Glycerol Feedstock Utilization for Oil-Based Fuel Manufacturing
The invention provides methods of manufacturing biodiesel and other oil-based compounds using glycerol and combinations of glycerol and other feedstocks as an energy source in fermentation of oil-bearing microorganisms. Methods disclosed herein include processes for manufacturing high nutrition edible oils from non-food feedstock materials such as waste products from industrial waste transesterification processes. Also included are methods of increasing oil yields by temporally separating glycerol and other feedstocks during cultivation processes. Also provided herein are oil-bearing microbes containing exogenous oil production genes and methods of cultivating such microbes on glycerol and other feedstocks.
1 . A method of biodiesel manufacturing, the method comprising:
(a) culturing a lipid-producing microorganism in a first microbial culture;
(b) recovering lipid from the biomass produced by the first microbial culture;
(c) subjecting the lipid to transesterification to produce fatty acid ester(s) and glycerol; and
(d) adding the glycerol to a second microbial culture.
2 . The method of claim 1 , wherein the first and second microbial cultures are cultures of the same species of microorganism.
3 . The method of claim 1 , wherein the microorganism is a microalgae.
4 . The method of claim 3 , wherein the microorganism is selected from the group consisting of the microalgae listed in Table 1.
5 . The method of claim 4 , wherein the microorganism is a species of the genus Chlorella.
6 . The method of claim 5 , wherein the microorganism is selected from the group consisting of Chlorella anitrata, Chlorella antarctica, Chlorella aureoviridis, Chlorella candida, Chlorella capsulata, Chlorella desiccata, Chlorella ellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var. vacuolata, Chlorella glucotropha, Chlorella infusionum, Chlorella infusionum var. Actophila, Chlorella infusionum var. Auxenophila, Chlorella kessleri, Chlorella luteoviridis, Chlorella luteoviridis var. aureoviridis, Chlorella luteoviridis var. Lutescens, Chlorella miniata, Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna, Chlorella parva, Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides, Chlorella pyrenoidosa, Chlorella regularis, Chlorella regularis var. minima, Chlorella regularis var. umbricata, Chlorella reisiglii, Chlorella saccharophila, Chlorella saccharophila var. ellipsoidea, Chlorella salina, Chlorella simplex, Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica, Chlorella stigmatophora, Chlorella vanniellii, Chlorella vulgaris, Chlorella vulgaris, Chlorella vulgaris f. tertia, Chlorella vulgaris var. airidis, Chlorella vulgaris var. vulgaris, Chlorella vulgaris var. vulgaris f. tertia, Chlorella vulgaris var. vulgaris f. viridis, Chlorella xanthella , and Chlorella zofingiensis.
7 . The method of claim 1 , wherein the second microbial culture comprises microorganisms selected from the group consisting of Parachlorella kessleri, Chlorella protothecoides, Bracteococcus medionucleatus, Prototheca moriformis, Chlorella minutissima, Chlorella sp., and Chlorella sorokiniana.
8 . The method of claim 1 , wherein the microorganism is an oleaginous yeast.
9 . The method of claim 8 , wherein the oleaginous yeast is selected from the group consisting of Cryptococcus curvatus, Cryptococcus terricolus, Candida sp., Lipomyces starkeyi, Lipomyces lipofer, Endomycopsis vernalis, Rhodotorula glutinis, Rhodotorula gracilis , and Yarrowia lipolytica.
10 . The method of claim 1 , wherein the microorganism is a fungus.
11 . The method of claim 10 , wherein the fungus is selected from the group consisting of a species of the genus Mortierella, Mortierrla vinacea, Mortierella alpine, Pythium debaryanum, Mucor circinelloides, Aspergillus ochraceus, Aspergillus terreus, Pennicillium iilacinum , a species of the genus Hensenulo , a species of the genus Chaetomium , a species of the genus Cladosporium , a species of the genus Malbranchea , a species of the genus Rhizopus , and a species of the genus Pythium.
12 . The method of claim 1 , wherein the microorganism contains at least one exogenous sucrose utilization gene.
13 . The method of claim 12 , wherein the sucrose utilization gene encodes a sucrose transporter, a sucrose invertase, a hexokinase, a glucokinase, or a fructokinase.
14 . The method of claim 1 , wherein the microorganism contains at least one exogenous gene encoding a lipid pathway enzyme.
