Reassessing the Progress in the Production of ... - ACS Publications

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Reassessing the Progress in the Production of Advanced Biofuels in the Current Competitive Environment and Beyond: What Are the Successes and Where Progress Eludes Us and Why Eleftherios T. Papoutsakis* Molecular Biotechnology Laboratory, Department of Chemical & Biomolecular Engineering, Department of Biological Sciences & the Delaware Biotechnology Institute, University of Delaware, Newark, Delaware 19711, United States ABSTRACT: The concept, science, and technology of biofuels has captured the public’s imagination as well as the attention of the academic and industrial R&D communities over the last 10 years. The 2006 US DOE report has been the basis for funding three US Bioenergy Centers, and has precipitated increased research funding worldwide. Despite the large funding resources, there is little evidence to support that processes for advanced biofuels (i.e., fuel molecules more dense than ethanol) are anywhere near achieving economic feasibility. In assessing the current status of the field for biology-based processes, there has been tremendous progress in metabolic and pathway engineering and the development of synthetic-biology tools that can be applied to engineer strains. However, the economic feasibility of what is currently possible remains in doubt, especially in light of the recent low prices of oil and natural gas. Issues liming the economic feasibility are assessed and strategies to take advantage of the successful achievements are suggested. Analysis suggests that for biofuel molecules larger than four carbons, purely biological processes cannot be currently justified, but hybrid biological/catalytic technologies may offer the necessary economies to produce such biofuels.

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for the rest of the world) is the celebrated 2006 DOE report3 (largely based on data up to 2005), that was the basis for the funding, starting in 2007, of three US Bioenergy Centers for 10 years.4−6 The report set 5- and 10-year technological targets for developing robust industrial processes to produce biofuels. Most significantly, it led to large investments in biofuel R&D not only in the US but throughout the world, it made biofuels an important policy focus for the governments of many countries, and it triggered a large debate worldwide on “food versus biofuel” and the associated issues of sustainability and biodiversity. In the process, “biofuel” became a household word. In what follows, with focus on biology-based processes, expectations back then are discussed in light of what has been achieved emphasizing largely science and technology and leaving aside the socio-political issues of the subject, which have been recently reviewed.1 An effort is also made to identify what have been the major advances and successes and why, 10 some years later, we are nowhere near to producing advanced biofuels economically at the industrial scale.

INTRODUCTION AND FOCUS The history of the production and associated governmental policies of biologically produced liquid fuels (the so-called biofuels) has been recently succinctly summarized in an exceptional report.1 Following two clearly identifiable energy (petroleum supply) crises in 1973−74 and 1978−79, and the large increases in oil prices after 2000, American policy and to some extent also policies in most economically developed nations that are not major producers of oil (petroleum), has set goals for the production of liquid fuels based on renewable resources and notably crops and biomass. The only significant biofuel used in the US and most of the Western world is ethanol, to which one would add the relatively modest quantities of biodiesel (produced from various seeds2). An estimated 220 billion gallons of liquid transportation fuels are consumed annually in the US. Of those, 13 billion gallons of ethanol (about 6% of the total transportation liquid fuel) is produced annually largely from corn, consuming about 30% of the US corn crop, 6% of all US cropland, and 1% of the total U.S. land area. The situation is of course different in countries like Brazil, where the use of sugar cane offers exceptional economies and makes the production of ethanol a more attractive proposition. However, the Brazilian case is not typical of most of the rest of the world. Given these figures and the rising oil prices in the early to mid 2000s, the US Department of Energy (DOE) has embarked into a renewed effort to support the science and technology for better economies of production of biofuels, with a large emphasis on the so-called “advanced biofuels”, practically meaning liquid-fuel molecules more energy dense than, and thus, other than and larger than ethanol. An important document that set technological targets for the production of biofuels for the US (but has also impacted policy © 2015 American Chemical Society

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BIOFUELS IN 2005 VERSUS NOW Ethanol Was and Remains the Dominant Biofuel. In the 2005/06 DOE report3 ethanol was the dominant biofuel molecule, which attracted extensive discussion especially in the context of its production from cellulosic substrates. It was then anticipated that cellulosic ethanol produced at high rates and Special Issue: Doraiswami Ramkrishna Festschrift Received: Revised: Accepted: Published: 10170

May 6, 2015 July 6, 2015 July 7, 2015 July 7, 2015 DOI: 10.1021/acs.iecr.5b01695 Ind. Eng. Chem. Res. 2015, 54, 10170−10182

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Industrial & Engineering Chemistry Research

Figure 1. Ehrlich pathway for the alpha-keto acid−based biosynthesis of i-butanol and i-amyl-alcohol in yeast. Figure was redrawn based in Figure 1 of Yoshizawa. Adapted with permission from ref 15. Copyright 1965 Taylor Francis.

