion gradient chamber

The diffusion gradient chamber (DGC) has proven to be a useful experimental tool for studying population-level microbial growth and chemotaxis. A mathematical model capable of reproducing the population-level patterns formed as a result of cellular growth and chemotaxis in the DGC has been developed. The model consists of coupled partial differential balance equations for cells, chemoattractants, and a nutrient, which are solved simultaneously by the alternating direction implicit method. Modeling simulation results were compared with population-level migration patterns of Escherichia coli growing on glycerol and responding to a gradient of the chemoattractant aspartate for two different initial conditions. To accurately reproduce the experimental results, a second chemoattractant equation was necessary. The second chemoattractant has been identified as oxygen by directly measuring oxygen gradients in the DGC. Important trends observed experimentally and reproduced by the model include the formation of a chemotactic wave, a reduction in the wave velocity as it encounters higher chemoattractant concentrations, and chemotaxis in response to two different chemoattractants simultaneously. The model was also used to study the relative magnitude of cell fluxes due to random motility and chemotaxis, and the suppression of chemotaxis due to receptor saturation.