15 . The method of claim 14 , wherein the lipid pathway enzyme is selected from the group consisting of a stearoyl-ACP desaturase, a glycerolipid desaturase, a pyruvate dehydrogenase, an acetyl-CoA carboxylase, an acyl carrier protein, and a glycerol-3 phosphate acyltransferase.
16 . A method of fermentation comprising culturing a microorganism in the presence of glycerol and at least one other fixed carbon source.
17 . The method of claim 16 , wherein the glycerol and the at least one other fixed carbon source are provided to the microorganism simultaneously at a predetermined ratio.
18 . The method of claim 17 , wherein all of the glycerol and the at least one other fixed carbon source are provided to the microorganism at the beginning of the fermentation.
19 . The method of claim 17 , wherein all of the glycerol and the at least one other fixed carbon source are fed to the microorganism at a predetermined rate over the course of the fermentation.
20 . The method of claim 16 , wherein
(a) glycerol is provided to the microorganism in the absence of the at least one other fixed carbon source for a first period of time;
(b) the at least one other fixed carbon source is provided at the end of the first period of time; and
(c) the microorganism is cultured for a second period of time in the presence of the at least one other fixed carbon source.
21 . The method of claim 20 , wherein the at least one other fixed carbon source is fed to the microorganism at a predetermined rate during the second period of time.
22 . The method of claim 20 , wherein all of the at least one other fixed carbon source is provided to the microorganism at the end of the first period of time.
23 . The method of claim 16 , wherein
(a) the at least one other fixed carbon source is provided to the microorganism in the absence of glycerol for a first period of time;
(b) glycerol is provided at the end of the first period of time; and
(c) the microorganism is cultured for a second period of time in the presence of glycerol.
24 . The method of claim 16 , wherein the microorganism is a microalgae.
25 . The method of claim 24 , wherein the microalgae is selected from Table 1.
26 . The method of claim 24 , wherein the microalgae is of the genus Chlorella.
27 . The method of claim 26 , wherein the microalgae is selected from the group consisting of Chlorella anitrata, Chlorella antarctica, Chlorella aureoviridis, Chlorella candida, Chlorella capsulata, Chlorella desiccata, Chlorella ellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var. vacuolata, Chlorella glucotropha, Chlorella infusionum, Chlorella infusionum var. Actophila, Chlorella infusionum var. Auxenophila, Chlorella kessleri, Chlorella luteoviridis, Chlorella luteoviridis var. aureoviridis, Chlorella luteoviridis var. Lutescens, Chlorella miniata, Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna, Chlorella parva, Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides, Chlorella regularis, Chlorella regularis var. minima, Chlorella regularis var. umbricata, Chlorella reisiglii, Chlorella saccharophila, Chlorella saccharophila var. ellipsoidea, Chlorella salina, Chlorella simplex, Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica, Chlorella stigmatophora, Chlorella vanniellii, Chlorella vulgaris, Chlorella vulgaris, Chlorella vulgaris f. tertia, Chlorella vulgaris var. airidis, Chlorella vulgaris var. vulgaris, Chlorella vulgaris var. vulgaris f. tertia, Chlorella vulgaris var. vulgaris f. viridis, Chlorella xanthella, and Chlorella zofingiensis.
28 . The method of claim 16 , wherein the microorganism is an oleaginous yeast.
29 . The method of claim 28 , wherein the oleaginous yeast is selected from the group consisting of Cryptococcus curvatus, Cryptococcus terricolus, Candida sp., Lipomyces starkeyi, Lipomyces lipofer, Endomycopsis vernalis, Rhodotorula glutinis, Rhodotorula gracilis , and Yarrowia lipolytica.
30 . The method of claim 16 , wherein the microorganism is a fungus.
31 . The method of claim 30 , wherein the fungus is selected from the group consisting of a species of the genus Mortierella, Mortierrla vinacea, Mortierella alpine, Pythium debaryanum, Mucor circinelloides, Aspergillus ochraceus, Aspergillus terreus, Pennicillium iilacinum , a species of the genus Hensenulo , a species of the genus Chaetomium , a species of the genus Cladosporium , a species of the genus Malbranchea , a species of the genus Rhizopus , and a species of the genus Pythium.
32 . The method of claim 16 , wherein the microorganism contains at least one exogenous sucrose utilization gene.
33 . The method of claim 32 , wherein the sucrose utilization gene encodes a sucrose transporter, a sucrose invertase, a hexokinase, a glucokinase, or a fructokinase.
34 . The method of claim 16 , wherein the microorganism contains at least one exogenous gene encoding a lipid pathway enzyme.
