Research of Sustainable Jet Fuel Production Using Microbes

Introduction
Atmospheric greenhouse gases (GHG) such as carbon-di-oxide (CO₂), methane, nitrous oxide, and fluorinated gases contribute to global climate change. Due to anthropogenic activities, GHS emission levels are increasing tremendously during the past few decades. The Environmental Protection Agency (EPA) report stated that in 2016 of all U.S. greenhouse gas emissions from human activities, the CO₂ emission accounted for about 81.6% while methane contributed about ten percent (10%), and nitrous oxide accounted for six percent (6%) [1].

Pacala and Socolow (2004) [2] reported that the CO₂ emission from fossil fuels at the global level was seven Gt (gigatonnes) of carbon per year. In the United States alone, between 1990 and 2016 the carbon dioxide emissions increased by about four percent (4%) [1]. This increase in emissions is attributed to the rapid increase in economy, population, and demand for air transport. The NASA climate change department reported that the CO₂ emission in March 2018 was 408ppm, which is significantly high [3]. Among the different industries that contributed to these GHG emissions in the U.S, the fossil fuel combustion in the transportation sector alone accounted for 34% of total CO₂ emission and 27% of total GHG emissions [1]. Within the transportation sector, the demand for air travel is increasing rapidly. Based on compound average growth rate (CAGR) of 3.7%, the International Air Transport Association has forecasted that the demand for air travel will double by 2035 [4].

Airlines are spending approximately 28% of their operating cost to fuel their fleets [5]. This high operating cost and the volatility in fuel prices are the driving force for the aviation industry to develop biofuel. Climate change has also necessitated the reduction of the carbon footprint at the global level, and as the aviation industry emits more CO₂, IATA has developed guidelines to reduce CO₂ emissions by improving fuel efficiency by one and a half (1.5%) which will aid in achieving carbon neutrality by 2020 and reduce the emissions by 50% in 2050 [6]. Sustainable bio-jet fuel offers low carbon emissions. The life cycle assessment (LCA) of biofuels from different renewable processes decreased the CO₂ emissions by approximately 80% based on the production technology [7]. The feedstocks used for biofuel production are biological in origin, namely jatropha, algae, camelina, halophytes, agricultural and forest residues, etc., and microbes play a significant role in biofuel production from these feedstocks. This paper will discuss specifically the biofuel production technologies, role of microbes in producing hydrocarbons (alkanes), and conclude by briefly reviewing the recent advancements in microbial engineering to enhance the biofuel production.

Alternative Aviation Fuels and Their Production Technologies
Aviation industry uses two types of jet fuel namely, Jet A and Jet A1 (kerosene-based fuel, which are mixtures of hydrocarbons, primarily alkanes) and Jet B (gasoline-based fuel). Among these two fuels types, kerosenebased fuels are mixtures of hydrocarbons, predominantly alkanes. Kallio et al., (2014) [8] reported that alkanes significantly reduce both the freezing and boiling points of the aviation fuel, and so it is a vital product to develop bio-jet fuels. Hence, most of the recent research works are targeting alkanes as an essential product for bio-jet fuel synthesis [9]. There are mainly two methodologies for producing alkanes; the first method is by producing triglycerides and fatty acids by utilizing oleaginous crops and then chemically converting that to alkanes; the second method is based on pyrolysis of biomass to produce syngas and then chemically converting that to alkanes [9]. Microorganisms have the exceptional ability to produce fatty acids and some microbial species are able to convert the fatty acids into alkanes/alkenes. The innovations and development of biotechnology and bioengineering have paved the way for more research in the field of alkane production through microbes.

The alternative aviation fuels that are gaining popularity are sustainable in nature as they are predominantly produced from renewable resources, and the related GHG emissions are also comparatively lesser than in the use of fossil fuels. Moreover, these alternative fuels are compatible with the conventional fuels. The different types of alternative aviation fuels that are being developed are Hydroprocessed Renewable Jet fuels (HRJs) or Hydroprocessed Esters and Fatty Acids (HEFA) [10], Fischer Tropsch fuels (FT fuels) [11], liquid bio-hydrogen and bio-methane [12], biodiesel [13], and bio-alcohols [14].

