Biorefinery system, methods and compositions thereof

Abstract

The present disclosure relates to bioengineering approaches for producing biofuel and, in particular, to the use of a C 1 metabolizing microorganism reactor system for converting C 1 substrates, such as methane or methanol, into biomass and subsequently into biofuels, bioplastics, or the like.

What is claimed is: 
     
         1 . A controlled culturing unit in which a C 1  substrate is delivered in a gas phase to a microbial biofilm in solid phase fermentation, and wherein the microbial biofilm comprises a recombinant C 1  metabolizing microorganism,
 wherein the C 1  metabolizing microorganism comprises a heterologous polynucleotide encoding a thioesterase, a malonyl CoA-acyl carrier protein transacylase, an acetyl-CoA carboxylase or any combination thereof, and wherein the C 1  metabolizing microorganism accumulates an increased level of fatty acids when grown on the C 1  substrate as compared to a wild-type C 1  metabolizing microorganism without the heterologous polynucleotide.   
     
     
         2 . The controlled culturing unit of  claim 1 , wherein the heterologous polynucleotide encoding the thioesterase is codon optimized for expression in the C 1  metabolizing microorganism. 
     
     
         3 . The controlled culturing unit of  claim 1 , wherein the heterologous polynucleotide encoding the malonyl CoA-acyl carrier protein transacylase is an  E. coli  fabD and wherein the heterologous polynucleotide is codon optimized for expression in the C 1  metabolizing microorganism. 
     
     
         4 . The controlled culturing unit of  claim 1 , wherein the heterologous polynucleotide encoding the acetyl-CoA carboxylase is an  E. coli  accA, accB, accC, accD, or any combination thereof and wherein the heterologous polynucleotide is codon optimized for expression in the C 1  metabolizing microorganism. 
     
     
         5 . The controlled culturing unit of  claim 1 , wherein the C 1  metabolizing microorganism further comprises a mutation that minimizes or eliminates fatty acid-CoA ligase activity, wherein the mutation is in an endogenous fatty acid-CoA ligase gene. 
     
     
         6 . The controlled culturing unit of  claim 1 , wherein the C 1  metabolizing microorganism is a C 1  metabolizing bacterium or yeast. 
     
     
         7 . The controlled culturing unit of  claim 6 , wherein the C 1  metabolizing bacterium is selected from the group consisting of  Methylomonas, Methylobacter, Methylococcus, Methylosinus, Methylocystis, Methylomicrobium, Methanomonas, Methylophilus, Methylobacillus,  and  Methylobacterium.    
     
     
         8 . The controlled culturing unit of  claim 6 , wherein the C 1  metabolizing bacterium is a methanotrophic bacterium or a methylotrophic bacterium. 
     
     
         9 . The controlled culturing unit of  claim 1 , wherein the biofilm comprises a methanotrophic bacterium and one or more heterologous bacteria. 
     
     
         9 . The controlled culturing unit of  claim 1 , wherein the C 1  metabolizing microorganism is a methanotrophic bacterium selected from the group consisting of  Methylococcus capsulatus  Bath,  Methylosinus trichosporium  OB3b,  Methylomonas  sp. 16a,  Methylosinus sporium, Methylocystis parvus, Methylomonas methanica, Methylomonas albus, Methylobacter capsulatus  Y,  Methylobacterium organophllum, Methylomonas  sp. AJ-3670,  Methylomicrobium alcaliphllum, Methylocella silvestris, Methylacidiphllum infernorum,  and  Methylibium petrolelphllum.    
     
     
         10 . The controlled culturing unit of  claim 1 , wherein the C 1  metabolizing microorganism is a methanotrophic bacterium selected from the group consisting of  Methylobacterium extorquens, Methylobacterium radiotolerans, Methylobacterium populi, Methylobacterium chloromethanicum,  and  Methylobacterium nodulans.    
     