titers would have been possible within 5 years (2010−11) utilizing tolerant mesophilic and thermophilic organisms, and in fact organisms that would enable efficient ethanol production in a single step (the so-called, consolidated bioprocessing concept, CBP). The 10-year (i.e., 2015−16) technical milestones anticipated development of strains with higher yields, ability to predictively engineer ethanol tolerance, and fully predictive pathway models to enable model-driven design of cellular biocatalytic systems. Assessing these milestones in midyear 2015, it is clear that hardly any of these milestones have been achieved. Cellulosic ethanol remains a technological challenge to be met, let alone using simple CBP or thermophilic systems. Fully predictive models of pathways and their engineering remain also a distant milestone for now. In the current market environment of inexpensive natural gas, it remains questionable if cellulosic ethanol is for now an economically viable option.7−10 In the US, ca. 90% of biofuel ethanol is still made from corn using grain-based technology. Cellulosic ethanol plants were anticipated since the early 2010s or even earlier, but as of mid 2014, it is reported that there are 6

commercial cellulosic ethanol plants under construction or nearing operation10 in the US, half of which are based on a thermochemical rather than enzymatic process. A few of these plants and a plant in Brazil are now operational, but their economic viability remains to be demonstrated.1 Importantly, however, these new cellulosic plants will be providing valuable process experience and data for a realistic assessment of process economics. Advanced Biofuels: A Summary of What Was Then and What Is Now. As stated above, advanced biofuels are commonly defined as fuel molecules more dense in energy than ethanol. The 2005/06 DOE report3 (starting on p. 147), contains a brief discussion to anticipate the production of such energy-rich fuels in which most of the discussion was focused on alkanes, longer-chain alcohols, and fatty acids. It correctly assessed the situation at the time that microbial metabolism by native, native-like, semisynthetic, or synthetic pathways offers tremendous opportunities for producing more complex molecules whether for biofuel, commodity, or specialty chemicals. It also anticipated the difficulty of producing these 10171

DOI: 10.1021/acs.iecr.5b01695 Ind. Eng. Chem. Res. 2015, 54, 10170−10182

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Figure 2. Clostridium pathways for the production of alcohols and carboxylic acids from various simple and complex carbohydrates (pentoses, hexoses, celluloses, and hemicelluloses (xylans)), syngas and waste gases, and 1-C substrates (methanol and formate). The upper left depicts the Wood−Ljungdahl pathway (WLP) employed by acetogens for chemotrophic growth using CO2/CO/H2 as substrates. The upper right is the standard glycolysis pathway and the associated pentose-phosphate pathway for utilizing 5-C sugars. The WLP and glycolysis can be energetically coupled to enable exceptional carbon and electron economies that lead to superior product yields. The lower left and lower right depict the core primary metabolism of solventogenic and related clostridia for the production of alcohols and carboxylic acids. Figure was based on redrawing Figure 2 of Tracy et al.,18 which should be consulted for detailed explanation of the enzymes deployed in these pathways. Figure adapted from ref 18. Copyright 2011 Elsevier.

to produce energy-rich molecules. Metabolic engineering at the time had not reached the strategic delineation of pathways and chemistries. For example, the acclaimed 2006 Science review11 did not discuss specific biofuels other than ethanol and

more complex, hydrophobic molecules at high titers and rates due to their high toxicity to cells. Regarding energy-rich biofuels, the 2005/06 DOE report and the literature at the time did not discuss specific molecules or likely biological pathways 10172


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Figure 3. Natural, synthetic, and semisynthetic pathways for the production of higher alcohols (upper and lower left), alkenes (upper right), and alkanes, fatty alcohols, and fatty-acid ethyl esters (lower right) starting from simple and complex carbohydrates, that is, celluloses, hemicelluloses (xylans), and starches. The upper left depicts the alpha-keto acid pathways for the production of short and medium-chain length alcohols. The lower right summarizes the Clostridium acetoacetyl-CoA pathway for the production of short and medium-chain length alcohols. The upper and lower right depicts the chemistries for the production of alkanes, alkenes, fatty alcohols, and fatty-acid ethyl esters via the mevalonate (MEV) and/or the deoxyxylulose (DXP or MEP) pathways (upper right), or fatty acid biosynthesis (lower right). Figure was based on combining and redrawing Figures 2 and 3 from ref 20. Copyright 2013 Elsevier. 10173


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field, and assess the current state of advanced biofuels and its economic feasibility.

