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Title: A novel integrated biorefinery process for diesel fuel blendstock production using lipids from the methanotroph, Methylomicrobium buryatense

Abstract

In light of the availability of low-cost methane (CH 4) derived from natural gas and biogas along with increasing concerns of the greenhouse gas emissions, the production of alternative liquid biofuels directly from CH 4 is a promising approach to capturing wasted energy. A novel biorefinery concept integrating biological conversion of CH 4 to microbial lipids together with lipid extraction and generation of hydrocarbon fuels is demonstrated in this study for the first time. An aerobic methanotrophic bacterium, Methylomicrobium buryatense capable of using CH 4 as the sole carbon source was selected on the basis of genetic tractability, cultivation robustness, and ability to accumulate phospholipids in membranes. A maximum fatty acid content of 10% of dry cell weight was obtained in batch cultures grown in a continuous gas sparging fermentation system. Although phospholipids are not typically considered as a good feedstock for upgrading to hydrocarbon fuels, we set out to demonstrate that using a combination of novel lipid extraction methodology with advanced catalyst design, we could prove the feasibility of this approach. Up to 95% of the total fatty acids from membrane-bound phospholipids were recovered by a two-stage pretreatment method followed by hexane extraction of the aqueous hydrolysate. The upgradingmore » of extracted lipids was then demonstrated in a hydrodeoxygeation process using palladium on silica as a catalyst. Lipid conversion in excess of 99% was achieved, with a full selectivity to hydrocarbons. Lastly, the final hydrocarbon mixture is dominated by 88% pentadecane (C 15H 32) based on decarbonylation/decarboxylation and hydrogenation of C16 fatty acids, indicating that a biological gas-to-liquid fuel (Bio-GTL) process is technically feasible.« less

Authors:
 [1];  [2];  [3];  [1];  [1];  [3];  [1]
  1. National Renewable Energy Lab. (NREL), Golden, CO (United States)
  2. Xi'an Jiaotong Univ., Xi'an (China)
  3. Johnson Matthey Technology Center, Cleveland (United Kingdom)
Publication Date:
Research Org.:
National Renewable Energy Lab. (NREL), Golden, CO (United States)
Sponsoring Org.:
USDOE Advanced Research Projects Agency - Energy (ARPA-E)
OSTI Identifier:
1392737
Alternate Identifier(s):
OSTI ID: 1347277
Report Number(s):
NREL/JA-5100-65377
Journal ID: ISSN 0196-8904
Grant/Contract Number:
AC36-08GO28308; 0670-5169
Resource Type:
Journal Article: Published Article
Journal Name:
Energy Conversion and Management
Additional Journal Information:
Journal Volume: 140; Journal Issue: C; Journal ID: ISSN 0196-8904
Publisher:
Elsevier
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; methahnotroph; fermentation; extraction; hydrotreating; biofuels; biorefinery

