Replacement of conventional petroleum fuels with renewable fuels reduces net emissions of carbon and greenhouse gases, and affords opportunities for increased domestic energy security. Here, we present alkyl dialkoxyalkanoates (or DAOAs) as a family of synthetic diesel and marine fuel candidates that feature ester and ether functionality. These compounds employ pyruvic acid and fusel alcohols as precursors, which are widely available as metabolic intermediates at high titer and yield. DAOA synthesis proceeds in high yield using a simple, mild chemical transformation performed under air that employs bioderived and/or easily recovered reagents and solvent. The scalability of the synthetic protocol was proven in continuous flow with in situ azeotropic water removal, yielding 375 g of isolated product. Chemical stability of DAOAs against aqueous 0.01 M H2SO4 and accelerated oxidative conditions is demonstrated. The isolated DAOAs were shown to meet or exceed widely accepted technical criteria for sustainable diesel fuels. In particular, butyl 2,2-dibutoxypropanoate (DAOA-2) has indicated cetane number 64, yield soot index 256 YSI per kg, lower heating value 30.9 MJ kg−1 and cloud point < −60 °C and compares favorably to corresponding values for renewable diesel, biodiesel and petroleum diesel.
Corynebacterium glutamicum has been successfully employed for the industrial production of amino acids and other bioproducts, partially due to its native ability to utilize a wide range of carbon substrates. We demonstrated C. glutamicum as an efficient microbial host for utilizing diverse carbon substrates present in biomass hydrolysates, such as glucose, arabinose, and xylose, in addition to its natural ability to assimilate lignin-derived aromatics. As a case study to demonstrate its bioproduction capabilities, L-lactate was chosen as the primary fermentation end product along with acetate and succinate. C. glutamicum was found to grow well in different aromatics (benzoic acid, cinnamic acid, vanillic acid, and p-coumaric acid) up to a concentration of 40 mM. Besides, 13C-fingerprinting confirmed that carbon from aromatics enter the primary metabolism via TCA cycle confirming the presence of β-ketoadipate pathway in C. glutamicum. 13C-fingerprinting in the presence of both glucose and aromatics also revealed coumarate to be the most preferred aromatic by C. glutamicum contributing 74 and 59% of its carbon for the synthesis of glutamate and aspartate respectively. 13C-fingerprinting also confirmed the activity of ortho-cleavage pathway, anaplerotic pathway, and cataplerotic pathways. Finally, the engineered C. glutamicum strain grew well in biomass hydrolysate containing pentose and hexose sugars and produced L-lactate at a concentration of 47.9 g/L and a yield of 0.639 g/g from sugars with simultaneous utilization of aromatics. Succinate and acetate co-products were produced at concentrations of 8.9 g/L and 3.2 g/L, respectively. Our findings open the door to valorize all the major carbon components of biomass hydrolysate by using C. glutamicum as a microbial host for biomanufacturing.
Ramirez-Corredores, M.M.; Vega-Montoto, Lorenzo; Monroe, Eric; Davis, Ryan W.
Decarbonizing the transportation sector is likely to require both electrification and increased incorporation of biofuels and/or bioblendstocks. While the social and environmental benefits of bioblendstocks are well understood, their real value for the fuel producers has not been established. This work considers prenol as a bioblendstock case study to identify sources of intrinsic value to fuel blenders by studying the properties of binary mixtures with gasoline components. The considered refinery blendstocks were samples of full range naphthas from the distillation, fluidized catalytic cracking, isomerization, alkylation, and reforming units. Octane numbers, Reid vapor pressure, distillation curves, and sulfur content were evaluated. Our results indicate the need for adjusting the formulation of the base fuel, depending on the interplay among the properties of the bioblendstock and those of the base fuel. Prenol increased research octane number (RON) and octane sensitivity (OS) of the base fuel, by up to 25 and 10 octane numbers, respectively. Additionally, 10 vol% prenol reduced RVP up to 2.2 psi, for the more volatile blendstock. Thus, considering prenol as a low volatility, RON/OS boosting bioblendstock, the composition of the preferred base fuel was proposed as containing reduced olefins and aromatics, and increase light fractions. The potential impact of this new gasoline formulation on refining processes and products gives rise to direct sources of value to the refiners, such as exporting products to the chemicals market, increasing the value of intermediate refinery streams, decreasing operating severity of certain refinery units, and broadening of the product suite.