35 . The method of claim 34 , wherein the lipid pathway enzyme is selected from the group consisting of a stearoyl-ACP desaturase, a glycerolipid desaturase, a pyruvate dehydrogenase, an acetyl-CoA carboxylase, an acyl carrier protein, and a glycerol-3 phosphate acyltransferase.
36 . The method of claim 16 , wherein the glycerol is a byproduct of a transesterification process.
37 . The method of claim 36 , wherein the glycerol is acidulated.
38 . The method of claim 36 , wherein the glycerol is non-acidulated.
39 . The method of claim 16 , wherein the at least one other fixed carbon source is glucose.
40 . The method of claim 16 , wherein the at least one other fixed carbon source is depolymerized cellulosic material.
41 . The method of claim 16 , wherein the at least one other fixed carbon source is sucrose.
42 . A fermentor comprising:
(a) a population of microorganisms;
(b) glycerol; and
(c) at least one sugar selected from the group consisting of xylose, glucose, and sucrose.
43 . The fermentor of claim 42 , wherein the glycerol is a byproduct of a lipid transesterification process.
44 . The fermentor of claim 42 , wherein the microorganisms are microalgae.
45 . The fermentor of claim 44 , wherein the microorganisms are selected from the group consisting of the microalgae listed in Table 1.
46 . The fermentor of claim 45 , wherein the microorganisms are a species of the genus Chlorella.
47 . The fermentor of claim 46 , wherein the microorganisms are selected from the group consisting of Chlorella anitrata, Chlorella antarctica, Chlorella aureoviridis, Chlorella candida, Chlorella capsulata, Chlorella desiccata, Chlorella ellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var. vacuolata, Chlorella glucotropha, Chlorella infusionum, Chlorella infusionum var. Actophila, Chlorella infusionum var. Auxenophila, Chlorella kessleri, Chlorella luteoviridis, Chlorella luteoviridis var. aureoviridis, Chlorella luteoviridis var. Lutescens, Chlorella miniata, Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna, Chlorella parva, Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides, Chlorella regularis, Chlorella regularis var. minima, Chlorella regularis var. umbricata, Chlorella reisiglii, Chlorella saccharophila, Chlorella saccharophila var. ellipsoidea, Chlorella salina, Chlorella simplex, Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica, Chlorella stigmatophora, Chlorella vanniellii, Chlorella vulgaris, Chlorella vulgaris, Chlorella vulgaris f. tertia, Chlorella vulgaris var. airidis, Chlorella vulgaris var. vulgaris, Chlorella vulgaris var. vulgaris f. tertia, Chlorella vulgaris var. vulgaris f. viridis, Chlorella xanthella , and Chlorella zofingiensis.
48 . The fermentor of claim 42 , wherein the microorganisms are selected from the group consisting of Parachlorella kessleri, Chlorella protothecoides, Bracteococcus medionucleatus, Prototheca moriformis, Chlorella minutissima, Chlorella sp., and Chlorella sorokiniana.
49 . The fermentor of claim 42 , wherein the microorganisms are oleaginous yeast.
50 . The fermentor of claim 49 , wherein the oleaginous yeast are selected from the group consisting of Cryptococcus curvatus, Cryptococcus terricolus, Candida sp., Lipomyces starkeyi, Lipomyces lipofer, Endomycopsis vernalis, Rhodotorula glutinis, Rhodotorula gracilis , and Yarrowia lipolytica.
51 . The fermentor of claim 42 , wherein the microorganisms are fungi.
52 . The fermentor of claim 51 , wherein the fungi are selected from the group consisting of a species of the genus Mortierella, Mortierrla vinacea, Mortierella alpine, Pythium debaryanum, Mucor circinelloides, Aspergillus ochraceus, Aspergillus terreus, Pennicillium iilacinum , a species of the genus Hensenulo , a species of the genus Chaetomium , a species of the genus Cladosporium , a species of the genus Malbranchea , a species of the genus Rhizopus , and a species of the genus Pythium.
53 . The fermentor of claim 42 , wherein the microorganisms contain at least one exogenous sucrose utilization gene.
54 . The fermentor of claim 53 , wherein the sucrose utilization gene encodes a sucrose transporter, a sucrose invertase, a hexokinase, a glucokinase, or a fructokinase.
55 . The fermentor of claim 42 , wherein the microorganisms contain at least one exogenous gene encoding a lipid pathway enzyme.
