Michigan State University

Michigan Biotechnology Institute

Joseph G Zeikus

Hyung-Hwan Hyun

Doo Hyun Park

Jurgen Thiele

Claire Vieille

Mahendra K Jain

Hyoun S Shin

Badal C Saha

Guoqiang Dong

Douglas S Burdette

Electrodes and configurations for electrochemical bioreactor systems that can use electrical energy as a source of reducing power in fermentation or enzymatic reactions and that can use electron mediators and a biocatalyst, such as cells or enzymes, to produce electricity are disclosed. Example electrodes in the system may comprise: (1) neutral red covalently bound to graphite felt; (2) a carboxylated cellulose bound to the graphite felt, neutral red bound to the carboxylated cellulose, NADbound to the graphite felt, and an oxidoreductase (e.g., fumarate reductase) bound to the graphite felt; or (3) a metal ion electron mediator bound to graphite. Various biocatalysts, such as an oxidoreductase, cells of, cells of, and sewage sludge, are suitable for use in the electrochemical bioreactor system.

Electrode compositions and configurations for electrochemical bioreactor systems

Disclosed are methods using neutral red to mediate the interconversion of chemical and electrical energy. Electrically reduced neutral red has been found to promote cell growth and formation of reduced products by reversibly increasing the ratio of the reduced:oxidized forms of NAD(H) or NADP(H). Electrically reduced neutral red is able to serve as the sole source of reducing power for microbial cell growth. Neutral red is also able to promote conversion of chemical energy to electrical energy by facilitating the transfer of electrons from microbial reducing power to a fuel cell cathode.

Electrochemical methods for generation of a biological proton motive force

Electrodes and configurations for electrochemical bioreactor systems that can use electrical energy as a source of reducing power in fermentation or enzymatic reactions and that can use electron mediators and a biocatalyst, such as cells or enzymes, to produce electricity are disclosed. Example electrodes in the system may comprise: (1) neutral red covalently bound to graphite felt (FIG.); (2) a carboxylated cellulose bound to the graphite fell, neutral red bound to the carboxylated cellulose, NADto the graphite fell, and an oxidoreductase (e.g., fumarate reductase) bound to the graphite fell; or (3) a metal ion electron mediator bound to graphite. Various biocatalysts, such as an oxidoreductase, cells ofcells ofand sewage sludge, are suitable for use in the electrochemical bioreactor system.

Electrochemical methods for generation of a biological proton motive force and pyridine nucleotide cofactor regeneration

This invention relates in general to alkaline phosphatase and, in particular, to alkaline phosphatase from a hyperthermophilic microorganism. The invention also relates to a method of purifying a hyperthermophilic alkaline phosphatase.

Purification and characterization of alkaline phosphates from Thermotoga

The adhB gene encoding Thermoanaerobacter ethanolicus 39E secondary-alcohol dehydrogenase (2.degree. Adh) was cloned, sequenced, and expressed in Escherichia coli. The 1056 bp gene encodes a homotetrameric recombinant enzyme consisting of 37.7 kDa subunits. The purified recombinant enzyme is optimally active above 90.degree. C. with a half-life of approximately 1.7 h at 90.degree. C. An NADP(H)-dependent enzyme, the recombinant 2.degree. Adh has 1400-fold greater catalytic efficiency in propan-2-ol versus ethanol oxidation. The enzyme was inactivated by chemical modification using dithionitrobenzoate (DTNB) and diethylpyrocarbonate, indicating that Cys and His residues are involved in catalysis. Zinc was the only metal enhancing 2.degree. Adh reactivation after DTNB modification, implicating the involvement of a strongly bound zinc in catalysis. Arrhenius plots for the oxidation of propan-2-ol by the native and recombinant 2.degree. Adhs were linear from 25.degree. C. to 90.degree. C. when the enzymes were incubated at 55.degree. C. prior to assay. Discontinuities in the Arrhenius plots for propan-2-ol and ethanol oxidations were observed, however, when the enzymes were preincubated at 0.degree. C. or 25.degree. C. The observed Arrhenius discontinuity, therefore, resulted from a temperature dependent, catalytically significant 2.degree. Adh structural change. Hydrophobic Cluster Analysis comparisons of both mesophilic versus thermophilic 2.degree. Adh and 1.degree. versus 2.degree. Adh amino acid sequences were performed. These comparisons predicted that specific proline residues may contribute to 2.degree. Adh thermostability and thermophilicity, and that the catalytic Zn ligands are different in 1.degree. Adhs (two Cys and a His) and 2.degree. Adhs (Cys, His, and Asp).

