Randy S. Lewis

Formation of ethanol from carbon monoxide via a new microbial catalyst

Abstract A recently discovered clostridial bacteria converts components of synthesis gas (CO, CO 2 , H 2 ) into liquid products such as ethanol, butanol and acetic acid. Isolated from an agricultural lagoon, the stability and productivity characteristics of the bacteria were studied in a continuous 4.5 l bubble column bioreactor at 37°C using artificial blends of CO, CO 2 , and N 2 . Preliminary results on the rates of cell growth, substrate utilization, product formation, and yields of products and cells from CO are discussed. At steady state, apparent yields (mole C in products per mole CO consumed) of ethanol, butanol, and acetic acid were 0.15, 0.075 and 0.025, respectively, and the cell yield was 0.25 g / mol CO. The theoretical yield of ethanol is 0.33 if CO is only utilized for the production of ethanol. The experimental yield of CO 2 from CO was approximately 60% compared to the theoretical yield of 67% with ethanol as the sole product. As a comparison with another ethanol-producing bacteria, the results of a similar fermentation study using batch-grown Clostridium ljungdahlii showed yields of 0.062 for ethanol and 0.094 for acetic acid and a cell yield of 1.378 g / mol .

Improved haemocompatibility of cysteine-modified polymers via endogenous nitric oxide.

A novel method for improving the haemocompatibility of biomedical materials through endogenous nitric oxide (NO) is presented. L-cysteine was covalently immobilized onto two biomedical polymers: polyurethane (PU) and polyethylene terephthalate (PET). The L-cysteine content on the polymers was approximately 5-8 nmol/cm2 as quantified via a chemiluminescence-based assay. The haemocompatibility of the modified polymers was evaluated in terms of the number of adhered platelets when exposed to a platelet suspension labeled with Cr51. Platelet adherence on the L-cysteine-modified polymers was reduced more than 50% as compared to the control (glycine-modified polymers) when the platelet suspension contained plasma constituents. No difference in platelet adhesion was observed in the absence of plasma constituents. Further experiments demonstrated that NO was easily transferred to the L-cysteine-modified polymers from S-nitroso-albumin in PBS buffer. The NO was then released from the polymer. NO transfer or release was not observed for the control. The results suggest that L-cysteine-modified polymers are effective in reducing platelet adhesion via the transfer of NO from endogenous S-nitrosoproteins in plasma to the polymer followed by the subsequent release of NO. Thus, exploiting endogenous NO is a viable option for improving the haemocompatibility of biomaterials.

A comparison of mass transfer coefficients between trickle-bed, hollow fiber membrane and stirred tank reactors.

Trickle-bed reactor (TBR), hollow fiber membrane reactor (HFR) and stirred tank reactor (STR) can be used in fermentation of sparingly soluble gasses such as CO and H2 to produce biofuels and bio-based chemicals. Gas fermenting reactors must provide high mass transfer capabilities that match the kinetic requirements of the microorganisms used. The present study compared the volumetric mass transfer coefficient (K(tot)A/V(L)) of three reactor types; the TBR with 3 mm and 6 mm beads, five different modules of HFRs, and the STR. The analysis was performed using O2 as the gaseous mass transfer agent. The non-porous polydimethylsiloxane (PDMS) HFR provided the highest K(tot)A/V(L) (1062 h(-1)), followed by the TBR with 6mm beads (421 h(-1)), and then the STR (114 h(-1)). The mass transfer characteristics in each reactor were affected by agitation speed, and gas and liquid flow rates. Furthermore, issues regarding the comparison of mass transfer coefficients are discussed.

A thermodynamic analysis of electron production during syngas fermentation.