The technologies used for producing renewable jet fuels are classified broadly into two types based on the type of processes utilized for its production viz. thermochemical process and biochemical process [15]. Thermochemical processes include Biomass to liquid process (BTL process), Fischer Tropsch process (FT process), and hydroprocessing. While the biochemical process consists of direct sugar to hydrocarbon process (DHSC) and alcohol to jet (ATJ) process [9]. Wang and Tao (2016) [16] classified the conversion pathways into four types based on raw materials (alcohol, oil, gas, and sugars) used for fuel production namely alcohol to jet (ATJ), oil to jet (OTJ), gas to jet (GTJ), and sugar to jet (STJ). These pathways are certified by American Society for Testing and Materials (ASTM) [17].

Alcohol to jet (ATJ) pathway is used to convert butanol and ethanol to jet fuels using processes like alcohol dehydration, oligomerization, and hydrogenation. HEFA/HRJ fuels are produced through oil to jet (OTJ) synthesis, which utilizes different process such as catalytic thermolysis or hydrothermal liquefaction and the hydro-treated depolymerized cellulosic jet process [8,16]. Gas to jet (GTJ) involves two pathways namely Fisher Tropsch Biomass to Liquid (Fischer-Tropsch Synthesis Paraffin Kerosene [FT-SPK]) and gas fermentation process. In the FT process, the biomass is first converted to syngas, then converted to liquid hydrocarbons through F-T synthesis while in gas fermentation pathway the biomass is transformed into syngas which is fermented to either ethanol or butanol and finally upgraded to jet fuel through the ATJ process. Sugar to jet (STJ) pathway includes the biological and catalytic conversion of sugars to hydrocarbons [17].

Role of Microorganisms, and Current Status of Microbial Biofuel Production
Among these technologies described, the utilization of lignocellulosic material to produce bioethanol contributes significantly towards the commercial production of sugars from renewable resources. This is a promising approach towards the sustainable bio-jet fuel production. This conversion is brought about either by the catalytic upgrading of sugars to hydrocarbons pathway [18] or by biological fermentation [16]. There are two types of fermentation, namely direct fermentation, which refers to the conversion of plant materials to sugar and then sugar to alcohol or indirect fermentation referring to the pyrolysis of plant materials followed by the conversion of syngas to ethanol by using acetogenic bacteria [19]. Indirect fermentation is rarely used.

When compared with the catalytic upgrading of sugars to hydrocarbon pathway, the biological fermentation method has more advantages. It does not require chemical catalysts, high energy/temperature reactions, and the conversion can be carried out in a single fermentation tank. Due to these reasons, STJ technology is regarded as a significantly promising pathway for the production of bio-jet fuels. STJ technology with special reference to biological conversion and the role of microbes in biofuel production will be further discussed briefly.

Biofuel production involves different process such as identifying the potential plant source, isolation of the microbial strains (fungal, and/or bacterial species), and designing the conversion pathway for efficient conversion of plant materials to sugars, and then converting that to ethanol using microbes either utilizing native strains or genetically engineered strains. The most important step in biofuel production is the conversion of the raw material to sugar.

Different plant materials such as agricultural crops, grasses, weeds, algae are used for biofuel production and their composition (cellulose, hemicellulose, lignin etc.) varies widely. Based on this composition of the plant material, different kinds of microbes, optimum conditions for their incubation, and other requirements for biofuel production, will also differ considerably [20]. The ideal microbe will possess a high tolerance to inhibitors, high metabolic fluxes, optimum utilization of substrates, and have deregulated pathways for sugar transport and produce a single fermentation product.

Fermentation (Ethanol Production)
Many research works are carried out to identify the potential microorganisms that are able to bring about these transformations and to develop a technologically and economically viable method based on this conversion. Irrespective of the plant material used, the starch, cellulose or hemicellulose were first degraded to pentose, and hexoses and later it is fermented into ethanol. There are different fermentation pathways (Example: hetereolactic acid fermentation by lactic acid bacteria such as Leuconostoc species and mixed acid fermentation by enteric bacteria) that produce ethanol as one of the end product. Nonetheless, ethanol has to be the primary product for the commercial production of biofuel. Especially, the yeast Saccharomyces cerevisiae and gram-negative bacteria Zymomonas mobilis produce two moles of ethanol per mole of hexose. Both these microbes convert pyruvate to alcohol through pyruvate decarboxylase/alcohol dehydrogenase enzymes. However, the conversion of hexoses to ethanol using S. Cerevisiae is widely adopted at an industrial level production [21]. Since, plant materials consist of both cellulose and hemicellulose (precursor of xylose, a pentose sugar), microbes that can efficiently and effectively metabolize these two sugars are required for the industrial production of alcohol. Several microorganisms such as the fungi Penicillium capsulatum and Talaromyces emersonii [22], the thermophilic actinomycete Thermomonospor fusca [23], the hyperthermophile Caldicellulosiruptor saccharolyticus [24] completely depolymerize hemicellulases to xylose. Usually, xylose is metabolized to pyruvate through the pentose phosphate pathway. Different microbes such as anaerobic fungi and different groups of mesophilic and thermophilic anaerobic bacteria are able to metabolize xylose [25].