Cloning and expression of the gene encoding thermoanaerobacter

The present invention describes the cloning and expression of a hyperthermostable xylose isomerase gene in an industrial host and the heat facilitated purification of the enzyme. The hyperthermostable enzyme has unique features sought by the fructose syrup industry.

Gene encoding hyperthermostable xylose isomerase from Thermotoga

An improved process for the biomethanation of an organic substrate includes treating the substrate in a first reactor to form organic acid anions, passing an aqueous preparation containing dissolved organic anions through an ion exchanger so that the organic acid anions are adsorbed and separated from the remainder of the aqueous preparation, desorbing; the organic acid anions and passing the desorbed acids to a second reactor containing methanogenic bacteria which convert the acids to methane. In a preferred embodiment, a bicarbonate solution is produced in the second reactor and it is used to desorb the organic acid anions and regenerate the anion exchanger into the bicarbonate form. A preferred apparatus first and second reactors, and a pair of anion exchangers connected by tubing to both reactors also is disclosed.

Apparatus for biomethanation

An improved method of preparing high maltose conversion syrups and other sweeteners from starch substrates comprises saccharifying the starch at higher temperatures than presently used with environmentally compatible thermostable .beta.-amylase and other thermostable enzymes.

Method for preparing high conversion syrups and other sweeteners

An improved process for the biomethanation of an organic substrate includes treating the substrate in a first reactor to form organic acid anions, passing an aqueous preparation containing dissolved organic anions through an anion exchanger so that the organic acid anions are adsorbed and separated from the remainder of the aqueous preparation, desorbing the organic acid anions and passing the desorbed acids to a second reactor containing methanogenic bacteria which convert the acids to methane. In a preferred embodiment, a bicarbonate solution is produced in the second reactor and it is used to desorb the organic acid anions and regenerate the anion exchanger into the bicarbonate form.

Method for biomethanation

A method is disclosed for producing catabolite repression-resistant mutant strains of C. thermosulfurogenes and C. thermohydrosulfuricum. The method comprises challenging a wild strain of the organism with nitrosoguanidine, followed by enrichment on 2-deoxyglucose and culturing an iodine stained starch-glucose containing agar. The colonies which convert starch most efficiently are catabolite repression-resistant. Pure cultures of the mutants and methods employing the mutants to prepare enzymes are also described.

Regulation and enhancement of enzyme production

A thermostable and thermoactive, extracellular .beta.-amylase is produced by Clostridium thermosulfurogenes. Methods of producing the .beta.-amylase and using it to convert starch to maltose are also disclosed.

Thermostable beta-amylase

A thermostable pullulanase and a thermostable glucoamylase are produced by Clostridium thermohydrosulfuricum. Methods of producing the enzymes and using them to hydrolyze starch are also disclosed.

Thermostable starch converting enzymes

Starch is converted directly to ethanol by growing a co-culture of Clostridium thermosulfurogenes and Clostridium thermohydrosulfuricum under anaerobic conditions on a starch containing substrate in a medium comprising essential vitamins, minerals and growth factors. The co-culture also produces the thermostable enzymes .beta.-amylase, glucoamylase and pullulanase.

Growing community of inventors

Okemos, MI, United States of America

Joseph G Zeikus