Currently, syngas fermentation is being developed as one option towards the production of biofuels from biomass. This process utilizes the acetyl-CoA (Wood-Ljungdahl) metabolic pathway. Along the pathway, CO and CO(2) are used as carbon sources. Electrons required for the metabolic process are generated from H(2) and/or from CO. This study showed that electron production from CO is always more thermodynamically favorable compared to electron production from H(2) and this finding is independent of pH, ionic strength, gas partial pressure, and electron carrier pairs. Additionally, electron production from H(2) may be thermodynamically unfavorable in some experimental conditions. Thus, it is unlikely that H(2) can be utilized for electron production in favor of CO when both species are present. Therefore, CO conversion efficiency will be sacrificed during syngas fermentation since some of the CO will provide electrons at the expense of product and cell mass formation.

Ethanol production during semi-continuous syngas fermentation in a trickle bed reactor using Clostridium ragsdalei.

An efficient syngas fermentation bioreactor provides a mass transfer capability that matches the intrinsic kinetics of the microorganism to obtain high gas conversion efficiency and productivity. In this study, mass transfer and gas utilization efficiencies of a trickle bed reactor during syngas fermentation by Clostridium ragsdalei were evaluated at various gas and liquid flow rates. Fermentations were performed using a syngas mixture of 38% CO, 28.5% CO2, 28.5% H2 and 5% N2, by volume. Results showed that increasing the gas flow rate from 2.3 to 4.6sccm increased the CO uptake rate by 76% and decreased the H2 uptake rate by 51% up to Run R6. Biofilm formation after R6 increased cells activity with over threefold increase in H2 uptake rate. At 1662h, the final ethanol and acetic acid concentrations were 5.7 and 12.3g/L, respectively, at 200ml/min of liquid flow rate and 4.6sccm gas flow rate.

Analysis of functionalized polyethylene terephthalate with immobilized NTPDase and cysteine.

Polyethylene terephthalate (PET) was functionalized to introduce carboxyl groups onto its surface by a carboxylation technique. Surface and bulk properties, such as possible surface deterioration, surface roughness and the mechanical strength of the carboxylated polymers, were studied and compared with those of aminolyzed and hydrolyzed PET. Atomic force microscopy studies showed that unlike aminolysis and hydrolysis, which increased the surface roughness significantly due to cracking and pitting, the surface roughness of unmodified and carboxylated PET were comparable. While hydrolysis and aminolysis of PET resulted in significant loss of strength, tensile testing revealed that unmodified and carboxylated polymers had similar strength. The development of mechanically stable, functionalized PET would vastly improve the biomedical applications of this polymer. To understand the potential for improving biomedical applications, biologically active molecules, namely nucleoside triphosphate diphosphohydrolase (NTPDase) and cysteine, were immobilized on the carboxylated PET using amide bonds. NTPDase was also immobilized to aminolyzed PET using imine bonds, while cysteine was immobilized on aminolyzed PET using both imine and amide bonds. Attachment of NTPDase and cysteine was verified by analyzing the NTPDase activity and the cysteine surface concentration. The stability of these immobilizations was also studied.

Design of a Novel Apparatus to Study Nitric Oxide (NO) Inhibition of Platelet Adhesion

AbstractNitric oxide (NO) is a simple biological molecule which inhibits adhesion and aggregation of platelets. A novel NO delivery device has been developed to quantitatively study the effects of NO concentration and flux on the adhesion of platelets to a surface. The slit-flow device is lined with a protein-coated membrane through which NO gas permeates into a perfusing platelet suspension. A model predicting spatial NO concentrations and fluxes within the flow slit was validated. At a wall shear rate of 250s-1, platelet adhesion was inhibited 87% relative to controls for exposures as low as 0.1 ppm NO. Corresponding model predictions of the aqueous NO concentration and fluxes at the surface were 0.15 nM, and between 0.5 and 1.1 nanomoles cm-2 s-1, respectively. Endo-thelial cells, which release NO to inhibit platelet adhesion in vivo, generate NO at an estimated flux similar to the above values. At a NO exposure of 0.02 ppm, platelet inhibition was only 10%. The delivery device is useful for other studies in which a knowledge of the spatial NO fluxes or concentrations is desired. Knowledge of these fluxes or concentrations is beneficial towards the design of biomaterials incorporating NO to inhibit platelet adhesion.