biodiesel, and no biological paths to synthesis of fuel molecules. It did discuss catalytic conversion of biomass to alkane molecules, a then nascent field of research. Detailing of core biochemical pathways (such as the mevalonate pathway or fatty-acid synthesis pathways, both of which would attract large attention staring in the late 2000s) were not discussed at the time in the context of biofuel production. Interestingly, both the 2005/06 DOE report and the 2006 Science review mentioned the likely importance of syngas (a mixture of CO, H2 and CO2 obtained from gasification reactions of municipal or agricultural wastes or from the partial CH4 oxidation) as a possible fermentation substrate for ethanol production, but without discussing any details of the celebrated Wood− Ljungdahl pathway (WLP)12 employed by acetogens to carry out these fermentations. Butanol, normal or its isoforms (i-butanol or 2-butanol), was not mentioned or anticipated in the 2005/06 DOE report. Butanol has become the poster child of advanced, more energydense biofuels after Butamax (a joint venture between DuPont and BP) announced in the summer of 2006 their development plans for biological production of butanol and notably ibutanol. A provisional patent13 had been filed by DuPont/ Butamax on October 26, 2005 describing the development of organisms (notably Escherichia coli and yeast) to produce ibutanol based on an amplified (or enhanced, by expressing non-native genes) Ehrlich pathway,14,15 a well-known biochemical pathway, with a history going back to the early 1900s, native to many yeasts, and, with variations, in several prokaryotes. The Ehrlich pathway is based on alpha-keto acids to produce so-called “fusel” (a German word for “bad liquor”) alcohols, including i-butanol, starting from the core metabolite pyruvate. Figure 1 displays a version of the Ehrlich pathway for the formation of i-butanol and i-amyl-alcohol from glucose as it was known back in 1965.15 Normal butanol has been produced biologically based on the ABE (acetone−butanol−ethanol) Clostridium fermentation since the early 1900s, a fermentation process widely known, taught, and discussed for several reasons: science, technology, patent law, and politics. ABE has been known since Pasteur’s times (Pasteur had discussed its characteristics), it had an enormous impact on the production of explosives (cordite from acetone) used in WWI and WWII, as well as in the development of the automotive industry (butanol for producing car lacquers).16 As detailed,16 a key inventor and innovator of ABE was Chaim Weizmann (who became the first president of the state of Israel), the process was nationalized in the UK in 1916 to ensure increased supply of acetone for producing explosives for WWI, and in 1923, it became the first industrial biological process to become a subject of patent litigation. Although not widely employed in the Western world since the late 1950s, research on the molecular biology and metabolic engineering of solventogenic Clostridium organisms (notably C. acetobutylicum) was an active field of research in 2005/06 with emphasis on butanol production as a commodity chemical or fuel.17 Since 2006, advances in the clostridial genetics and process developments have rejuvenated the industrial interest in the ABE fermentation as discussed below. While new production facilities have been started since then (all outside the US), all pertain to the production of n-butanol not as biofuel but rather as a chemical, which has a considerably higher market value and a large market worldwide. In the sections below, key developments over the last 10 years are discussed aiming to capture the many successes in the

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LARGE PROGRESS IN PATHWAY ENGINEERING The last 9−10 years since the DOE 2005/06 report have seen great progress in developing synthetic or semisynthetic routes/ pathways for the biological production of advanced biofuels. Key pathways are briefly summarized below based on molecule classes, notably, higher alcohols (C3 and above), fatty acids as precursors to alkane synthesis, and direct hydrocarbon synthesis. There exist several molecules, such as higher carboxylic acids that can be converted to hydrocarbons by catalytic means. These are briefly reviewed below, as well. Higher Alcohols. Many organisms natively produce alcohols with 3 or 4 carbons (3-C or 4-C), such as propanols and butanols, and some produce, at low levels, 6-C alcohols (hexanols). These organisms include yeast cells and prokaryotes such as Clostridium class and genus organisms. Clostridia use a variety of substrates (hexoses, pentoses, other oligo- and polysaccharides, but also CH3OH, and gases such as CO2, H2, and CO, the latter by organisms known as acetogens and employing the Wood−Ljungdahl pathway)18 (Figure 2). The Clostridium pathways depart from acetyl-CoA to form acetoacetyl-CoA on the way to butyryl-CoA, and hexanoylCoA synthesis that are then reduced to the corresponding alcohols. Amplified and semisynthetic pathways based on these core Clostridium pathways have been proposed and developed to produce short- and medium-chain alcohols (C3−C8).19−22 Yeasts produce a large spectrum of short and medium length chain “fusel” alcohols at low concentrations through the Ehrlich or alpha-keto acid pathway (Figure 1).14 These alcohols and the corresponding aldehydes and their derivatives are to a large extent responsible for the complex flavor of alcoholic beverages (beer, wine) produced from various carbohydrate substrates. The Ehrich pathway is the basis for the amplified semisynthetic (same enzymatic steps, different genes/enzymes) pathways of the celebrated i-butanol biosynthesis.13,23 A broad spectrum of medium chain alcohols can be thus generated through the alpha-keto acid pathway as depicted in Figure 3.20 The mevalonate (MEV) and/or the deoxyxylulose (DXP) pathways can be also engaged to synthesize a range of higherchain alcohols such as i-pentanol and farnesol.19−21 A summary of the alcohol biosynthesis pathways is depicted in Figure 3. Alkanes and Other Hydrocarbons. Alkanes, alkenes, and other hydrocarbons can be produced directly from biological processes or by combining biological processes with nonbiological processing such as dehydration and chemical catalysis. The main pathways for producing hydrocarbons are the isoprenoid pathways, namely the mevalonate (MEV) and/ or the deoxyxylulose (DXP) pathways and fatty-acid biosynthesis pathways (Figure 3).19−22 Isoprenoids are formed by the successive condensation of the 5-carbon isopentenyl-pyrophosphate (IPP) (or its isomer, dimethyl-allyl pyrophosphate (DMAP)), which is synthesized by the MEV or DXP pathways. These pathways lead to the synthesis of such complex molecules such as farnesene and pinene. Fatty acids can be synthesized from several precursors (Figure 3). One pathway is from malonyl-CoA by fatty-acid synthases. Malonyl-CoA is made from acetyl-CoA by the action of the acetyl-CoA carboxylase. The growing fatty acids are attached to acyl-carrier proteins (ACP). Thioesterases cleave fatty acids off the ACP. Fatty acids can be directly converted to 10174