Consolidated Bioprocessing (CBP)
CBP is another significant strategy adopted at the industrial level to produce biofuel in an economically efficient way. CBP refers to the microbial conversion of plant materials to biofuels without enzymatic treatments [26]. This conversion is brought about by either a single microbe or a combination of microbes. The anaerobic, thermophilic bacterium Clostridium thermocellum is one of the significant candidates used in CBP. Another potential candidate from the same genus is C. Bescii and it was actually engineered to produce ethanol [27].

Similarly, the mesophilic and filamentous fungus Trichoderma reesei has also been engineered to produce ethanol [28]. Yamada, Taniguchi, Tanaka, Ogino, Fukuda, and Kondo (2010) [29] suggested that an alternative efficient way is to engineer an ethanol producing microbe to digest cellulose. Microbes such as S. Cerevisiae, E. Coli and Z. Mobilis were engineered to digest cellulose through the expression of heterologous cellulolytic enzymes. Lynd, Currie, Ciazza, Herring, and Orem (2008) [30] utilized thermophilic grampositive Firmicutes that belongs to the orders Clostridiales and Thermoanaerobiales and their research proved that Clostridium thermocellum has the highest cellulose degradation ability among the order Clostridiales. Clostridium phytofermentans is a gram-positive, anaerobic bacteria that ferments a wide variety of polysaccharides, oligosaccharides, and monomeric sugars to produce mainly ethanol [31]. Clostridium Acetobutylicum is another interesting species which predominantly produces butanol, along with acetone, and ethanol [32]. In general, several clostridia species are able to produce sugar by degrading the cellulose and then convert sugar to ethanol. Though promising, the amount of ethanol produced (on a w/v scale) rarely exceeds 5% and CBP using thermophilic anaerobes have not yet been commercialized. Geobacillus thermoglucosidans has the ability to convert lignocellulosic biomass to bioethanol [33].

Microbes Producing Alkane/Alkene (Fermentation of Sugar to Alkane/Alkene)
Fu, Chi, Ma, Zhou, Liu, Lee, and Chi (2015) [34] stated that most of the eubacteria synthesize low amount of alkanes. As it is synthesized intracellularly, its separation and purification from the cells are quite difficult. On the other hand, few prokaryotes are able to produce alkanes both intracellularly and extracellularly. Among the prokaryotes, anaerobic bacteria especially the species belonging to the genus Clostridium, and Desulfovibrio have exhibited significant results in producing alkanes mainly C₂₅ to C₃₅ [35]. In cyanobacteria, alkanes are found in lipid droplets and are actually packed with quite a lot of hydrophobic energy-dense compounds [36].

Han, McCarthy, Calvin, and Benn (1968) [37] reported that several species of cyanobacteria are able to produce a hydrocarbon that is within the appropriate range for jet fuel (C₁₅ to C₁₉). But the biosynthesis pathway of alkane in cyanobacteria was not well known for a long time. Only in 2010, Schirmer, Rude, Li, Popova, and Cardayre, [38] explained the alkane biosynthesis pathway in cyanobacteria. In this cyanobacterial pathway, fatty acyl-ACP is converted to alkane/alkene (C₁₅ to C₁₉ especially C₁₇). Beller, Goh, and Keasling, (2010) [39] studied another pathway in which alkene is produced by Micrococcus luteus, a gram-positive bacteria. They reported that in this pathway alkane/alkene is produced as long chain hydrocarbon (C₂₃ to C₃₃) with at least one double bond. This is referred to as head-to-head condensation of fatty acids. It begins with the formation of fatty acyl-CoA (or ACP) which is converted to β-ketoacyl-CoA by acyl-CoA dehydrogenase, an enoyl-CoA hydratase, and a 3-hydroxy acyl-CoA dehydrogenase in a sequential way and ultimately it leads to the alkene biosynthesis. Park, Tanabe, Hirata, and Miyamoto (2001) [40] reported that Vibrio furnissii synthesizes alkanes between C₁₅ and C₂₄. This pathway starts with fatty acid which is then converted into alkane and is referred to as hypothetical vibrio pathway.