56 . The fermentor of claim 55 , wherein the lipid pathway enzyme is selected from the group consisting of a stearoyl-ACP desaturase, a glycerolipid desaturase, a pyruvate dehydrogenase, an acetyl-CoA carboxylase, an acyl carrier protein, and a glycerol-3 phosphate acyltransferase.
57 . A method of fermenting a microorganism comprising providing byproduct glycerol from a transesterification process as a sole source of fixed carbon energy.
58 . The method of claim 57 , wherein no light energy is provided to the microorganism.
59 . The method of claim 57 , wherein light energy is provided to the microorganism.
60 . The method of claim 57 , wherein the microorganism is a microalgae.
61 . The method of claim 60 , wherein the microorganism is selected from the group consisting of the microalgae listed in Table 1.
62 . The method of claim 61 , wherein the microorganism is a species of the genus Chlorella.
63 . The method of claim 62 , wherein the microorganism is selected from the group consisting of Chlorella anitrata, Chlorella antarctica, Chlorella aureoviridis, Chlorella candida, Chlorella capsulata, Chlorella desiccata, Chlorella ellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var. vacuolata, Chlorella glucotropha, Chlorella infusionum, Chlorella infusionum var. Actophila, Chlorella infusionum var. Auxenophila, Chlorella kessleri, Chlorella luteoviridis, Chlorella luteoviridis var. aureoviridis, Chlorella luteoviridis var. Lutescens, Chlorella miniata, Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna, Chlorella parva, Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides, Chlorella regularis, Chlorella regularis var. minima, Chlorella regularis var. umbricata, Chlorella reisiglii, Chlorella saccharophila, Chlorella saccharophila var. ellipsoidea, Chlorella salina, Chlorella simplex, Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica, Chlorella stigmatophora, Chlorella vanniellii, Chlorella vulgaris, Chlorella vulgaris, Chlorella vulgaris f. tertia, Chlorella vulgaris var. airidis, Chlorella vulgaris var. vulgaris, Chlorella vulgaris var. vulgaris f. tertia, Chlorella vulgaris var. vulgaris f. viridis, Chlorella xanthella , and Chlorella zofingiensis.
64 . The method of claim 57 , wherein the microorganism is selected from Parachlorella kessleri, Chlorella protothecoides, Bracteococcus medionucleatus, Prototheca moriformis, Chlorella minutissima, Chlorella sp., and Chlorella sorokiniana.
65 . The method of claim 57 , wherein the microorganism is an oleaginous yeast.
66 . The method of claim 65 , wherein the oleaginous yeast is selected from the group consisting of Cryptococcus curvatus, Cryptococcus terricolus, Candida sp., Lipomyces starkeyi, Lipomyces lipofer, Endomycopsis vernalis, Rhodotorula glutinis, Rhodotorula gracilis , and Yarrowia lipolytica.
67 . The method of claim 57 , wherein the microorganism is a fungus.
68 . The method of claim 67 , wherein the fungus is selected from the group consisting of a species of the genus Mortierella, Mortierrla vinacea, Mortierella alpine, Pythium debaryanum, Mucor circinelloides, Aspergillus ochraceus, Aspergillus terreus, Pennicillium iilacinum , a species of the genus Hensenulo , a species of the genus Chaetomium , a species of the genus Cladosporium , a species of the genus Malbranchea , a species of the genus Rhizopus , and a species of the genus Pythium.
69 . The method of claim 57 , wherein the microorganism contains at least one exogenous sucrose utilization gene.
70 . The method of claim 69 , wherein the sucrose utilization gene encodes a sucrose transporter, a sucrose invertase, a hexokinase, a glucokinase, or a fructokinase.
71 . The method of claim 57 , wherein the microorganism contains at least one exogenous gene encoding a lipid pathway enzyme.
72 . The method of claim 71 , wherein the lipid pathway enzyme is selected from the group consisting of a stearoyl-ACP desaturase, a glycerolipid desaturase, a pyruvate dehydrogenase, an acetyl-CoA carboxylase, an acyl carrier protein, and a glycerol-3 phosphate acyltransferase.
73 . The method of claim 1 , wherein the culturing occurs in a fermentor.
74 . The method of claim 16 , wherein the culturing occurs in a fermentor.
75 . The method of claim 20 , wherein the microorganism is cultured for the second period of time in the absence of a fixed nitrogen source.
76 . The method of claim 23 , wherein the microorganism is cultured for the second period of time in the absence of a fixed nitrogen source.