- Open Access
Bioconversion of distillers’ grains hydrolysates to advanced biofuels by an Escherichia coli co-culture
© The Author(s) 2017
- Received: 8 June 2017
- Accepted: 31 October 2017
- Published: 9 November 2017
First generation bioethanol production utilizes the starch fraction of maize, which accounts for approximately 60% of the ash-free dry weight of the grain. Scale-up of this technology for fuels applications has resulted in a massive supply of distillers’ grains with solubles (DGS) coproduct, which is rich in cellulosic polysaccharides and protein. It was surmised that DGS would be rapidly adopted for animal feed applications, however, this has not been observed based on inconsistency of the product stream and other logistics-related risks, especially toxigenic contaminants. Therefore, efficient valorization of DGS for production of petroleum displacing products will significantly improve the techno-economic feasibility and net energy return of the established starch bioethanol process. In this study, we demonstrate ‘one-pot’ bioconversion of the protein and carbohydrate fractions of a DGS hydrolysate into C4 and C5 fusel alcohols through development of a microbial consortium incorporating two engineered Escherichia coli biocatalyst strains.
The carbohydrate conversion strain E. coli BLF2 was constructed from the wild type E. coli strain B and showed improved capability to produce fusel alcohols from hexose and pentose sugars. Up to 12 g/L fusel alcohols was produced from glucose or xylose synthetic medium by E. coli BLF2. The second strain, E. coli AY3, was dedicated for utilization of proteins in the hydrolysates to produce mixed C4 and C5 alcohols. To maximize conversion yield by the co-culture, the inoculation ratio between the two strains was optimized. The co-culture with an inoculation ratio of 1:1.5 of E. coli BLF2 and AY3 achieved the highest total fusel alcohol titer of up to 10.3 g/L from DGS hydrolysates. The engineered E. coli co-culture system was shown to be similarly applicable for biofuel production from other biomass sources, including algae hydrolysates. Furthermore, the co-culture population dynamics revealed by quantitative PCR analysis indicated that despite the growth rate difference between the two strains, co-culturing didn’t compromise the growth of each strain. The q-PCR analysis also demonstrated that fermentation with an appropriate initial inoculation ratio of the two strains was important to achieve a balanced co-culture population which resulted in higher total fuel titer.
The efficient conversion of DGS hydrolysates into fusel alcohols will significantly improve the feasibility of the first generation bioethanol process. The integrated carbohydrate and protein conversion platform developed here is applicable for the bioconversion of a variety of biomass feedstocks rich in sugars and proteins.
- Distillers’ grains with solubles (DGS)
- Microbial co-culture
- One-pot bioconversion
- Fusel alcohol
- Algae hydrolysate
Global bioethanol production reached 25.7 billion gallons in 2015 , with further increase in annual production projected. During the ethanol refining process, starch in the grain flour is converted into ethanol and the remainder of the grain components, such as proteins, lipids and fibers comprise a residual coproduct, commonly known as distillers’ grains with solubles (DGS) . It is estimated that in the dry milling process, the utilization of a bushel of corn (56 lb) results in 2.8 gallon of ethanol and 18 lb of DGS . In 2015, 40 million metric tons of DGS were produced from US ethanol biorefineries . DGS is considered as a rich source of cellulosic polysaccharides (52–57%), protein (27–31%), oil (10–12%) and other nutrients  and has long been marketed as a ruminant feed adjunct. However, due to the variability in nutrient content and digestibility issues as well as other concerns such as mycotoxins, antibiotic residues, sulphur content and the risk of introducing bacterial pathogens , acceptance of DGS in the feed industry has been limited. Alternatively, because of its vast supply and sugar and protein content, DGS is a potentially promising biomass source for upgrading to valuable fuel products using bioconversion strategies that are compatible with the established starch ethanol process. Therefore, efficient valorization of DGS to produce value-added products would significantly improve the techno-economic feasibility of the established starch bioethanol process.
Recent advances in synthetic biology, metabolic engineering, and systems biology, have enabled rapid progress in developing microbial factories [6–8] and novel enzyme cascade systems [9–11] for the synthesis of biofuels and other chemicals. When considering a microbial system for biomass conversion, although there are successful examples in developing ‘superbugs’ capable of multiple functions, engineering a single microbe to simultaneously perform multiple tasks is still quite challenging and bioenergetically costly under most situations, especially when utilizing complex substrates or performing complicated biosynthesis. Alternatively, well-designed microbial consortia involving two or more microbes that can take advantage of individual microbes and their interactions to realize synergistic division of labor and more efficient utilization of biochemical substrates, and therefore exhibit better properties than monocultures, should provide enhanced productivity, stability or metabolic efficiency [12–14].
