Microbial Respiration of Chloroxyanions as a Source of Oxygen for Bioprocessing

Abstract

Aerobic microbial processes comprise culturing microbes comprising a (per)chlorate respiration pathway in a bioreactor in a bioprocess employing microbial respiration of chloroxyanions as a source of oxygen for the bioprocess, in the absence of external addition of molecular oxygen.

Claims

1. A method of aerobic microbial processing comprising: culturing microbes comprising a (per)chlorate respiration pathway in a bioreactor in a bioprocess employing microbial respiration of chloroxyanions as a source of oxygen for the bioprocess, in the absence of external addition of molecular oxygen.

2. The method of claim 1, wherein the bioprocess comprises production of drugs, commercial or industrial enzymes, bioplastics or bioplastic precursors, biofuels or biofuel precursors, commodity chemicals, cosmetics, foods, such as plant-protein based meat substitutes, or food additives, such as citric acid.

3. The method of claim 1, wherein the microbes are obligately aerobic methanotrophic, and the bioprocessing converts the greenhouse gas methane (CH.sub.4) into the biopolymer polyhydroxybutyrate (PHB), polyhydroxyalkanoate (PHA), or polylactic acid (PLA).

4. The method of claim 1, wherein the microbes are obligately aerobic eukaryotic fungi, and the bioprocess utilizes complex lignin-celluosic feedstocks.

5. The method of claim 1, wherein the microbial respiration comprises a pathway comprising: (i) reduction of (per)chlorate to chlorite (ClO.sub.2.sup.−) by perchlorate reductase; (ii) dismutation of chlorite into chloride and molecular oxygen (O.sub.2) by chlorite dismutase; and (iii) reduction of molecular oxygen by cytochrome oxidase.

6. The method of claim 1, wherein the microbial respiration comprises a pathway comprising: (i) reduction of chlorate to chlorite (ClO.sub.2.sup.−) by a protein belonging to the DMSO reductase superfamily of molybdopterin oxidoreductases; and (ii) dismutation of chlorite into chloride and molecular oxygen (O.sub.2) by chlorite dismutase; and (iii) reduction of molecular oxygen by cytochrome oxidase.

7. The method of claim 1, wherein the microbial (per)chlorate respiration pathway is engineered.

8. The method of claim 1, wherein the microbes are genetically engineered to comprise an operative microbial (per)chlorate respiration pathway encoding: (i) a perchlorate reductase which effects reduction of (per)chlorate to chlorite (ClO.sub.2.sup.−); (ii) a chlorite dismutase which effects dismutation of the chlorite into chloride and molecular oxygen (O.sub.2); and (iii) a cytochrome oxidase which effects reduction of the molecular oxygen; wherein the pathway enables the engineered microbe to grow in the bioreactor with (per)chlorate as its sole electron acceptor in the absence of oxygen.

9. The method of claim 1, wherein the microbes are genetically engineered to comprise an operative microbial (per)chlorate respiration pathway encoding: (i) a protein belonging to the DMSO reductase superfamily of molybdopterin oxidoreductases which effects reduction of (per)chlorate to chlorite (ClO.sub.2.sup.−); (ii) a chlorite dismutase which effects dismutation of the chlorite into chloride and molecular oxygen (O.sub.2); and (iii) a cytochrome oxidase which effects reduction of the molecular oxygen; wherein the pathway enables the engineered microbe to grow in the bioreactor with (per)chlorate as its sole electron acceptor in the absence of oxygen.

10. A bioreactor configured for a method aerobic microbial processing comprising: culturing microbes comprising a (per)chlorate respiration pathway in a bioreactor in a bioprocess employing microbial respiration of chloroxyanions as a source of oxygen for the bioprocess, in the absence of external addition of molecular oxygen.

11. The bioreactor of claim 10, wherein the bioprocess comprises production of drugs, commercial or industrial enzymes, bioplastics or bioplastic precursors, biofuels or biofuel precursors, commodity chemicals, cosmetics, foods, such as plant-protein based meat substitutes, or food additives, such as citric acid.

12. The bioreactor of claim 10, wherein the microbes are obligately aerobic methanotrophic, and the bioprocessing converts the greenhouse gas methane (CH.sub.4) into the biopolymer polyhydroxybutyrate (PHB), polyhydroxyalkanoate (PHA), or polylactic acid (PLA).

13. The bioreactor of claim 10, wherein the microbes are obligately aerobic eukaryotic fungi, and the bioprocess utilizes complex lignin-celluosic feedstocks.

14. The bioreactor of claim 10, wherein the microbial respiration comprises a pathway comprising: (i) reduction of (per)chlorate to chlorite (ClO.sub.2.sup.−) by perchlorate reductase; (ii) dismutation of chlorite into chloride and molecular oxygen (O.sub.2) by chlorite dismutase; and (iii) reduction of molecular oxygen by cytochrome oxidase.

15. The bioreactor of claim 10, wherein the microbial (per)chlorate respiration pathway is engineered.

16. The bioreactor of claim 10, wherein the microbes are genetically engineered to comprise an operative microbial (per)chlorate respiration pathway encoding: (i) a perchlorate reductase which effects reduction of (per)chlorate to chlorite (ClO.sub.2.sup.−); (ii) a chlorite dismutase which effects dismutation of the chlorite into chloride and molecular oxygen (O.sub.2); and (iii) a cytochrome oxidase which effects reduction of the molecular oxygen; wherein the pathway enables the engineered microbe to grow in the bioreactor with (per)chlorate as its sole electron acceptor in the absence of oxygen.