     
         11 . The controlled culturing unit of  claim 1 , wherein the C 1  metabolizing microorganism is an obligate C 1  metabolizing bacterium. 
     
     
         12 . A method of producing a biomass oil composition, comprising culturing methanotroph bacteria in a controlled culturing unit in the presence of a feedstock comprising a C 1  substrate under conditions and for a time sufficient to produce a biomass oil composition, wherein said methanotroph bacteria comprise a heterologous polynucleotide and wherein said biomass oil composition comprises whole and/or lysed cells of methanotroph bacteria and more than about 50% w/w terpenoid or isoprenoid compounds. 
     
     
         13 . The method of  claim 12 , wherein said methanotroph bacteria are cultured in a controlled culture unit selected from the group consisting of a fermentor, a bioreactor, a hollow fiber cell, a packed bed bioreactor, and combinations thereof. 
     
     
         14 . The method of  claim 12 , wherein said feedstock comprising a C 1  substrate is natural gas or methane. 
     
     
         15 . The method of  claim 12 , wherein said biomass comprises (a) a culture of said methanotroph bacteria together with a culture media in which said methanotroph bacteria were grown; (b) said methanotroph bacteria recovered from said culture media; or (c) a spent media composition recovered from said culture media comprising said methanotroph bacteria. 
     
     
         16 . The method of  claim 12 , wherein said methanotroph bacteria is selected from the group consisting of  Methylomonas  sp. 16a (ATCC PTA 2402),  Methylosinus trichosporium  OB3b (NRRL B-11,196),  Methylosinus sporium  (NRRL B-11,197),  Methylocystis parvus  (NRRL B-11,198),  Methylomonas methanica  (NRRL B-11,199),  Methylomonas albus  (NRRL B-11,200),  Methylobacter capsulatus  Y (NRRL B-11,201),  Methylococcus capsulatus  Bath (NCIMB 11132),  Methylobacterium organophilum  (ATCC 27,886),  Methylomonas  sp. AJ-3670 (FERM P-2400),  Methylomicrobium alcaliphilum, Methylocella silvestris, Methylacidiphilum infernorum, Methylibium petrolelphilum, Methylobacterium populi,  and any combination thereof. 
     
     
         17 . The method of  claim 12 , wherein said methanotroph bacteria are cultured in a bioreactor comprising balanced media. 
     
     
         18 . The method of  claim 12 , wherein said methanotroph bacteria are cultured in a bioreactor comprising unbalanced media having limiting quantities of phosphorus, nitrogen, trace elements, oxygen relative to balanced media, or any combination thereof. 
     
     
         19 . The method of  claim 12 , wherein said biomass oil composition comprises more than about 50% w/w isoprenoid compounds or comprises more than about 50% w/w terpenoid compounds. 
     
     
         20 . The method of  claim 19 , wherein said terpenoid compound is farnesene, limonene or both. 
     
     
         21 . A methanotroph bacteria, wherein said methanotroph bacteria comprises a heterologous polynucleotide encoding an enzyme capable of promoting the production of terpenoid or isoprenoid compounds, and wherein said methanotroph bacteria accumulates an increased level of terpenoid or isoprenoid compounds when grown on a C 1  substrate as a carbon source when compared to a wild-type methanotroph bacteria without said heterologous polynucleotide and grown under the same conditions. 
     
     
         22 . The methanotroph bacteria of  claim 21 , wherein said methanotroph bacteria comprise more than about 50% w/w terpenoid compounds. 
     
     
         23 . The methanotroph bacteria of  claim 21 , wherein said terpenoid compound is farnesene, limonene or both. 
     
     
         24 . A methanotroph bacteria, wherein said methanotroph bacteria comprises a heterologous polynucleotide encoding an enzyme capable of promoting the production of isobutanol, and wherein said methanotroph bacteria accumulates an increased level of isobutanol when grown on a C 1  substrate as a carbon source when compared to a wild-type methanotroph bacteria without said heterologous polynucleotide and grown under the same conditions.