Strains and plasmids
Escherichia coli strain B (ATCC 11303) was purchased from ATCC. E. coli AY3 was previously developed in our lab . The mutant strain E. coli B01 with single deleted gene Δldh::cam+ was constructed using the technique of one-step disruption of chromosomal genes  using primers 5′-GGATGGCGATACTCTGCCATCCGTAATTTTTACTCCACTTCCTGCCAGTTTGTGTAGGCTGGAGCTGCTTC-3′ and 5′-CGCTATTCTAGTTTGTGATATTTTTTCGCCACCACAAGGAGTGGAAAATGTGACATGGGAATTAGCCATGGTCC-3′ from E. coli B strain.
To construct pLF101, part of the ilvD gene was PCR amplified using primers 5′-GTAAAAAATATGTTCCGCGCAGGTCC-3′ and 5′-TTTATTTGATGCCTCTAGCACGCGTACGCGTTTAACCCCCCAGTTTC-3′ using pYX90  (generously provided by Professor James C. Liao from University of California, Los Angeles) as the template. The rrnB T1 terminator was amplified using primers 5′-ACGCGTGCTAGAGGCATCAAATAAAAC-3′ and 5′-AGTGAGCGAGGAAGCGGAATATATC-3′ using pYX90 as the template. Then the two fragments were assembled with SbfI and AvrII digested pYX90 to achieve pLF101-alaS-ilvC-ilvD using In-Fusion® HD Cloning Kit (Clontech, CA) following the manufacture’s protocol. To construct pLF102, part of the AmpR gene and pLacO1 region was amplified using primers 5′-GCAAAAAAGCGGTTAGCTCCTTCG-3′ and 5′-CTCCTACTGTATACATGGTATATCTCCTTGTCGACAATGAATTCGGTCAGTGCGTCCTG-3′. The PCR fragment was assembled with PvuI and SalI digested pYX97  (generously provided by Professor James C. Liao from University of California, Los Angeles) using In-Fusion® HD Cloning Kit (Clontech, CA). The DNA sequences of the constructs were confirmed by DNA sequencing. The plasmids pLF101 and pLF102 were co-transformed into the E. coli strain B01, which generated the production strain E. coli BLF2.
Medium and culture conditions
M9 medium containing 40 g/L glucose or 40 g/L xylose or 20 g/L glucose and 20 g/L xylose, 5 g/L yeast extract, 100 µg/mL ampicillin, 34 µg/mL chloramphenicol, and 25 µg/mL spectinomycin, and 1000th dilution of Trace Metal Mix A5 (Sigma-Aldrich, MO) was used for cell growth. The cells were inoculated in 3 mL medium in the test tube and incubated at 37 °C overnight at 250 rpm. 200 µL of the overnight culture was inoculated into 20 mL fresh medium in the shake flask and incubated at 37 °C, 250 rpm. 1 mM isopropyl-β-d-thio-galactoside (IPTG) was added when OD600 reached 0.8. Then the culture was grown at 30 °C and 250 rpm for 2 days. Samples were collected at regular time intervals for further analysis.
Biomass pretreatment and fermentation
The distillers’ grains samples (35% solids) were provided by Aemetis, Inc. (Cupertino, CA) and pretreated following the protocols from the National Renewable Energy Laboratories. Briefly, DGS were mixed with 4% dilute sulfuric acid to a final concentration of 8.5% (w/v) solid. Then the mixture was incubated in the 90 °C water bath for 5 h and neutralized with Ca(OH)2 solids until pH reached 6.5. The resulting slurry was subsequently hydrolyzed with 1.5 mg/mL Pronase (Sigma-Aldrich, MO) following the manufacture’s protocol. After enzymatic digestion, the slurry was centrifuged and the supernatant was sterilized by filtration through the 0.22 µm PTFE membrane (Fisher Scientific, CA). Nannochloropsis sp. algae samples were pretreated similarly but incubated with 10% sulfuric acid. The resulting hydrolysates were used directly as the medium for cell growth and fusel alcohol production with no additional supplements.