Engineering Microbes for Alkane Production
Although a lot of microorganisms are able to produce alkanes naturally, the production level is not so high. Especially, being photosynthetic in nature cyanobacteria caught the attention of the biofuel industry as it is able to synthesize alkanes from sunlight and carbon dioxide. Several research works are being carried out with different methodologies by altering the growth and environmental conditions, and microbes are genetically engineered to increase the alkane production level to a commercial level. Wang, Liu, and Lu (2013) [41] were able to increase the alkane production in Synechocystis sp. PCC 6803 by 8.3 times higher than the wild strain. This high production rate was achieved by genetically modifying Synechocystis strain by deleting the β ketolyase phaA, and overexpressing the native FAR (Fatty acyl-ACP reductase) and ADO (Aldehyde-deformylating oxygenase) enzymes. Kageyama, Waditee-Sirisattha, Sirisattha, Tanaka, Mahakhant, and Takabe (2015) [42] were able to double the alkane production in the nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120 by exposing it either to nitrogen deficiency or salt stress. While Peramuna, Morton, and Summers (2015) [43] genetically modified the Nostoc punctiforme cells and exposed them to high light conditions in order to overexpress the genes that encoded FAR, ADO, and a lipase to increase the production of alkanes. They were able to increase the production from 1.12% to 12.9% of CDW (cell dry weight). Despite the developments attained, the cyanobacterial alkane production amount is still low and the biofuel industry shifted its attention to identify new microbes.

Due to the limitations related to the alkane production using cyanobacterial species, biofuel industry is attempting to develop new microbial platforms to produce alkanes. The industry specifically prefers certain microbes such as Escherichia coli, Bacillus subtilis, Aspergillus nidulans, and the yeast, Saccharomyces cerevisae either due to their production abilities or suitability for industrial production. The first attempt to engineer the bacterial platform to express the cyanobacteria pathway in E. Coli was made by Schirmer et al. (2010) [38]. By expressing FAR and ADO from S. Elongates PCC7942 the engineered E. Coli MG 1655 were able to produce 25mg/l of pentadecane and heptadecene. When the S. Elongates ADO was replaced with ADO from N. Punctiforme PCC 73102 the alkane/alkene production went up to 80 mg/l. When a modified medium was utilized the alkane/alkene (tridecane, pentadecene, pentadecane and heptadecene) production increased up to 300 mg/l and this methodology was patented by the biofuel company LS9 [44]. Harger, Zheng, Moon, Ager, An, Choe, et al. (2013) [45] engineered E. Coli to produce different alkane/alkene ranging from C₁₃ to C₁₇. By overexpressing the FAR and ADO enzymes from S. Elongates, and fabH2 from B. Subtilis, the engineered E. Coli BL 21 was able to produce alkane at a level of 98.3 mg/l [45]. Choi and Lee (2013) [46] developed a new E. Coli strain by deleting fadE, fadR and overexpressing fadD. This new strain was able to produce alkanes such as C₉, C₁₂, C₁₃, and C₁₄. Choi and Lee (2013) [46] reported a production level of 580.8 mg/l. Alkanes (C₁₃ to C₁₇) were produced directly from FFA (free fatty acids) by overexpressing the reductase complex from Photorhabdus luminiscens and ADO from N. Punctiforme in E. Coli [47]. This method produced up to 8 mg/l of alkanes and the Shell Company patented this method [47]. Song, Yu, and Zhu (2016) [48] engineered E. Coli to overexpress FAR and ADO from S. Elongatus PCC 7942, the transcription factor fadR and mutated the gene that encodes aldehyde reductase, yqhD. This engineered E. Coli BL 21 predominantly produced alkanes/alkene between C₁₅ and C₁₇ at a rate of 255.6 mg/l. Thus several E. Coli strains have been engineered to produce advanced biofuel such as isopropanol, n-butanol, n-propanol, isobutanol, 2-methyl-1-butanol and 3- methyl-1-butanol [49].