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at the genome scale, thus their use has been frequently referred to as genome engineering.33 Use of synthetic (constitutive or regulated) promoters is an obvious target for optimizing pathways and program at the subgenome as well the genome scale. One of the earliest reported cases was when synthetic promoters were tested in Lactococcus lactis for tuning gene expression.34 Ribosome binding sites (RBS) are also well-suited targets for genome engineering using either random libraries35 or through a rational design approach.36 Similarly, libraries with tunable intergenic regions (TIGRs; such as “mRNA secondary structures, RNase cleavage sites, and RBS sequestering sequences”) can be used to optimize the expression of multiple genes.37 And indeed there have been several successful examples of using such approaches for pathway optimization. A notable example is that of modular optimization of pathways for fatty-acid biosynthesis in E. coli using both transcriptional and translational optimization which resulted in the highest reported titers and rates of synthesis in this organism.38 As the cost of DNA synthesis decreases, and novel assembly techniques such as circular polymerase extension cloning (CPEC) are developed,39 construction of complex libraries utilizing TIGRs can be readily constructed to optimize expressions of entire pathways. Expression of multiple genes in various locations on the chromosome can be optimized using multiplex automated genome engineering (MAGE),40 which utilizes synthetic oligonucleotides that are inserted into target regions across the chromosome to increase genomic diversity. Similarly, using trackable multiplex recombineering (TRMR),41 synthetic cassettes can be inserted in the E. coli genome alongside molecular barcodes, to alter gene expression. Other approaches have also been used to engineer whole cellular programs, for example, simultaneous and tuned overexpression of multiple heat shock proteins (HSPs) has been used to engineer increased protein solubility in strains of E. coli producing recombinant proteins.42,43 Similar approaches were used to express a tunable set of HSPs aiming to generate strains that tolerate high levels of metabolite stress.44−46 Other approaches employed RNAs, which play important roles in post-transcriptional regulation by various mechanisms, ranging from structural composition of the mRNA transcript itself (hairpins in mRNA molecules47) to trans- or cis-acting small noncoding RNAs48−52 or RNA molecules with catalytic activity53 (riboswitches). To sum, synthetic-biological approaches are most certainly going to have a large impact in optimizing pathways and cellular traits impacting the production of biofuels and commodity chemicals, but much remains to be done to bring these tools to impact the industrial production of these molecules. Platform Organisms, Then and Now. The last 8−10 years have seen a renewed interest in developing production strains via metabolic engineering based on well-established industrial organisms such as E. coli, yeast, and Clostridium organisms, and at the same time develop new cell platforms, such as algae, cyanobacteria, and lactobacilli. Although, as reviewed below, E. coli and yeast systems remain the dominant platforms, the last 10 years have seen substantial advances in the exploration of alternative if niche organisms. Yeast-based systems remain dominant for now in fermentation processes to produce biofuel molecules and commodity chemicals. Beyond the ethanol fermentation, well-established processes based on a yeast platform include the production of lactic acid (NatureWorks, LLC),54,55 and succinic acid.55 Two other major processes are at the demonstration or early