Escherichia coli BLF2 cells were cultivated in 10 mL LB medium and grown at 37 °C, 250 rpm. The overnight culture was centrifuged at 4000 rpm for 10 min and the cell pellets were collected and washed with corresponding hydrolysates twice and resuspended in 1 mL hydrolysates. 0.8 mL of the mixture was inoculated into 20 mL DGS or algae hydrolysates supplemented with 100 µg/mL ampicillin, 34 µg/mL chloramphenicol, and 25 µg/mL spectinomycin. The culture was incubated at 37 °C, 250 rpm and induced with 1 mM IPTG when the OD600 reached 0.8. The flasks were cap-sealed and cultured for another 48 h at 30 °C, 250 rpm for fusel alcohol production. Samples were taken at the beginning and end of the fermentation for further analysis.
For co-culture fermentation, E. coli strains AY3 and BLF2 were cultivated in 10 mL LB medium separately. The overnight culture was centrifuged and the final cell pellets were individually re-suspended into 0.5–1 mL hydrolysates and were both adjusted to the same OD600. Then various ratios of AY3 and BLF2 cells (0.5:1, 1:1, 1.5:1, 2:1, etc.) were inoculated into the DGS or algae hydrolysates at a final concentration of 20% (v/v). The induction and fermentation was performed as described above.
To determine the concentrations of glucose, xylose and arabinose in the medium, as well as the products such as isobutanol and ethanol, culture of the grown cells was centrifuged at 13,000 rpm for 10 min and 5 mL of the supernatant was injected into an Agilent HPLC system (1100 Series) equipped with the Rezex ROA-Organic Acid Sugar column (Phenomenex, CA). Other fusel alcohols, including 2-methyl-1-butanol, 3-methyl-1-butanol, 2-phenylethanol were extracted with ethyl acetate at the ratio of 1:1 (fermentation broth: ethyl acetate) with 2-methyl-1-pentanol as the internal reference. The ethyl acetate layer was collected for GC–MS analysis. One microliter of sample was injected into the injection port (250 °C) of an Agilent gas chromatography 6890N equipped with a 30 m × 0.25 mm DB-WAXetr capillary column with a film thickness of 0.5 µm. The temperature of the column was programmed as follows: 40 °C for 4 min, increasing to 65 °C at 10 °C/min and holding for 10 min, then increasing to 300 °C at 65 °C/min and holding for 5 min. The carrier gas was ultra-high purity helium at a constant flow rate of 1.5 mL/min. The chromatograph was coupled to a quadrupole MS 5975B. Spectral components were searched against the Wiley275 mass spectral library.
The total amino acids and proteins in the pretreated DGS and Nannochloropsis sp. hydrolysates before and after fermentation were determined using the ninhydrin assay . The total carbohydrates in algae hydrolysates were determined by the phenol–sulfuric acid method  using a glucose standard.
Real time quantitative PCR
Primers for the species-specific sequences of BLF2 and AY3 strains were designed for the quantitative PCR reaction. Primers 5′-GCTTTAATGAGTGGAATCGCC-3′ and 5′-GATGCAATGTTCTGGCTAACG-3′ were used to specifically amplify the agaE gene of E. coli BLF2 strain and primers 5′-GTGGAAAGAGGGCGATAAGAG-3′ and 5′-TCATGACGTTGGTAGAAGCG-3′ were used for the specific amplification of the malB gene of AY3 strain.
The q-PCR assays were carried out with the CFX96 Real-time PCR system with a C1000 Thermal Cycler (Bio-Rad, CA). The reaction mixture of 20 µL final volumes contained 1 µL DNA template, 0.15 µM each respective primer, and 10 µL of SYBR Green Master Mix (Bio-Rad, CA). All amplifications were carried out in optical grade 96 well plates (Fisher Scientific, MA) with an initial step at 98 °C for 3 min followed by 35 cycles of 98 °C for 15 s, 59 °C for 30 s. At the completion of each run, melting curves for the amplicons were measured by raising the temperature 0.5 °C from 65 to 95 °C while monitoring fluorescence. The specificity of the PCR amplification was checked by examining the melting curve for Tm and the lack of non-specific peaks. All tests were conducted in triplicate.
Cell number determination in the co-culture
The cell numbers of E. coli BLF2 and AY3 in the co-culture were determined by the PCR-based multiple species cell counting method as described by Huang et al. . To prepare the reference mixed samples, E. coli BLF2 and AY3 were grown overnight in 3 mL LB medium respectively. Then their individual colony forming units per mL (CFU/mL) were determined using serial dilutions and plating method. The genomic DNA of the individual samples was extracted using the Quick-DNA Fungal/Bacterial Miniprep Kit (Zymo Research, CA) and the same amount (by volume) of DNA solution extracted from the two species was mixed and the threshold cycle C T,R was determined by quantitative PCR. For the unknown mixed samples, the genomic DNA of 2 mL fermentation culture was extracted and q-PCR was performed to determine the C T,X as described above.
