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 C 1  metabolizing non-photosynthetic microorganism, wherein said C 1  metabolizing non-photosynthetic 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 said C 1  metabolizing non-photosynthetic microorganism accumulates an increased level of fatty acids when grown on a C 1  substrate as a carbon source when compared to a wild-type C 1  metabolizing non-photosynthetic microorganism without said heterologous polynucleotide and grown under the same conditions. 
     
     
         2 . The C 1  metabolizing non-photosynthetic microorganism of  claim 1 , wherein said C 1  metabolizing non-photosynthetic microorganism is a bacteria or a yeast. 
     
     
         3 . The C 1  metabolizing non-photosynthetic microorganism of  claim 2 , wherein said recombinant C 1  metabolizing bacteria is a methylotroph. 
     
     
         4 . The C 1  metabolizing non-photosynthetic microorganism of  claim 3 , wherein said methylotroph is selected from the group consisting of  Methylobacterium extorquens, Methylobacterium radiotolerans, Methylobacterium populi, Methylobacterium chloromethanicum, Methylobacterium nodulans,  and a combination thereof. 
     
     
         5 . The C 1  metabolizing non-photosynthetic microorganism of  claim 2 , wherein said C 1  metabolizing bacteria is a syngas metabolizing bacteria. 
     
     
         6 . The C 1  metabolizing non-photosynthetic microorganism of  claim 5 , wherein said syngas metabolizing bacteria is selected from the group consisting of  Clostridium autoethanogenum, Clostridium ljungdahli, Clostridium ragsdalei, Clostridium carboxydivorans, Butyribacterium methylotrophicum, Clostridium woodii, Clostridium neopropanologen,  and a combination thereof. 
     
     
         7 . The C 1  metabolizing non-photosynthetic microorganism of  claim 1 , wherein said heterologous polynucleotide encoding said thioesterase is codon optimized for expression in said C 1  metabolizing non-photosynthetic microorganism. 
     
     
         8 . The C 1  metabolizing non-photosynthetic microorganism of  claim 1 , wherein said heterologous polynucleotide encoding said malonyl CoA-acyl carrier protein transacylase is an  E. coli  fabD and wherein said heterologous polynucleotide is codon optimized for expression in said C 1  metabolizing non-photosynthetic microorganism. 
     
     
         9 . The C 1  metabolizing non-photosynthetic microorganism of  claim 1 , wherein said heterologous polynucleotide encoding said acetyl-CoA carboxylase is an  E. coli  accA, accB, accC, accD, or any combination thereof and wherein said heterologous polynucleotide is codon optimized for expression in said C 1  metabolizing non-photosynthetic microorganism. 
     
     
         10 . The C 1  metabolizing non-photosynthetic microorganism of  claim 1 , wherein said C 1  metabolizing non-photosynthetic microorganism further comprises a mutation that minimizes or eliminates fatty acid-CoA ligase activity, wherein said mutation is in an endogenous fatty acid-CoA ligase gene. 
     
     
         11 . A method for making an oil composition, comprising converting a biomass into said oil composition, wherein said biomass comprises (a) a culture of said C 1  metabolizing non-photosynthetic microorganism of  claim 1  together with a culture media in which said C 1  metabolizing non-photosynthetic microorganism were grown; (b) said C 1  metabolizing non-photosynthetic microorganism of  claim 1  recovered from said culture media; or (c) a spent media composition recovered from said culture media comprising the C 1  metabolizing non-photosynthetic microorganism of  claim 1 . 
     
     
         12 . The method of  claim 11 , wherein said C 1  substrate is selected from the group consisting of natural gas, unconventional natural gas, methane, a methylamine, a methylthiol, a methylhalogen, and any combination thereof. 
     
     
         13 . The method of  claim 11 , wherein said biomass comprises said C 1  metabolizing non-photosynthetic microorganism recovered from said culture media. 
     
     
         14 . The method of  claim 11 , wherein said biomass comprises said recovered spent media composition and said recovered spent media composition is converted into said oil composition by extraction or concentration of said recovered spent media composition. 
     
     
         15 . The method of  claim 11 , wherein said oil composition is extracted from said biomass contains cell membranes of said C 1  metabolizing non-photosynthetic microorganism, is extracted from a culture supernatant, or a combination thereof. 
     
     
         16 . The method of  claim 11 , wherein said C 1  metabolizing non-photosynthetic microorganism is cultured in a controlled culture unit selected from the group of a fermentor, a bioreactor, a hollow fiber cell, a packed bed bioreactor, and any combination thereof. 
     
     
         17 . The method of  claim 16 , wherein said C 1  metabolizing non-photosynthetic microorganism is cultured in a bioreactor comprising balanced media or cultured in a bioreactor comprising unbalanced media having limiting quantities of phosphorus, nitrogen, trace elements, oxygen relative to a balanced media, or any combination thereof. 
     
     
         18 . The method of  claim 11 , wherein said biomass is converted into said oil composition by extraction. 
     
     
         19 . The method of  claim 18 , wherein said extraction is by high-shear contact with an organic solvent and a conditioning agent. 
     
     
         20 . The method of  claim 18 , wherein said extraction is selected from the group consisting of wet extraction, supercritical fluid extraction, dry extraction, thermal extraction, enzymatic hydrolysis extraction, pulsed electric field extraction, microbubble extraction, and hollow fiber extraction. 
     
     
         21 . The method of  claim 11 , wherein said oil composition comprises at least 50% w/w fatty acids. 
     
     
         22 . The method of  claim 21 , wherein said fatty acids are free fatty acids. 
     
     
         23 . The method of  claim 21 , wherein said fatty acids comprise a mixture of diacylglycerides and triacylglycerides. 
     
     
         24 . The method of  claim 21 , wherein a majority of said fatty acids comprise carbon chain lengths of C14 to C18. 
     
     
         25 . The method of  claim 21 , wherein a majority of said fatty acids comprise carbon chain lengths of C16 to C18. 
     
     
         26 . The method of  claim 21 , wherein a majority of said fatty acids comprise carbon chain lengths of less than C16. 
     
     
         27 . The method of  claim 11 , wherein said oil composition comprises at least 50% w/w terpenoid compounds, isoprenoid compounds, or a combination thereof. 
     
     
         28 . The method of  claim 27 , wherein said terpenoid is farnesene. 
     
     
         29 . The method of  claim 27 , wherein said terpenoid is limonene. 
     
     
         30 . The method of  claim 11 , further comprising hydrotreatment to produce light hydrocarbons, wherein said light hydrocarbons are selected from the group consisting of methane, methanol, ethane, ethanol, propane, propanol, butane, pentane, butanol, and isobutanol. 
     
     
         31 . The method of  claim 30 , wherein said light hydrocarbon is methane, ethane, propane, butane, or pentane. 
     
     
         32 . The method of  claim 30 , wherein said light hydrocarbon is butanol or isobutanol.