alkanes, or alternatively the fatty acyl-ACP can be converted to fatty aldehydes that upon decarboxylation give rise to alkanes. Long-chain alkenes can be also synthesized from fatty acylACP. Reverse beta-oxidation offers an alternative pathway that uses CoA as a carrier molecule and acetyl-CoA to elongate the growing fatty-acid chain. In a more recently study,24 alkanes can be produced from fatty aldehydes that can be derived from fatty acyl-ACP converted to free fatty acid and then to fatty acylCoA. Hydrocarbon molecules can be also obtained from alcohol dehydration, the products of which can then be converted to more complex molecules such as jet fuel. For example, i-butanol can be dehydrated into i-butylene, which can be converted into paraffinic kerosene (jet fuel).25,26 Several simple and complex hydrocarbons can be generated catalytically with high selectivity from short-chain fatty acids and notably butyric acid. A recent example of efficient conversion of fermentation products into alkanes suitable as gasoline or jet fuel involves the catalytic conversion of products of the Clostridium ABE fermentation.27 Acetone contains a nucleophilic alpha-carbon, which is amenable to C−C bond formation with the electrophilic alcohols, n-butanol and ethanol, leading to the formation of long-chain alkanes. To sum, there have been exceptional advances in the development of semisynthetic or synthetic pathways to biologically produce fuel molecules, many celebrated in highend journal publications, but none of these advances have been translated into economically viable processes. Possible reasons are discussed below, but the issue boils down to low yields, titers, and expensive separation costs. The chemistries are clearly here now and for the taking, but how would one use these chemistries in a way to achieve the yields and titers that would make these processes economically viable remains an unsolved, multifaceted problem, that has been minimally addressed and discussed in the literature for several reasons. First, it resists the development of a systematic strategy to solve it, and second, by its nature, it will not lead to high-end publications that appear to be a central driver of most researchers active in the field. Synthetic Biology and Genome Engineering: A Major Advance That Could Not Have Been Anticipated Ten Years Ago. Synthetic biology as it is understood today, as a more complex and molecularly detailed, synthetic facet of metabolic or cell engineering, was not on the radar screen of many when the 2005/06 DOE report was published. It is now a well-accepted discipline with its own rules, parlance, journals, and following, and is expected to play a major role in the development of new technologies for producing biofuels and chemicals from renewable or fossil resources. The level of sophistication that the field of synthetic biology has achieved in a short period of time is admirable, yet there are no obvious impacts in the development of processes that have reached the economic industrial production scale. A quick assessment of the field in the narrow context of producing biofuels and commodity chemicals is beneficial in assessing the changing landscape in this arena. Many cellular programs are frequently coded in clusters with complex cis and/or trans regulations,28 and this hinders efforts to effectively engineer them. Thus, de novo engineering of such programs without the complex regulation is a synthetic biology strategy to overcome this problem, although such synthetic programs must be well balanced to effectively operate.29−32 Synthetic biology tools have been used to alter gene expression 10175


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US if not longer. Since on a carbon basis, carbon is much cheaper from natural gas (such as carbon from CH3OH or synthesis gas produced from CH4) compared even from the most inexpensive biomass (e.g., biomass in Brazil or Malaysia), the competition from natural gas should be taken seriously in the development of biomass utilization technologies. Instead of fighting it as a competitor, natural gas and its derivatives should be viewed as a friendly yield and value enhancer for biomassderived carbohydrates.71 For example, beyond the lower cost on a carbon basis, CH3OH is more reduced than all major sugars deriving from biomass, and as a result, its extra reducing power when utilized as a cosubstrate can be used to boost the yields of several oxygenated molecules such as alcohols, but also the yields of more reduced molecules, including hydrocarbons.71

production stage: production of farnesene by Amyris, and production of i-butanol by Gevo, Butamax, and Butalco.26,55 Yeast genetics and quantitative biology tools are some of the most advanced and as a result, given the long industrial experience with yeast fermentations, the yeast platform will grow in importance in the industrial production of biofuels and commodity chemicals. E. coli remains a popular strain platform, largely based on the exceptional genetic and metabolic engineering/synthetic biology tools available, which are widely accepted as the best among any known organism. Additional advantages include fast growth, large industrial experience from recombinant-protein production, plus its use for the production of a commodity chemical, notably PDO (1,3-propanediol), based on the wellknown DuPont process.56 More recently, a new E. coli process for production of another commodity chemical, 1,4-butanediol was developed and scaled for industrial production.57 Thus, the E. coli platform remains a top choice for production of biofuels and commodity chemicals. It is somewhat surprising that despite the fact that many E. coli strains have been reported for producing several advanced biofuel molecules (such as ibutanol, n-butanol, alkanes, etc; see section above), none of these strains have been explored yet for industrial scale-up in which yeast remains the dominant platform. Clostridium organisms are growing in importance as strains for production of biofuel molecules and commodity chemicals. Some are solventogenic clostridia (notably C. acetobutylicum), which, with strain development by genetic or classical tools, have become attractive again for the industrial production of butanol or a mixture of solvents such as acetone−butanol− ethanol (ABE)58−60 or i-propanol−butanol−ethanol (IBE).61 New industrial plants have been built in China and other countries overseas,58 and a few US companies are contemplating new industrial plans for n-butanol production. Clostridium organisms have been also scaled up for butyrate production62 by Korean and Chinese companies. Of note, butyrate can be efficiently converted to short-chain hydrocarbons by hydrothermal reforming with industrially relevant efficiencies.63 Significantly, in the last 2−3 years, major progress has been reported in the industrial scale production of ethanol from waste gases, gasified biomass, or syngas using Clostridium acetogens that employ the Wood−Ljungdahl pathway (WLP).10,64−66 Major efforts are under way to develop effective genetic and metabolic engineering tools for engineering Clostridium acetogens aiming to broaden the spectrum of biofuel molecules that can be synthesized from waste gases at rates and titers of industrial importance. There have been several literature reports on other potential platform organisms for the production of biofuels and commodity chemicals,67 a list that includes lactobacilli,44 Corynobacterium glutamicum,68 and cyanobacteria,69 but none of these efforts have advanced enough to contemplate industrial scale up for now. Photosynthetic algal and bacterial systems have also attracted large attention, largely based on their ability to accumulate (i) lipids that can be directly converted to fuel molecules or (ii) carbohydrate-rich biomass that can be used as a fermentation substrate for producing fuels and commodity chemicals.70 An Unexpected Factor: The Competition from the Natural Gas Bonanza. The 2005/06 DOE report did not anticipate the current overabundance of natural gas in the US and many other parts of the world. Natural-gas overabundance is expected to continue for at least the next 30−50 years in the