Bacterial strains and plasmids used in this study
pSC101 ori; ChlR; PrrnB; ilvE-ilvA-sdaB
p15A ori; SpectR; P LlacO1; alsS-ilvC-ilvD-avtA
ColE1 ori; AmpR; P LlacO1; leuDH-kivd-yqhD
p15A ori; SpectR; P LlacO1; alsS-ilvC-ilvD
ColE1 ori; AmpR; P LlacO1; kivd-yqhD
E. coli DH5α
E. coli YH40
BW25113/F′ [traD36, proAB +, lacIq ZΔM15] derivative with enhanced ability of amino acid utilization and with ΔglnA, ΔgdhA, ΔlsrA
E. coli AY3
E. coli YH40 with plasmids pYX68, pYX90 with the mutant genes and pYX97 with the mutant genes
E. coli B
E. coli B01
E. coli B Δldh:: cam+
E. coli BLF2
E. coli B01 with plasmids pLF101 and pLF102
Escherichia coli strain B (ATCC 11303) was selected as the wild type in this study for constructing the fusel alcohol production strain for carbohydrate utilization, because of its natural ability to metabolize glucose as well as xylose sugars . Therefore, this strain offers the opportunity to convert both hexose and pentose sugars present in the DGS hydrolysates. First, the gene encoding lactate dehydrogenase (ldh) was deleted from the chromosome of E. coli strain B using the technique of one-step disruption of chromosomal genes and was replaced with the chloramphenicol resistance gene (CmR) from the plasmid pKD3 . The resulting strain E. coli B01 had resistance to chloramphenicol, which enabled it to be co-cultured with the protein conversion strain E. coli AY3 that requires three antibiotic selectable markers (CmR, AmpR, SmR) to retain the plasmids. Two plasmids for introducing the pathway into E. coli B01 strain for isobutanol production from 2-keto acid precursors were constructed. Plasmid pLF101(SmR) contained the genes encoding for acetolactate synthase (AlsS) from Bacillus subtilis, acetohydroxy acid isomeroreductase (IlvC) and dihydroxyacid dehydratase (IlvD) from E. coli  and the second plasmid pLF102 (AmpR) contained the genes encoding for 2-ketoacid decarboxylase (Kdc) from Lactococcus lactis and alcohol dehydrogenase (Adh) from E. coli . These two plasmids were co-transformed into E. coli B01 strain and the resulting strain. Escherichia coli BLF2 (Table 1) overexpressed the five genes involved in the isobutanol production pathway. Therefore, pyruvate produced from glucose and xylose is converted by AlsS, IlvC and IlvD to 2-ketoisovalerate (KIV) which is further converted to isobutanol by Kdc and Adh (Additional file 1: Figure S1). Although Kdc from L. lactis has the highest specific activity towards 2-ketoisovalerate, it can also utilize several other 2-keto acids as substrates with lower specific activities . Therefore, besides isobutanol, other fusel alcohols such as 2-methyl-1-butanol and 3-methyl-1-butanol may also be produced from other 2-keto acid precursors such as 2-ketoisocaproate (KIC) and 2-ketomethylvalerate (KMV) respectively by Kdc and Adh (Additional file 1: Figure S1).
Fermentation of glucose and xylose sugars by E. coli BLF2
DGS fermentation by E. coli BLF2
‘One-pot’ bioconversion of DGS hydrolysate by E. coli–E. coli co-cultures
Based on the techno-economic impact of reducing unit operations and increasing net conversion yields of the whole biomass hydrolysate, we investigated the feasibility of simultaneous bioconversion of protein and carbohydrate fractions in a ‘one-pot’ fermentation by co-culturing the two strains E. coli BLF2 and AY3. In the co-culture, E. coli BLF2 was dedicated for conversion of hexose and pentose sugars in DGS hydrolysates into C4 and C5 fusel alcohols and E. coli AY3 was designated to convert DGS proteins into C4 and C5 fusel alcohols (Fig. 1).