17. The bioreactor of claim 10, wherein the microbes are genetically engineered to comprise an operative microbial (per)chlorate respiration pathway encoding: (i) a protein belonging to the DMSO reductase superfamily of molybdopterin oxidoreductases which effects reduction of (per)chlorate to chlorite (ClO.sub.2.sup.−); (ii) a chlorite dismutase which effects dismutation of the chlorite into chloride and molecular oxygen (O.sub.2); and (iii) a cytochrome oxidase which effects reduction of the molecular oxygen; wherein the pathway enables the engineered microbe to grow in the bioreactor with (per)chlorate as its sole electron acceptor in the absence of oxygen.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG. 1A. Polyhydroxybutyrate (PHB): overview

[0027] FIG. 1B. PHB (C.sub.4H.sub.6O.sub.2)n from methane: reactions

[0028] FIG. 2A. Perchlorate reduction: three-step process

[0029] FIG. 2B. Engineered anaerobic methane metabolism coupled to perchlorate

[0030] FIG. 2C. Direct O.sub.2-dependent oxidation with ClO.sub.4.sup.−

DESCRIPTION OF PARTICULAR EMBODIMENTS OF THE INVENTION

[0031] Unless contraindicated or noted otherwise, in these descriptions and throughout this specification, the terms “a” and “an” mean one or more, the term “or” means and/or. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein, including citations therein, are hereby incorporated by reference in their entirety for all purposes.

[0032] This invention takes advantage of the unique oxygen biogenesis biochemical pathway associated respiration of the chloroxyanions chlorate (ClO.sub.3.sup.−) and perchlorate (ClO.sub.4) [collectively denoted (per)chlorate]. The invention provides for aerobic microbial processes in the absence of external addition of molecular oxygen. Canonical microbial (per)chlorate respiration is an energetically favorable process that involves three steps: (i) the reduction of (per)chlorate to chlorite (ClO.sub.2.sup.−) by the perchlorate reductase; (ii) disputation of chlorite into chloride and molecular oxygen (O.sub.2) by the chlorite dismutase; and (iii) reduction of molecular oxygen by the cytochrome oxidase. In this way (per)chlorate respiring organisms can enzymatically produce oxygen under anoxic conditions. This provides organisms endowed with this pathway with a unique metabolic versatility, allowing them to use (per)chlorate not only as an electron acceptor but also as a co-substrate in oxygenase driven reactions in the absence of externally added oxygen. This pathway is well characterized with known biochemistry and genes. We have previously engineered this pathway into Shewanella species allowing the engineered strain to grow with chlorate as its sole electron acceptor in the absence of oxygen. An exemplary application of this invention is shown in FIGS. 1A-B and FIG. 2A-C, where the (per)chlorate pathway is engineered into an obligately aerobic methanotrophic organism to convert the greenhouse gas methane (CH.sub.4) into the biopolymer polyhydroxybutyrate (PHB), a component of bioplastics. The bioplastics 2025 bioplastics predicted annual market value is $27.9 billion. Methanotrophs are intrinsically efficient at producing PHB and have been shown to accumulate as much as 67% of the cell biomass in the form of PHB. This often translates into titers of greater than 2 g PHB.L.sup.−1 of culture which is sufficient for industrial production. In this process oxygen is used both as an electron acceptor and as a co-substrate for both cell growth and PHB production (FIGS. 1A-B and FIG. 2A-C). As such, these cultures often become oxygen limited requiring complex reactor designs and high energy inputs. Furthermore, these bioreactors are often operated at pressure of up to 3 atmospheres which represents an explosive hazard for mixed gasses of CH.sub.4 and O.sub.2. Perchlorate can overcome these limitations, as it is chemically stable, highly soluble (>67% by mass), and can be easily metabolized to provide both the energy and oxygen requirements of methanotrophy. Furthermore, perchlorate can be produced easily from renewable energy through electrochemical oxidation of sodium chloride (NaCl; table salt).

[0033] FIGS. 1A-1B: PHB production from methane is an oxygen intensive metabolism requiring 2 moles of O.sub.2 per mole of CH.sub.4 to grow the cells and 7 moles of O.sub.2 per 8 moles of CH.sub.4 to produce PHB.

[0034] 67% dry wt biomass possible e.g. Wendlandt et al. (J Biotechnol. 2001 Mar. 30; 86(2):127-33) showed Methylocystis trichosporium sp. GB25 on methane at 3 atm yielded 4 g L.sup.−1H.sup.−1 biomass and 2 g L.sup.−1 PHB with a yield of 0.55 g g.sup.−1 CH.sub.4. Glucose gives a yield of 0.3 to 0.4 gm PHB per gm glucose.

[0035] General Principle: 4n/x C.sub.xH.sub.yO.sub.z.fwdarw.(C.sub.4H.sub.6O.sub.2)n+n[2y/x−3] H.sub.2O+n[1/2−(y−2z)/x]O.sub.2

[0036] CH.sub.4=>X=1; y=4; z=0

[0037] 4n CH.sub.4+[n+n5/2]O.sub.2.fwdarw.(C.sub.4H.sub.6O.sub.2)n+n5 H.sub.2O

[0038] For example, If n=2 then:

[0039] 8 CH.sub.4+7O.sub.2.fwdarw.(C.sub.4H.sub.6O.sub.2).sub.2+10 H.sub.2O

[0040] Features: oxygen (energy) intensive process often leading to high risk (explosive) pressurized conditions in a bioreactor

[0041] FIGS. 2A-2C: Perchlorate easily satisfies the oxygen requirement of methanotrophy and PHB production (or any other aerobic microbial process) through the unique biochemical pathway in which ClO.sub.4.sup.− is converted into O.sub.2 which then becomes available to the organism as an electron acceptor and co-substrate for by-product production. Features: Perchlorate is well characterized Perchlorate is highly soluble Perchlorate is chemically stable Perchlorate can be produced from renewable energy.