Citation Formats

Dong, Tao, Fei, Qiang, Genelot, Marie, Smith, Holly, Laurens, Lieve M. L., Watson, Michael J., and Pienkos, Philip T. A novel integrated biorefinery process for diesel fuel blendstock production using lipids from the methanotroph, Methylomicrobium buryatense. United States: N. p., 2017. Web. doi:10.1016/j.enconman.2017.02.075.
Dong, Tao, Fei, Qiang, Genelot, Marie, Smith, Holly, Laurens, Lieve M. L., Watson, Michael J., & Pienkos, Philip T. A novel integrated biorefinery process for diesel fuel blendstock production using lipids from the methanotroph, Methylomicrobium buryatense. United States. doi:10.1016/j.enconman.2017.02.075.
Dong, Tao, Fei, Qiang, Genelot, Marie, Smith, Holly, Laurens, Lieve M. L., Watson, Michael J., and Pienkos, Philip T. Wed . "A novel integrated biorefinery process for diesel fuel blendstock production using lipids from the methanotroph, Methylomicrobium buryatense". United States. doi:10.1016/j.enconman.2017.02.075.
@article{osti_1392737,
title = {A novel integrated biorefinery process for diesel fuel blendstock production using lipids from the methanotroph, Methylomicrobium buryatense},
author = {Dong, Tao and Fei, Qiang and Genelot, Marie and Smith, Holly and Laurens, Lieve M. L. and Watson, Michael J. and Pienkos, Philip T.},
abstractNote = {In light of the availability of low-cost methane (CH4) derived from natural gas and biogas along with increasing concerns of the greenhouse gas emissions, the production of alternative liquid biofuels directly from CH4 is a promising approach to capturing wasted energy. A novel biorefinery concept integrating biological conversion of CH4 to microbial lipids together with lipid extraction and generation of hydrocarbon fuels is demonstrated in this study for the first time. An aerobic methanotrophic bacterium, Methylomicrobium buryatense capable of using CH4 as the sole carbon source was selected on the basis of genetic tractability, cultivation robustness, and ability to accumulate phospholipids in membranes. A maximum fatty acid content of 10% of dry cell weight was obtained in batch cultures grown in a continuous gas sparging fermentation system. Although phospholipids are not typically considered as a good feedstock for upgrading to hydrocarbon fuels, we set out to demonstrate that using a combination of novel lipid extraction methodology with advanced catalyst design, we could prove the feasibility of this approach. Up to 95% of the total fatty acids from membrane-bound phospholipids were recovered by a two-stage pretreatment method followed by hexane extraction of the aqueous hydrolysate. The upgrading of extracted lipids was then demonstrated in a hydrodeoxygeation process using palladium on silica as a catalyst. Lipid conversion in excess of 99% was achieved, with a full selectivity to hydrocarbons. Lastly, the final hydrocarbon mixture is dominated by 88% pentadecane (C15H32) based on decarbonylation/decarboxylation and hydrogenation of C16 fatty acids, indicating that a biological gas-to-liquid fuel (Bio-GTL) process is technically feasible.},
doi = {10.1016/j.enconman.2017.02.075},
journal = {Energy Conversion and Management},
number = C,
volume = 140,
place = {United States},
year = {Wed Mar 08 00:00:00 EST 2017},
month = {Wed Mar 08 00:00:00 EST 2017}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record at 10.1016/j.enconman.2017.02.075