Bioconversion of the algae hydrolysates by E. coli co-cultures
Dynamics of the co-culture by q-PCR analysis
To differentiate BLF2 and AY3 strain in the co-culture and to monitor the cell number of each species during fermentation, specific primers targeting the unique genes in the chromosome of BLF2 and AY3 strain were designed. Although the E. coli strains have high nucleotide sequence homology and similar genome organization, BLF2 was engineered from wild-type B strain while AY3 was derived from the K-12 strain. E. coli B strain is deficient for malB gene encoding for the maltose high affinity receptor which is present in the K-12 strain , while K-12 strain lacks the IID domain of the N-acetyl-galactosamine transporter (agaE) . Therefore, the primers specific for malB and agaE were used to specifically target AY3 and BLF2 respectively. The specificity of the primers and validation of the q-PCR test was confirmed (Additional file 1: Figure S2). The parameters needed for calculating the cell numbers of BLF2 and AY3 in the co-culture as described in “Methods” section were also determined (Additional file 1: Figure S3, Table S1).
Individual populations of BLF2 and AY3 in the co-culture at the end of fermentation of DGS hydrolysates based on q-PCR analysis
Initial BLF2/AY3 inoculation ratio
Average cell number (cell/mL) in the co-culture at 52 h
Final BLF2/AY3 ratio in the co-culture at 52 h
3.2 × 1010
1.8 × 1010
4.1 × 108
2.7 × 1010
6.9 × 108
8.2 × 109
5.6 × 109
1.9 × 109
1.6 × 109
3.1 × 109
Individual populations of BLF2 and AY3 in the co-culture at the end of fermentation of algae hydrolysates based on q-PCR analysis
Initial BLF2/AY3 inoculation ratio
Average cell number (cell/mL) in the co-culture at 48 h
Final BLF2/AY3 ratio in the co-culture at 48 h
2.0 × 109
3.5 × 109
5.9 × 107
2.8 × 109
8.8 × 107
1.9 × 109
9.3 × 107
1.5 × 109
2.5 × 108
1.5 × 109
4.0 × 108
9.6 × 108
2.8 × 108
1.5 × 109
4.6 × 108
1.2 × 109
6.5 × 108
7.4 × 108
DGS, the major coproduct from the bioethanol industry, is produced in large and increasing quantities annually. Efficient valorization of DGS to support starch bioethanol process viability requires processes to convert both of the major DGS biochemical pools—proteins and carbohydrates—to value-added products. In this study, we developed a microbial factory to convert both the protein and carbohydrate fractions of DGS to advanced biofuels. These results should support improvement of the techno-economic feasibility and net energy return of the first-generation bioethanol process since up to ~ 30% more fuel products can be produced from the same amount of corn. This integrated carbohydrate and protein conversion platform is versatile for the bioconversion of other carbohydrate and protein rich biomass, which was demonstrated using microalgae biomass. The mixed fusel alcohols that were produced contained primarily isobutanol and other higher carbon numbers alcohols, including 2-methyl-1-butanol, 3-methyl-1-butanol and 2-phenylethanol. It was previously shown that mixed alcohol forms (especially C3–C5) provide increased energy densities and other improved physical properties (e.g. reduced water solubility and corrosivity) than ethanol which can provide increased combustion efficiencies, reduced emission profiles, and improved compatibility with the existing liquid fuels infrastructure . Therefore, mixed fusel alcohols have promising potential applications as a fuel blendstock in gasoline, diesel, jet fuel, heating oil or as a neat fuel of itself.
The microbial co-culture developed here, specifically the carbohydrate conversion strain and the protein conversion strain, allows the microbes to utilize multiple substrates and accomplish complex biosynthesis that is difficult to achieve by a single cell. Also it allows division of labor and reduction of the metabolic burden on each cell type. The isobutanol produced from glucose by the carbohydrate conversion strain E. coli BLF2 is higher than that which has been reported from a previous other study where the E. coli production strain included deletion of six genes involved in byproducts formation , suggesting superior capacity of E. coli strain B as a host for isobutanol production. We envision that the isobutanol yield from E. coli BLF2 strain can be further increased by optimizing the process conditions and strain engineering, e.g. deleting the competing pathways for the byproducts and removing any bottlenecks from the pathway.