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WHY ARE WE NOT THERE YET (AND ARE NOT LIKELY TO BE THERE SOON) There are several issues that confound progress in bringing new products and processes to the industrial setting. While much progress has been made at the academic setting in identifying potential products (chemicals as fuel molecules or commodity or specialty chemicals) and pathways to synthesize these products or their precursors, major barriers to the industrial success of these processes remain. In perspective, the great and widely acknowledged progress in the development of new pathways (chemistries) and tools (in metabolic/pathway engineering and synthetic biology tools) may be the easy part on the road to the development of economically sustainable processes for biofuel production. Our opinion is that these tremendous advances over the last 10 years or so will not remain without industrial impact. But that will be in the production of commodity and specialty chemicals, rather than biofuel molecules, which are practically subcommodity chemicals: very low value, and thus hard to justify new industrial investments. If there is one sentence one could capture the reason for lack of new industrial bioprocesses for the production of advanced biofuel molecules, it would be the following. The titers and product yields are too low, and the separation costs f rom aqueous solutions too high to justify producing molecules as biof uels other than ethanol for now. Separating organic molecules out of dilute aqueous solutions is an unforgiving problem. It is understood that the production economics of all new advanced biofuel processes must improve by 2 to 4 fold to become economically competitive, in the US at least. In some ways, this has been an anticipated problem and it trumps all the successes in the pathway engineering that have been achieved. The recent drop in oil prices and the large supplies of natural gas have made matters worse. Still, examining the issues in some detail would be beneficial as it may identify more targeted strategies for breaking the Gordian Knot of advanced biofuels. Parameters that Affect Process Economics. First, there is the issue of choosing the product to be targeted for production using a biological process or a combination of biological and nonbiological process. While DOE and several literature publications have listed a large number of target molecules as fuels or commodity chemicals, this list may incomplete and may miss a set of important molecules, especially molecules that can be produced by combining biological with nonbiological processes. The choice of target molecules also depends on whether chemistries and technologies that would use the molecule exist, thus guaranteeing a 10176



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WHAT BIOFUEL MOLECULES MAKE SENSE THROUGH PURELY-BIOLOGICAL ROUTES? While very reduced biofuel molecules such as alkanes, longhydrocarbon chain alcohols or similar compounds can be produced from sugars as already discussed, is a biological route the most efficient and effective way to do so? Ethanol, butanols, propanols, and the corresponding carboxylic acids (as biofuel precursors) can be directly produced from sugars with the excess oxygen from the sugar being removed and released as CO2 (and H2O, e.g., in the case of butanols). Biological routes to alkanes, terpenes, and generally biofuel chemicals with hydrocarbon chains of more than 4 or 5 carbons require further removal of oxygen from the sugar and formation of new C−C bonds. This is typically accomplished by metabolizing additional sugar to provide reducing equivalents (electrons; effectively an aqueous reforming process) and energy generation (ATP; thioester bonds) for C−C bond formation. Thus, product yields on sugars are negatively impacted. This point is illustrated by examining the theoretical yields for various products from the MEV, DXP, and fatty-acid pathways of Figure 3. Most of the multicarbon (number of carbons >5− 7) and highly reduced products have low product yields (mole or gram of product per mole or gram of sugar) due to ATP and electron limitations. For example, the maximum theoretical yield for farnesene (C15H24; Figure 3 and ref 77) is 0.22 mol/ mol glucose or 0.25 g/g glucose assuming that acetyl-CoA is generated through glycolysis. For this theoretical yield, for every mol of farnesene, 9 mol of glucose must be used and 12 mol of CO2 released. But can even such low theoretical yields be achieved in practical fermentations? In most cases, to achieve these theoretical yields, it is necessary to use the aerobic electron transport chain to generate the needed ATP for building C−C bonds, while at the same time achieving a highly reduced cellular environment (typical of anaerobic fermentations) that would enable the use of available electrons to generate the highly reduced product. As a result, such theoretical yields, in contrast to those of simpler metabolites like ethanol and butanol, are unlikely to be achieved under practical fermentation conditions. Practically achievable yields are significantly lower than theoretical yields, even for naturally produced molecules, for which the cells have evolutionarily tuned cellular regulation to favor their production under normal conditions to benefits the cells. For example, data on the production of long-chain fatty acids and their derivatives show product yields typically less than 20−30% of theoretical.78,79 Data on novel pathways (from which the cell derives no benefit for growth or survival, such as for farnesene production) are hard to find in the literature, but the complexity and high cost of media and the culture conditions reported for such products are suggestive of product yields significantly lower than the low theoretical yields. If one combines these considerations with the severe toxicity of such products or their intermediates (e.g., ref 80), production costs would exceed all reasonable biofuel target costs by several fold if not an order of magnitude. Yet, this argument seems to have been lost on both privately as well as government-funded R&D programs. It should be emphasized, however, that for products of high value, such as specialty chemicals, nutrichemicals, and pharmaceuticals, the fermentation-only approach is rational and useful as many established applications demonstrate. Fermentation processes makes sense if used in combination with chemical and catalytic processes. Thermal energy is less