High-protein algal biomass is an important bio-commodity that has the potential to provide a new source of sustainable protein products. Herein is a critical review that identifies (1) the most relevant sustainability findings related to the processing of proteinaceous algal biomass to higher value protein products and (2) the potential pathways to improve life cycle assessment (LCA) and techno-economic analysis (TEA) metrics, including life-cycle carbon dioxide equivalent (CO2eq), life cycle energy, and minimum selling price (MSP) of these products. The critical review of the literature revealed a large variation in model input parameters relating to these metrics. Therefore, a Monte Carlo analysis was conducted to assess the risk associated with these input variations. To understand the uncertainties that propagate into high-protein algae to products' systems, we reviewed more than 20 state-of-the-art unit operations for algal biomass processing., including cell disruption, protein solubilization, protein precipitation and purification, and protein concentration. We evaluated displacement of proteinaceous products by algal-bioproducts, including ruminant feed, aquaculture feed, protein tablets, and biopolymers and biopolyesters, with prices in the market ranging from 1.9 to 120 $ kg―1 protein. This review realized that the MSP of ruminant and non-ruminant feed ranges from 0.65 ± 0.56 to 2.9 ± 1.1 $ kg―1 protein, and bioplastics' MSP ranges from 0.97 to 7.0 $ kg―1 protein. Regarding LCA metrics, there is limited research on life cycle energy in proteinaceous biomass concentration and bioproduct systems, reported at 32.7 MJ kgprotein―1, for animal feed displacement. Animal feed emissions in the literature report negative fluxes, representing environmental benefits, as low as ―3.7 kgCO2eq kg―1 protein and positive fluxes, i.e., global warming potential, as high as 12.8 kgCO2eq kg―1 protein. There is limited research on bioplastics life cycle emissions reported at 0.6 kgCO2eq kg―1 protein. In general, the studies to date of algae-derived protein bioproducts showed similar life cycle emissions to soybean meals, nylon, polymers, and polystyrenes. Our risk analysis realized that more than 50% of scenarios can result in negative-net life cycle CO2eq emissions. This review and risk analysis assess and demonstrate the scenarios that improve economic and environmental sustainability metrics in high-protein algal bioproduct systems.
Sarnaik, Aditya; Mhatre, Apurv; Faisal, Muhammad; Smith, Dylan; Davis, Ryan W.; Varman, Arul M.
Ultra-low temperature (ULT) storage of microbial biomass is routinely practiced in biological laboratories. However, there is very little insight regarding the effects of biomass storage at ULT and the structure of the cell envelope, on cell viability. Eventually, these aspects influence bacterial cell lysis which is one of the critical steps for biomolecular extraction, especially protein extraction. Therefore, we studied the effects of ULT-storage (-80°C) on three different bacterial platforms: Escherichia coli, Bacillus subtilis and the cyanobacterium Synechocystis sp. PCC 6803. By using a propidium iodide assay and a modified MTT assay we determined the impact of ULT storage on cellular viability. Subsequently, the protein extraction efficiency was determined by analyzing the amount of protein released following the storage. The results successfully established that longer the ULT-storage time lower is the cell viability and larger is the protein extraction efficiency. Interestingly, E. coli and B. subtilis exhibited significant reduction in cell viability over Synechocystis 6803. This indicates that the cell membrane structure and composition may play a major role on cell viability in ULT storage. Interestingly, E. coli exhibited concomitant increase in cell lysis efficiency resulting in a 4.5-fold increase (from 109 μg/ml of protein on day 0 to 464 μg/ml of protein on day 2) in the extracted protein titer following ULT storage. Furthermore, our investigations confirmed that the protein function, tested through the extraction of fluorescent proteins from cells stored at ULT, remained unaltered. These results established the plausibility of using ULT storage to improve protein extraction efficiency. Towards this, the impact of shorter ULT storage time was investigated to make the strategy more time efficient to be adopted into protocols. Interestingly, E. coli transformants expressing mCherry yielded 2.7-fold increase (93 μg/mL to 254 μg/mL) after 10 mins, while 4-fold increase (380 μg/mL) after 120 mins of ULT storage in the extracted soluble protein. We thereby substantiate that: (1) the storage time of bacterial cells in-80°C affect cell viability and can alter protein extraction efficiency; and (2) exercising a simple ULT-storage prior to bacterial cell lysis can improve the desired protein yield without impacting its function.