Effects of nitric oxide (NO) and soluble nucleoside triphosphate diphosphohydrolase (NTPDase) on inhibition of platelet deposition in vitro.

Vascular thrombosis is regulated via the release of several constituents from the vascular endothelium, including nucleoside triphosphate diphosphohydrolases (NTPDases or ectonucleotidases), nitric oxide (NO), and eicosanoids. Currently, it is unknown how these constituents interact in the inhibition of platelet aggregation and adhesion. To investigate the combined effects of NO and NTPDase on platelet deposition sequestration, an in vitro study was performed to compare inhibition of platelet deposition to a biomaterial by NO in the absence or presence of soluble NTPDase. Results of the platelet inhibition studies with NO and NTPDase conclusively show that the inhibitory effects of NTPDase and NO are additive. The platelet inhibitory potency in the presence of NO was enhanced by NTPDase in a dose-dependent manner, for a given NO exposure. This augmentation is independent of aspirin; the ability of NTPDase or NO alone to inhibit platelet deposition is also independent of aspirin. Clearly, NO and NTPDase independently contribute to platelet inhibition via different mechanisms. The inaction of NO on the activity of NTPDase confirmed that NO or reaction products in the presence of O(2) do not interact with NTPDase directly.

Nitric oxide, superoxide, and peroxynitrite effects on the insulin secretion and viability of βTC3 cells

AbstractThe onset of insulin-dependent diabetes mellitus (IDDM) is often associated with the infiltration of pancreatic cells by macrophages. Upon activation, macrophages release nitric oxide (NO) and superoxide (O2-). These species or their reactive intermediates can be cytoxic, mutagenic, or carcinogenic. Previous studies have reported both positive and negative effects of extracellularly generated NO on insulin secretion and viability of pancreatic cells. Inherent problems of several previous studies assessing the effects of NO on insulin secretion include unsteady state NO concentration exposures and the generation of other potentially damaging species. In this study, these problems were eliminated by using a modified experimental system in which NO delivery was achieved via diffusion across a gas-permeable tube and O2- delivery was maintained using an enzymatic reaction. The delivery rates were constant, leading to steady state concentrations of NO and O2- in the experimental system. Based on reaction kinetics, a model was developed to predict NO, O2- and peroxynitrite ONOO- concentrations during the experiment. This study showed that NO, O2- and ONOO- at predicted concentrations as high as 2.8 μM, 0.25 μM, and 0.1 nM, respectively, do not affect the insulin secretion rates of βTC3 pancreatic cells over short times.

Free radical profiles in an encapsulated pancreatic cell matrix model.

AbstractThe survival of encapsulated pancreatic cells or islets is often limited because of nutrient deficiency, fibrotic overgrowth, and immune attack. Activated immune cells, such as macrophages, release nitric oxide (NO) and superoxide O2- These species or their reactive intermediates, such as peroxynitrite, can be cytotoxic, mutagenic, and/or carcinogenic. The transport of these free radicals to encapsulated pancreatic cells cannot be impeded by the present immunoisolation technology. A model has been developed simulating free radical profiles within an encapsulation matrix due to macrophage immune cells attached to the surface of an encapsulation matrix. The model incorporates the transport and reactions of NO,O2- O2 and total peroxynitrite (PER). The model predictions of NO, O2- and PER concentrations to which pancreatic cells are potentially exposed are in the range of 8–42 μM, 0.5–8 nM, and 0.1–0.8 μM, respectively, for a 100–500 μm radius encapsulation matrix. The results demonstrate that the potential exists for free radical damage of encapsulated pancreatic cells and also demonstrates that additional exposure studies may be necessary for assessing free radical effects on pancreatic cell function. Also, care must be taken in assuming that encapsulated cell systems are completely protected from immunological action.