Metabolic engineering in chemolithoautotrophic hosts for the production of fuels and chemicals

Nybo, S. Eric; Khan, Nymul E.; Woolston, Benjamin M.; Curtis, Wayne R.

doi:10.1016/j.ymben.2015.04.008

Cited by 87 publications

(45 citation statements)

References 125 publications

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“…In the quest for a cheaper feedstock, synthesis gas offers attractive features. Also, steel mill gas and natural gas can be used as a cost-effective feedstock (12,(17)(18)(19). As such, a syngas-toliquids process represents an infrastructure-compatible platform that will be important for reducing dependence on fossil fuels.…”

Section: Discussionmentioning

confidence: 99%

Integrated bioprocess for conversion of gaseous substrates to liquids

Chakraborty

Kumar

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

155

130

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In the quest for inexpensive feedstocks for the cost-effective production of liquid fuels, we have examined gaseous substrates that could be made available at low cost and sufficiently large scale for industrial fuel production. Here we introduce a new bioconversion scheme that effectively converts syngas, generated from gasification of coal, natural gas, or biomass, into lipids that can be used for biodiesel production. We present an integrated conversion method comprising a two-stage system. In the first stage, an anaerobic bioreactor converts mixtures of gases of CO 2 and CO or H 2 to acetic acid, using the anaerobic acetogen Moorella thermoacetica. The acetic acid product is fed as a substrate to a second bioreactor, where it is converted aerobically into lipids by an engineered oleaginous yeast, Yarrowia lipolytica. We first describe the process carried out in each reactor and then present an integrated system that produces microbial oil, using synthesis gas as input. The integrated continuous bench-scale reactor system produced 18 g/L of C16-C18 triacylglycerides directly from synthesis gas, with an overall productivity of 0.19 g·L −1 ·h −1 and a lipid content of 36%. Although suboptimal relative to the performance of the individual reactor components, the presented integrated system demonstrates the feasibility of substantial net fixation of carbon dioxide and conversion of gaseous feedstocks to lipids for biodiesel production. The system can be further optimized to approach the performance of its individual units so that it can be used for the economical conversion of waste gases from steel mills to valuable liquid fuels for transportation.two-stage bioprocess | lipid production | microbial fermentation | gas-to-liquid fuel | CO 2 fixation C oncerns over diminishing oil reserves and climate-changing greenhouse gas emissions have led to calls for clean and renewable liquid fuels (1). One promising direction has been the production of microbial oil from carbohydrate feedstocks. This oil can be readily converted to biodiesel and recently there has been significant progress in the engineering of oleaginous microbes for the production of lipids from sugars (2-5). A major problem with this approach has been the relatively high sugar feedstock cost. Alternatively, less costly industrial gases containing CO 2 with reducing agents, such as CO or H 2 , have been investigated. In one application, anaerobic Clostridia have been used to convert synthesis gas to ethanol (6), albeit at low concentration requiring high separation cost. Here we present an alternative gas-to-lipids approach that overcomes the drawbacks of previous schemes.We have shown previously that acetate in excess of 30 g/L can be produced from mixtures of CO 2 and CO/H 2 , using an evolved strain of the acetogen Moorella thermoacetica, with a substantial productivity of 0.55 g·L −1 ·h −1 and yield of 92% (7). We also have demonstrated that the engineering of the oleaginous yeast Yarrowia lipolytica can yield biocatalysts that can produce lipids from...

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Section: Discussionmentioning

confidence: 99%

Integrated bioprocess for conversion of gaseous substrates to liquids

Chakraborty

Kumar

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

155

130

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“…In addition to building a symbiosis with ANME to enable methane consumption, sulfate‐reducing bacteria can remediate heavy metals (García, Moreno, Ballester, Blázquez, & González, ; Joo, Choi, Kim, Kim, & Oh, ) and produce plastic precursor storage molecules (Hai, Lange, Rabus, & Steinbüchel, ; Wang, Yin, & Chen, ). Sulfide generated through this process can be used as feedstock for genetically tractable chemoautotrophs (Kernan, West, & Banta, ; Nybo, Khan, Woolston, & Curtis, ), which can make high‐value chemical products (Kernan et al, ). Finding a conductive physical scaffold to enable industrially‐relevant reactions within the context of an ecophysiologicaly sustainable system—whereby microbial communities can take advantage of natural electrical and chemical gradients (e.g., Pfeffer et al, )—would decrease transport and scale‐up costs while building a robust, customizable system.…”

Section: Introductionmentioning

confidence: 99%

“…| 1451 Rabus, & Steinbüchel, 2004;Wang, Yin, & Chen, 2014). Sulfide generated through this process can be used as feedstock for genetically tractable chemoautotrophs (Kernan, West, & Banta, 2017;Nybo, Khan, Woolston, & Curtis, 2015), which can make high-value chemical products (Kernan et al, 2016). Finding a conductive physical scaffold to enable industrially-relevant reactions within the context of an ecophysiologicaly sustainable systemwhereby microbial communities can take advantage of natural electrical and chemical gradients (e.g., Pfeffer et al, 2012)-would decrease transport and scale-up costs while building a robust, customizable system.…”