market for the target molecule. Using as a case example lactate, the market for biologically produced lactic acid and it lactatebased polymers (e.g., the NatureWorks, LLC, technology54) took several years of development to reach the point of sustainable profitability. The more recent case is that of ibutanol, a chemical with a small chemical market until recently requiring the development of processes based on it, or the ability to produce it at a cost that would make it a competitive biofuel. The latter appears to be unattainable for now. Thus, it appears unlikely that there exists a systematic way to rationally identify target molecules beyond those that have been already identified. Identification of additional target molecules will rest with individual companies and investigators. Part of minimizing costs is the ability to carry out the fermentation under largely nonaseptic or minimally protected fermentation conditions to avoid contamination as is the case of the yeast ethanol fermentation in Brazil.72 Although aside from these ethanol fermentations there is no technological experience with running industrial-scale fermentations aseptically, this is an issue that deserves careful consideration and perhaps some fresh solutions in order to minimize the cost of preparing and running the fermentors under strict aseptic conditions. While not an issue in fermentations to produce higher-value products, the low production costs that biofuel molecules demand makes this an issue worthy of careful consideration. Assuming that target products have been identified, there are many barriers until a process to produce such products industrially becomes a profitable proposition. These barriers derive from not being able to achieve important process characteristics/metrics that are necessary for industrial success. While many process parameters will affect the industrial viability of the process, the most important ones include the following: • ability of the organism to use inexpensive substrates at high rate • ability to carry out the fermentation under nonstrict aseptic conditions • achieving high product-formation rates, titers, yields and selectivity (i.e., the ability to produce only the desirable product without byproducts, thus increasing the yield of the desirable product) • ease of continuous-like or semicontinuous operations • process integration with separation technologies for postfermentation product purification • industrial experience with the chosen production organism • freedom to operate, as it relates to intellectual-property protection Significantly, it is the integration of all these parameters, several of which are interdependent, that makes it very difficult to predict the likely success of a process. At the end, however, for biofuel molecules and commodity chemicals, it boils down to low substrate cost, high rates of product formation and product titers, and low separation costs. Technologies to separate organic molecules from dilute aqueous solutions and process integration schemes have been extensively reviewed (se, e.g., refs 73−76), so these aspects will not be discussed here. Instead, the focus on what follows are biological issues that affect substrate costs, process yields, and titers. 10177


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choice and development that impact substrate utilization and costs. Engineering broader substrate utilization capabilities and flexibility is not a simple matter, as the well-known example of engineering xylose utilization capabilities in yeast demonstrates, where it took more than 35 years of research by many groups worldwide and an inordinate amount of funding to engineer ethanol producing S. cerevisiae to utilize xylose, just one of the nonglucose components of cellulosic biomass. Biomass-derived substrates contain a mixture of different sugars, and thus there exists the possibility of Carbon Catabolite Repression (CCR), in which the presence of a preferred substrate, such as glucose, will suppress the utilization of other sugars.82 Biomass composition varies with plant source, location, plant age and time of the year. It is made up of the fermentable cellulose and hemicelluloses/xylans and lignin. Typical sugar composition of hardwood (spruce, douglas fir, pine, poplar) biomass83−86 is 43−45% glucose, 2−14% mannose, and 2.5−15% xylose, with arabinose and galactose being minor components ( 60% of the overall production cost81) in biofuel and commodity-chemical production is substraterelated costs. Many issues impact the costs associated with substrates and their utilization: substrate sourcing and availability; variability in substrate composition and purity; extent of necessary pretreatment; cellular flexibility in efficient and simultaneous utilization of various substrates with season or source-dependent composition variability; but also product yield, and complete substrate utilization. These issues largely define the “true product cost”, which is beyond the cost of a single type or source of biomass. Strain-development technologies to address the profound impact of the “true substrate cost” have not been given the attention they deserve. The issues and importance of biomass supply and logistics as well as of biomass pretreatment and saccharification and their impact on process economics have been most recently expertly reviewed.1 What follows focuses then on aspects of strain 10178