The first recombinant yeast S. Cerevisiae that produced alkanes was reported by Bernard, Domergue, Pascal, Jetter, Renne, Faure, et al. (2012) [50]. Buijs, Zhou, Siewers, and Nielsen (2015) [51] tested the cyanobacterial pathway of alkane production in the yeast S. Cerevisiae. They engineered S. Cerevisiae. by overexpressing FAR and ADO from S. Elongatus PCC 7942, overexpression of the E. Coli ferredoxin and the ferredoxin NADP+ reductase (Fdx and FNR) and deleting hfd1. The engineered strain produced alkanes such as C₁₃, C₁₅, and C₁₇ and the production level was 22 μg/l CDW. Foo, Susanto, Keasling, Leong, and Chang (2016) [52] used FFA instead of glucose to produce alkanes. The alkane production was about 70 μg/l. This methodology was further improved by Zhou, Buijs, Zhu, Qin, Siewers, and Nielsen (2016) [53] by substituting glucose as a substrate instead of FFA and they achieved a production of 10.4 g/l of FFA.

Beopoulos, Cescut, Haddouche, Uribelarrea, Molina-Jouve, and Nicaud (2009) [54] reported that the oleaginous yeast Yarrowia lipolytica is capable of storing up to 36% of CDW as lipids and it is considered as a potential strain for the industrial biofuel production. Liu, Pan, Spofford, Zhou, and Alper (2015) [55] reported that the engineered strain of Yarrowia lipolytica is able to accumulate 39.1 g/l of lipid. The black yeast Aerobasidium pullulans var melanogenunm is able to accumulate up to 66.3% of CDW as lipids (26.7 % palmitic acid, 44.5 % oleic acid and 21 % linoleic acids) [56]. Kohn and Kim (2015) [57] isolated approximately 46 microbial isolates that are able to produce C₃ to C₈ hydrocarbons. They identified species of Actinomyces sp., Enterococcus Faecium, E. Hirae, E. Coli, Clostridium Glycolicum, Proteus sp. and Tissirella sp. Oleaginous yeast is a promising candidate but requires more research to be completed with regard to its metabolic pathway, and genetics to commercialize its utilization in the alkane production. The current demand for sustainable fuels can be fulfilled only when these alternative microbes which have a high potential to produce alkane is characterized fully.

Conclusion
As the global consumption of sustainable biofuel increases and the aviation sector relies on the utilization of bio-jet fuel to achieve its target in reducing the GHG emission, it is essential to discover new methodologies to produce renewable jet fuel. There are still a lot of challenges associated with the commercialization of sustainable bio-jet fuels. ICAO and the regional aviation authorities should develop policies to bridge the price gap between bio-jet and conventional jet fuel and should provide incentives to encourage the biofuel production. The entire supply chain from feedstock production to the distribution of bio jet fuel will have to be well developed.

Among the production pathways discussed earlier, the majority of the biofuel produced today is through HEFA pathway. Out of all the commercial operation, only AltAir facility in California produces chiefly bio jet fuel using Ecofining technology. The others produce mainly biodiesel and a small quantity of bio jet fuel. The operational capacity of all the HEFA facilities accounts only for 4.3 bln L/y. In HEFA pathway Ecofining and NEXBTL are the two main production technologies that are commercialized so far. NEXBTL technology is used by Neste Company in Finland, Singapore, and the Netherlands while Ecofining technology is used by AltAir, Emerald Biofuels, Diamond Green Diesel, Renewable Energy Grouo (US), and ENI (Italy) [58].

Other methods that utilize biomass, lignocellulosic and algal sources, to produce bio-jet fuel are still in demonstration stage with promising results. This conversion uses FT, ATJ, STJ pathways. This paper discussed the microbial production systems that converted the sugars to alkane based jet fuel. Though these engineered microbial systems are not yielding the productivity at a commercial level, in the future as the supply of fossil fuel decreases, and the applied bioengineering, biotechnological, and microbiological research improves, this will definitely provide a competitive alternative for the shift towards sustainable bio jet fuel.

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