Section: Introductionmentioning

confidence: 99%

Harnessing a methane‐fueled, sediment‐free mixed microbial community for utilization of distributed sources of natural gas

Marlow¹,

Kumar²,

Enalls³

et al. 2018

Biotech & Bioengineering

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Harnessing the metabolic potential of uncultured microbial communities is a compelling opportunity for the biotechnology industry, an approach that would vastly expand the portfolio of usable feedstocks. Methane is particularly promising because it is abundant and energy‐rich, yet the most efficient methane‐activating metabolic pathways involve mixed communities of anaerobic methanotrophic archaea and sulfate reducing bacteria. These communities oxidize methane at high catabolic efficiency and produce chemically reduced by‐products at a comparable rate and in near‐stoichiometric proportion to methane consumption. These reduced compounds can be used for feedstock and downstream chemical production, and at the production rates observed in situ they are an appealing, cost‐effective prospect. Notably, the microbial constituents responsible for this bioconversion are most prominent in select deep‐sea sediments, and while they can be kept active at surface pressures, they have not yet been cultured in the lab. In an industrial capacity, deep‐sea sediments could be periodically recovered and replenished, but the associated technical challenges and substantial costs make this an untenable approach for full‐scale operations. In this study, we present a novel method for incorporating methanotrophic communities into bioindustrial processes through abstraction onto low mass, easily transportable carbon cloth artificial substrates. Using Gulf of Mexico methane seep sediment as inoculum, optimal physicochemical parameters were established for methane‐oxidizing, sulfide‐generating mesocosm incubations. Metabolic activity required >∼40% seawater salinity, peaking at 100% salinity and 35 °C. Microbial communities were successfully transferred to a carbon cloth substrate, and rates of methane‐dependent sulfide production increased more than threefold per unit volume. Phylogenetic analyses indicated that carbon cloth‐based communities were substantially streamlined and were dominated by Desulfotomaculum geothermicum. Fluorescence in situ hybridization microscopy with carbon cloth fibers revealed a novel spatial arrangement of anaerobic methanotrophs and sulfate reducing bacteria suggestive of an electronic coupling enabled by the artificial substrate. This system: 1) enables a more targeted manipulation of methane‐activating microbial communities using a low‐mass and sediment‐free substrate; 2) holds promise for the simultaneous consumption of a strong greenhouse gas and the generation of usable downstream products; and 3) furthers the broader adoption of uncultured, mixed microbial communities for biotechnological use.

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“…Its metabolic capability has led to its utilization in bioprocesses including copper bioleaching and desulfurization of coal and sour gases (Monticello and Finnerty, 1985). We have been investigating these cells for use as a unique biotechnology platform enabling biofuels and biomass to be produced from carbon dioxide and electricity (termed electrofuels) (Conrado et al, 2013;Ishii et al, 2012;Li and Liao, 2013;Nybo et al, 2015). We have reported the genetic modification of A. ferrooxidans to produce exogenous chemicals (isobutyric acid [IBA] or heptadecane) from carbon dioxide (Kernan et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

Enhancing isobutyric acid production from engineered Acidithiobacillus ferrooxidans cells via media optimization

West²,

Banta³

2015

Biotech & Bioengineering

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The chemolithoautotrophic bacterium Acidithiobacillus ferrooxidans has previously been genetically modified to produce isobutyric acid (IBA) from carbon dioxide while obtaining energy from the oxidation of ferrous iron. Here, a combinatorial approach was used to explore the influence of medium composition in both batch and chemostat cultures in order to improve IBA yields (g IBA/mol Fe(2+)) and productivities (g IBA/L/d). Medium pH, ferrous concentration (Fe(2+)), and inclusion of iron chelators all had positive impact on the IBA yield. In batch experiments, gluconate was found to be a superior iron chelator because its use resulted in smaller excursions in pH. In batch cultures, IBA yields decreased linearly with increases in the final effective Fe(3+) concentrations. Chemostat cultures followed similar trends as observed in batch cultures. Specific cellular productivities were found to be a function of the steady state ORP (Oxidation-reduction potential) of the growth medium, which is primarily determined by the Fe(3+) to Fe(2+) ratio. By operating at low ORP, chemostat cultures were able to achieve volumetric productivities as high as 3.8 ± 0.2 mg IBA/L/d which is a 14-fold increase over the previously reported value.

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Metabolic engineering in chemolithoautotrophic hosts for the production of fuels and chemicals

Cited by 87 publications

References 125 publications

Integrated bioprocess for conversion of gaseous substrates to liquids

Integrated bioprocess for conversion of gaseous substrates to liquids

Harnessing a methane‐fueled, sediment‐free mixed microbial community for utilization of distributed sources of natural gas

Enhancing isobutyric acid production from engineered Acidithiobacillus ferrooxidans cells via media optimization

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