We demonstrated production of a superior performance biodiesel referred to here as fatty acid fusel alcohol esters (FAFE) – by reacting fusel alcohols (isobutanol, 3-methyl-1-butanol, and (S)-(-)-2-methyl-1-butanol) with oil (glyceryl trioleate) using lipase from Aspergillus oryzae. Reaction conditions corresponding to a molar ratio of 5:1 (fusel alcohols to oil), enzyme loading of 2% w/w, reaction temperature of 35 °C, shaking speed of 250 rpm, and reaction time of 24 h achieved >97% conversion to FAFE. Further, FAFE obtained from reacting a fusel alcohol mixture with corn oil were evaluated for use as a fuel for diesel engines. FAFE mixtures showed superior combustion and cold-flow properties, with the derived cetane numbers up to 4.8 points higher, cloud points up to −6 °C lower, and the heat of combustion up to 2.1% higher than the corresponding FAME samples, depending on the fusel mixture used. This represents a significant improvement for all three metrics, which are typically anti-correlated. FAFE provides a new opportunity for expanded usage of biodiesel by addressing feedstock limitations, fuel performance, and low temperature tolerance.
Davis, Ryan W.; Liu, Fang; Derose, Katherine; Simmons, Blake A.; Quinn, Jason C.
Distiller's grains are a byproduct of corn ethanol production and provide an opportunity for increasing the economic viability and sustainability of the overall grain-to-fuels process. Typically, these grains are dried and sold as a ruminant feed adjunct. This study considers utilization of the residuals in a novel supplementary fermentation process to produce two products, enriched protein and fusel alcohols. The value-added proposition and environmental impact of this second fermentation step for distiller's grains are evaluated by considering three different processing scenarios. Techno-economic results show the minimum protein selling price, assuming fusel alcohol products are valued at $0.79 per liter gasoline equivalent, ranges between $1.65-$2.48 kg protein-1 for the different cases. Environmental impacts of the systems were evaluated through life cycle assessment. Results show a baseline emission results of 17 g CO2-eq (MJ fuel)-1 for the fuel product and 10.3 kg CO2-eq kg protein-1 for the protein product. Sensitivity to allocation methods show a dramatic impact with results ranging between -8 to 140 g CO2-eq (MJ fuel)-1 for the fuel product and -0.3 to 6.4 kg CO2-eq kg protein-1 for the protein product. The discussion is focused on the potential impact of the technology on corn ethanol production economics and sustainability.
More efficient engines enabled by better fuels derived from biomass could increase the fuel economy of the light duty (LD) fleet by 10% over current technology and planned developments. This report identifies top LD boosted spark ignition (BSI) biofuel candidates for further development and commercialization identified using a fuel property basis. The BSI merit function was used to evaluate the performance of candidate bio-blendstocks in improving engine efficiency. This report is aimed at biofuel researchers looking to better understand the efficiency implications of biofuels under development, as well as engine researchers who are interested in future biofuels with properties that enable more efficient engine design and operation.
