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dc.rights.licenseRestricted to current Rensselaer faculty, staff and students. Access inquiries may be directed to the Rensselaer Libraries.
dc.contributorBelfort, Georges
dc.contributorCollins, Cynthia H.
dc.contributorBelfort, Marlene
dc.contributorTessier, Peter M.
dc.contributorKilduff, James
dc.contributor.authorGrimaldi, Joseph
dc.date.accessioned2021-11-03T08:18:35Z
dc.date.available2021-11-03T08:18:35Z
dc.date.created2015-03-09T11:40:42Z
dc.date.issued2014-12
dc.identifier.urihttps://hdl.handle.net/20.500.13015/1316
dc.descriptionDecember 2014
dc.descriptionSchool of Engineering
dc.description.abstractFuture plans should employ either in silico design to add surface charges, or directed evolution to develop more active and stable ADH and KdcA mutants. In addition, a stable mutant or fusion protein of FDH will need to be developed so all three enzymes are immobilized. Finally, the fully immobilized enzyme reaction system can be scaled up and combined with the new pervaporation membrane system to create a continuous production system.
dc.description.abstractLimitations on biofuel production using cell culture (Escherichia coli, Clostridium, Saccharomyces cerevisiae, brown microalgae, blue-green algae and others) include low product (alcohol) concentrations (2-5 vol%) due to feedback inhibition, instability of cells, and lack of economical product recovery processes. To overcome these challenges, we propose an alternate simplified biofuel production scheme based on a cell-free immobilized enzyme system. Optimized immobilized enzymes can be more stable and productive than free enzyme in solution. We measured the activity of two multi-domain enzymes, alcohol dehydrogenase (ADH) and keto-acid decarboxylase (KdcA), after immobilization onto multiple surface curvatures and chemistries. β-galactosidase was used as a model enzyme, with a colorimetric assay, to help develop an immobilization strategy. Several solid supports including silica nanoparticles (convex), mesopourous silica (concave), carbon diatoms (concave), and methacrylate (concave) were tested as a function of external/internal porosities.
dc.description.abstractThis alcohol production scheme is attractive because (i) there are no cells that may experience product feedback inhibition, (ii) enzymes are produced at high titer using standard expression, (iii) protein engineering can be used to stabilize the two enzymes, (iv) enzymes are stabilized through immobilization, and (v) the reaction is driven towards product using continual removal (recovery).
dc.description.abstractHigh conversion rates and low protein leaching was achieved by covalent immobilization of enzymes on methacrylate resin. ADH exhibited long-term stability and efficient conversion of aldehyde to alcohol over multiple batch cycles. KdcA fusion proteins (fKdcA) were developed to stabilize the enzyme. In addition to the two enzymes involved in the production of isobutanol, formate dehydrogenase (FDH) was needed to recycle the cofactor (NADH) using an inexpensive substrate, formate. The complete reaction scheme was demonstrated by immobilizing ADH, immobilized fKdcA and using FDH free in solution. The new system without in situ removal of isobutanol achieved a 55% conversion of ketoisovaleric acid to isobutanol. To provide a removal strategy, new pervaporation membranes were developed using plasma grafting techniques. These membranes were used to selectively remove isobutanol from simulated reaction system. The new grafted brush membrane had a flux of 0.8 ± 0.15 lmh (L/m2-h); this is comparable to the fluxes for commercial polydimethylsiloxane (PDMS) membranes (J = 0.7 ± 0.06 lmh and 1 ± 0.11 lmh). However, the separation factor for the brush membrane was 10.1 ± 0.86 compared with 6.7 ± 0.11 and 6.7 ± 0.05 for the commercial PDMS membranes. This is a 1.5 times increase in the selectivity. Additionally, the new brush membrane was not only better at selectively removing isobutanol, but also retained essential reactive compounds.
dc.language.isoENG
dc.publisherRensselaer Polytechnic Institute, Troy, NY
dc.relation.ispartofRensselaer Theses and Dissertations Online Collection
dc.subjectChemical engineering
dc.titleCell free production of a liquid fuel
dc.typeElectronic thesis
dc.typeThesis
dc.digitool.pid174826
dc.digitool.pid174827
dc.digitool.pid174828
dc.rights.holderThis electronic version is a licensed copy owned by Rensselaer Polytechnic Institute, Troy, NY. Copyright of original work retained by author.
dc.description.degreePhD
dc.relation.departmentDept. of Chemical and Biological Engineering


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