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CONCLUDING REMARKS There has been remarkable progress made in engineering new synthetic or semisynthetic pathways or in improving natural pathways for producing biofuel molecules. Synthetic biology tools and approaches have been developed that will likely enhance our ability to develop new pathways and organisms that display superior production characteristics. For now, however, beyond ethanol production, producing advanced biofuels does not appear economically feasible due to the low titers, yields, and rate of these more energy-dense molecules. This prevents the development of separation technologies that could be deployed for the economic production of these subcommodity chemicals so that they could compete in the market place with conventional petroleum-derived fuels or natural gas. The recent startup of a few cellulosics-based ethanol plants will undoubtedly be crucial in developing expertise and data that would be necessary to more accurately assess the economics of producing advanced biofuels from biomass and waste gases. At the same time, the large advances made in pathway engineering and synthetic biology can be put to use in developing biological processes for producing commodity and specialty chemicals, which have target production costs that exceed those of biofuel molecules by at least 2−3 fold. That will generate valuable process know-how and, with experience, may lead to efficiencies that can be utilized for the production of biofuels. At the end, the combination of new, more robust and productive strains with process integration may hold the key to success in the biofuels technologies, but that seems unlikely in the immediate future especially in the current environment of low petroleum prices and abundant natural gas.

also formed. These include alcohols, organic acids, aldehydes (furfural and hydroxymethyfurfural), and ketones, all of which are inhibitory to cells, and thus strains with increased tolerance to such chemicals must be deployed.44,95−99 Thus, developing microbial tolerance to a broad spectrum of chemicals is a problem of significant industrial importance. Importantly, one must also ascertain that the tolerant strains are capable of producing the desirable toxic metabolites are higher rates, and titers, which would translate to the ultimate benefit of reduced production and separation costs thus leading to an industrially feasible process. The last 10 years have witnessed a large activity in tolerance engineering.44,96−99,101−103 In most studies, however, the improvement in tolerance have been small to modest, and also, these improvements have not been tested if they would result in higher titers and production rates of the toxic metabolite. Moreover, the work has been fragmented, and with few exceptions (e.g., refs 45,100, and 104), only one or two genetic determinants have been employed for improved tolerance. There has been no systematic effort to combine genetic determinants or presumably orthogonal programs to generate a stronger tolerant phenotype. There is a clear need to develop a new action plan to establish dramatically improved tolerance that would meet industrial expectations. Could we possibly reach practical butanol titers of 30 g/L or higher, or practical ethanol titers of 200 g/L? Are there biological limits that may preclude the achievement of such goals,105 what are those, and how can they be overcome or bypassed? Strain Stability, Resistance to Phage Infection, And the Importance of Minimally Aseptic Fermentation Conditions. As already mentioned, strain stability is of paramount importance for robust process development. Strain stability typically refers to genetic stability as it pertains to growth and product-formation characteristics. Genetic stability refers to DNA changes that affect cell growth and product formation and could be on either or both native and transgene DNA. For example, for new or altered pathways, the stability of the transgene constructs is essential and a long-standing problem when dealing with engineered strains. Strain stability also includes stability and reproducibility of key fermentation characteristics that may not be necessarily determined by genetic changes. Growth and product formation may be very sensitive to small changes in fermentation conditions (e.g., media composition, pH, pO2, T, mixing characteristics), and can thus result in undesirable variability in fermentation performance. It is also becoming increasingly evident that epigenetic effects (changes in gene expression and cell metabolism without changes in the cell’s genomic DNA) play a role in cell metabolism and product formation as in the case of the Clostridium ABE fermentation.106−108 Epigenetic effects106 may be related to DNA methylation or other DNA modifications, the role of small noncoding RNAs and their stochastic expression,109,110 or complex regulation resulting in bistability and epigenetic inheritance.106,111 Phage infection has been a well-documented problem in industrial fermentations for over 100 years,16 and remains an essential concern in strain and process development, but has attracted relatively little attention in modern prokaryotic strain development and biotechnology. Finally, as already discussed, strains that make it possible to run the fermentation process under nonstrict aseptic conditions and thus reduce the cost of bioprocessing are especially desirable for processes that produce biofuel molecules.

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AUTHOR INFORMATION

Corresponding Author

*Phone: (302) 831-8376. Fax: (302) 831-4841. E-mail: epaps@ udel.edu. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The author acknowledges financial support by the US Department of Energy (DOE) ARPA-E program through contract No. DE-AR0000432 during the preparation of this manuscript. The author thanks the DOE for allowing him to use parts of his contribution to the UD Department of Energy 2015 Report “Lignocellulosic Biomass for Advanced Biofuels and Bioproducts: Workshop Report”112 in this manuscript. Discussions with Prof. Ryan Gill of the University of Colorado during the aforementioned DOE workshop are gratefully acknowledged as inspiration for parts of this work. Finally, the author thanks an anonymous reviewer whose comments inspired the section “What Biofuel Molecules Make Sense through Purely-Biological Routes?”

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REFERENCES

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