This work describes the first documented case of an effect defined herein as “octane hyperboosting” by an oxygenated fuel compound, 3-methyl-2-buten-1-ol (prenol). Octane hyperboosting is characterized by the Research Octane Number (RON) of a mixture (e.g. an oxygenate biofuel blended into gasoline) exceeding the RON of the individual components in that mixture. This finding counters the widely held assumption that interpolation between the RON values of a pure compound and the base fuel provides the bounds for the RON performance of the blend. This is clearly distinct from the more commonly observed synergistic blending of oxygenates with gasoline, where the RON never exceeds the performance of the highest performing component. Octane hyperboosting was observed for blends of prenol and six different gasoline fuels with varying composition. Testing of compounds chemically similar to prenol yielded no qualitatively similar instances of octane hyperboosting, which suggests that the effect may not be widespread among fuel candidates. The phenomenon suggests an unexplored aspect of autoignition kinetics research for fuel blends, and may provide a new mechanism for significantly increasing fuel octane number, which is necessary for increasing combustion efficiency in spark ignition engines. This phenomenon also increases the potential candidate list of biofuels, as compounds hitherto discounted due to their lower pure component RON may exhibit hyperboosting behavior, and thereby enhanced performance, in blends.
Background: Engineering strategies to create promoters that are both higher strength and tunable in the presence of inexpensive compounds are of high importance to develop metabolic engineering technologies that can be commercialized. Lignocellulosic biomass stands out as the most abundant renewable feedstock for the production of biofuels and chemicals. However, lignin a major polymeric component of the biomass is made up of aromatic units and remains as an untapped resource. Novel synthetic biology tools for the expression of heterologous proteins are critical for the effective engineering of a microbe to valorize lignin. This study demonstrates the first successful attempt in the creation of engineered promoters that can be induced by aromatics present in lignocellulosic hydrolysates to increase heterologous protein production. Results: A hybrid promoter engineering approach was utilized for the construction of phenolic-inducible promoters of higher strength. The hybrid promoters were constructed by replacing the spacer region of an endogenous promoter, P emrR present in E. coli that was naturally inducible by phenolics. In the presence of vanillin, the engineered promoters P vtac, P vtrc, and P vtic increased protein expression by 4.6-, 3.0-, and 1.5-fold, respectively, in comparison with a native promoter, P emrR. In the presence of vanillic acid, P vtac, P vtrc, and P vtic improved protein expression by 9.5-, 6.8-, and 2.1-fold, respectively, in comparison with P emrR. Among the cells induced with vanillin, the emergence of a sub-population constituting the healthy and dividing cells using flow cytometry was observed. The analysis also revealed this smaller sub-population to be the primary contributor for the increased expression that was observed with the engineered promoters. Conclusions: This study demonstrates the first successful attempt in the creation of engineered promoters that can be induced by aromatics to increase heterologous protein production. Employing promoters inducible by phenolics will provide the following advantages: (1) develop substrate inducible systems; (2) lower operating costs by replacing expensive IPTG currently used for induction; (3) develop dynamic regulatory systems; and (4) provide flexibility in operating conditions. The flow cytometry findings strongly suggest the need for novel approaches to maintain a healthy cell population in the presence of phenolics to achieve increased heterologous protein expression and, thereby, valorize lignin efficiently.
Recent studies have revealed that caryophyllene and its stereoisomers not only exhibit multiple biological activities but also have desired properties as renewable candidates for ground transportation and jet fuel applications. This study presents the first significant production of caryophyllene and caryolan-1-ol by an engineered E. coli with heterologous expression of mevalonate pathway genes with a caryophyllene synthase and a caryolan-1-ol synthase. By optimizing metabolic flux and fermentation parameters, the engineered strains yielded 449 mg/L of total terpene, including 406 mg/L sesquiterpene with 100 mg/L caryophyllene and 10 mg/L caryolan-1-ol. Furthermore, a marine microalgae hydrolysate was used as the sole carbon source for the production of caryophyllene and other terpene compounds. Under the optimal fermentation conditions, 360 mg/L of total terpene, 322 mg/L of sesquiterpene, and 75 mg/L caryophyllene were obtained from the pretreated algae hydrolysates. The highest yields achieved on the biomass basis were 48 mg total terpene/g algae and 10 mg caryophyllene/g algae and the caryophyllene yield is approximately ten times higher than that from plant tissues by solvent extraction. The study provides a sustainable alternative for production of caryophyllene and its alcohol from microalgae biomass as potential candidates for next generation aviation fuels.
Background: 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. Results: 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. Conclusions: 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.