In terms of protein conversion, up to 30% of proteins from both the DGS and algae hydrolysates were converted by the co-culture. The incomplete protein conversion in both hydrolysates is probably due to several facts. Firstly, the pretreated hydrolysates were directly used as the fermentation broth which may lack of some of the trace nutrients as in synthetic medium such as LB broth/; Additionally, potential fermentation inhibitors such as weak acids and furan derivatives  present in the hydrolysates may have inhibitory effect on the E. coli strain. Secondly, the protein conversion strain AY3 can only utilize 13 individual amino acids as the sole carbon source  which leads to the incomplete consumption of the proteins in the hydrolysates. Moreover, the carbohydrates present in the hydrolysates were also utilized as the carbon source for growth by AY3, which may reduce the consumption rate of proteins in the hydrolysates by the protein conversion strain AY3 compared with when only protein was available as the sole carbon source. This could be improved by using different inoculation strategies, i.e., inoculating AY3 following BLF2 in the co-culture when sugars in the hydrolysates are mostly consumed by the carbohydrate conversion strain BLF2 during fermentation. However, the fusel alcohol amino acid preference favors high abundance, low value amino acids such as glutamate and alanine ; therefore, isolation of the unutilized high value amino acids such as lysine, methionine and phenylalanine may provide a path toward a secondary high value co-product stream to further facilitate the process economics.
The q-PCR based cell quantification method developed here is a versatile tool for rapidly tracking the individual population in the mixed culture during fermentation. The protein conversion strain AY3 has much lower growth rate than the carbohydrate conversion strain BLF2, which is probably due to the fact that AY3 has more genetic modifications than BLF2, including deletion of several genomic genes [17, 19]. Previous studies have showed that multiple genomic deletions could cause decreases in the cell growth rate of E. coli . When co-culturing two strains whose growth rates differ substantially, it’s likely that one species become the dominant population and therefore the population ratios often have to be optimized to obtain a stable culture so that one cell type does not eliminate the other . In the case of our co-culture, although BLF2 cells did grow faster than AY3, the co-culturing of the two species didn’t eliminate the growth of AY3 (Fig. 8). This is probably due to the fact that BLF2 and AY3 don’t compete for pentose sugar and proteins as carbon source for growth, although they can both utilize glucose. Therefore, their substrate specificity allows the two strains to establish a stable co-culture system. Furthermore, the co-cultures at certain initial BLF2/AY3 inoculation ratios produced higher amount of fusel alcohols from the hydrolysates than others (Figs. 6a, 7a); q-PCR analysis clearly indicated that the difference between the cell numbers of BLF2 and AY3 in these co-cultures was minimized. The population dynamics analysis of the co-culture in this study demonstrated that changing the initial inoculation ratio is a simple and effective way to tune the co-culture population and that an optimized co-culture population is vital to achieve higher production yield by the engineered E. coli consortium.
We demonstrated ‘one-pot’ bioconversion of the DGS hydrolysate into fusel alcohols using a microbial co-culture strategy incorporating two engineered E. coli strains. The carbohydrate conversion strain E. coli BLF2 was constructed from the wild type strain B and showed improved capability to produce fusel alcohols from hexose and pentose sugars compared to previous efforts. The co-culture with an inoculation ratio of 1:1.5 of E. coli BLF2 and AY3 achieved the highest total fuel titer of up to 10.3 g/L from DGS hydrolysates. Moreover, the integrated carbohydrate and protein conversion platform is also applicable for the bioconversion of other multi-substrate biomass such as algae hydrolysates. The detailed population dynamics study suggested that an optimized co-culture population ratio lead to more efficient ‘one-pot’ bioconversion of multiple substrates in the hydrolysates.
FL designed and carried out the experiments, analyzed the data and prepared the manuscript. WW gave valuable suggestions in the experiments and manuscript editing. MBT, JDJ and XZ participated in the experiments. RWD supervised the research and revised the manuscript. All authors read and approved the final manuscript.
This research was conducted as part of the Co-Optimization of Fuels & Engines (Co-Optima) Project sponsored by the Bioenergy Technologies and Vehicle Technologies Offices, Office of Energy Efficiency and Renewable Energy (EERE), US Department of Energy (DOE). Co-Optima is a collaborative project of multiple national laboratories initiated to simultaneously accelerate the introduction of affordable, scalable, and sustainable biofuels and high-efficiency, low emission vehicle engines. Support for R.W. Davis is acknowledged from DOE-EERE BioEnergy Technologies Office (BETO) under agreement 26336. Sandia National Laboratories is a multimission laboratory managed and operated by National Technology and Engineering Solutions of Sandia LLC., a wholly owned subsidiary of Honeywell International Inc. for the US Department of Energy’s National Nuclear Security Administration under contract DE-NA0003525.
The authors declare that they have no competing interests.
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