Citation Metrics:
Cited by: 1work
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  • In light of the availability of low-cost methane (CH 4) derived from natural gas and biogas along with increasing concerns of the greenhouse gas emissions, the production of alternative liquid biofuels directly from CH 4 is a promising approach to capturing wasted energy. A novel biorefinery concept integrating biological conversion of CH 4 to microbial lipids together with lipid extraction and generation of hydrocarbon fuels is demonstrated in this study for the first time. An aerobic methanotrophic bacterium, Methylomicrobium buryatense capable of using CH 4 as the sole carbon source was selected on the basis of genetic tractability, cultivation robustness,more » and ability to accumulate phospholipids in membranes. A maximum fatty acid content of 10% of dry cell weight was obtained in batch cultures grown in a continuous gas sparging fermentation system. Although phospholipids are not typically considered as a good feedstock for upgrading to hydrocarbon fuels, we set out to demonstrate that using a combination of novel lipid extraction methodology with advanced catalyst design, we could prove the feasibility of this approach. Up to 95% of the total fatty acids from membrane-bound phospholipids were recovered by a two-stage pretreatment method followed by hexane extraction of the aqueous hydrolysate. The upgrading of extracted lipids was then demonstrated in a hydrodeoxygeation process using palladium on silica as a catalyst. Lipid conversion in excess of 99% was achieved, with a full selectivity to hydrocarbons. Lastly, the final hydrocarbon mixture is dominated by 88% pentadecane (C 15H 32) based on decarbonylation/decarboxylation and hydrogenation of C16 fatty acids, indicating that a biological gas-to-liquid fuel (Bio-GTL) process is technically feasible.« less
  • Aerobic methanotrophs oxidize methane at ambient temperatures and pressures and are therefore attractive systems for methane-based bioconversions. In this work, we developed and validated genetic tools for Methylomicrobium buryatense, a haloalkaliphilic gammaproteobacterial (type I) methanotroph. M. buryatense was isolated directly on natural gas and grows robustly in pure culture with a 3-h doubling time, enabling rapid genetic manipulation compared to many other methanotrophic species. As a proof of concept, we used a sucrose counterselection system to eliminate glycogen production in M. buryatense by constructing unmarked deletions in two redundant glycogen synthase genes. We also selected for a more genetically tractablemore » variant strain that can be conjugated with small incompatibility group P (IncP)-based broad-host-range vectors and determined that this capability is due to loss of the native plasmid. These tools make M. buryatense a promising model system for studying aerobic methanotroph physiology and enable metabolic engineering in this bacterium for industrial biocatalysis of methane.« less
  • Methane is a feedstock of interest for the future, both from natural gas and from renewable biogas sources. Methanotrophic bacteria have the potential to enable commercial methane bioconversion to value-added products such as fuels and chemicals. A strain of interest for such applications is Methylomicrobium buryatense 5GB1, due to its robust growth characteristics. But, to take advantage of the potential of this methanotroph, it is important to generate comprehensive bioreactor-based datasets for different growth conditions to compare bioprocess parameters. The datasets of growth parameters, gas utilization rates, and products (total biomass, extracted fatty acids, glycogen, excreted acids) were obtained formore » cultures of M. buryatense 5GB1 grown in continuous culture under methane limitation and O2 limitation conditions. Additionally, experiments were performed involving unrestricted batch growth conditions with both methane and methanol as substrate. All four growth conditions show significant differences. The most notable changes are the high glycogen content and high formate excretion for cells grown on methanol (batch), and high O2:CH4 utilization ratio for cells grown under methane limitation. The results presented here represent the most comprehensive published bioreactor datasets for a gamma-proteobacterial methanotroph. This information shows that metabolism by M. buryatense 5GB1 differs significantly for each of the four conditions tested. O2 limitation resulted in the lowest relative O2 demand and fed-batch growth on methane the highest. Future studies are needed to understand the metabolic basis of these differences. However, these results suggest that both batch and continuous culture conditions have specific advantages, depending on the product of interest.« less
  • Methane utilization by methanotrophic bacteria is an attractive application for biotechnological conversion of natural or biogas into high-added-value products. Haloalcaliphilic methanotrophic bacteria belonging to the genus Methylomicrobium are among the most promising strains for methane-based biotechnology, providing easy and inexpensive cultivation, rapid growth, and the availability of established genetic tools. A number of methane bioconversions using these microbial cultures have been discussed, including the derivation of biodiesel, alkanes, and OMEGA-3 supplements. These compounds are derived from bacterial fatty acid pools. Here, we investigate fatty acid biosynthesis in Methylomicrobium buryatense 5G(B1). Most of the genes homologous to typical Type II fattymore » acid biosynthesis pathways could be annotated by bioinformatics analyses, with the exception of FA transport and regulatory elements. Different approaches for improving fatty acid accumulation were investigated. These studies indicated that both fatty acid degradation and acetyl- and malonyl-CoA levels are bottlenecks for higher level fatty acid production. The best strain generated in this study synthesizes 111 ± 2 mg/gDCW of extractable fatty acids, which is ~20% more than the original strain. A candidate gene for FA-biosynthesis regulation, farE, was identified and studied. Its deletion resulted in drastic changes to the FA profile, leading to an increased pool of C18-fatty acid methyl ester. The FarE-regulon was further investigated by RNA-seq analysis of gene expression in farE-knockout mutants and farE-overexpressing strains. These gene profiles highlighted a novel set of enzymes and regulators involved in fatty acid biosynthesis. As a result, the gene expression and fatty acid profiles of the different farE-strains support the hypothesis that metabolic fluxes upstream of fatty acid biosynthesis restrict fatty acid production in the methanotroph.« less
  • Many methylotrophs, microorganisms that consume carbon compounds lacking carbon–carbon bonds, use two different systems to oxidize methanol for energy production and biomass accumulation. The MxaFI methanol dehydrogenase (MDH) contains calcium in its active site, while the XoxF enzyme contains a lanthanide in its active site. The genes encoding the MDH enzymes are differentially regulated by the presence of lanthanides. In this study, we found that the histidine kinase MxaY controls the lanthanide-mediated switch in Methylomicrobium buryatense 5GB1C. MxaY controls the transcription of genes encoding MxaFI and XoxF at least partially by controlling the transcript levels of the orphan response regulatormore » MxaB. We identify a constitutively active version of MxaY, and identify the mutated residue that may be involved in lanthanide sensing. Finally, we find evidence to suggest that tight control of active MDH production is required for wild-type growth rates.« less