The Impact of Biotechnological Advances on the Future of US Bioenergy†

Perspective The impact of biotechnological advances on the future of US bioenergy†

Brian H. Davison, Craig C. Brandt, Adam M. Guss, Udaya C. Kalluri, Antony V. Palumbo, Richard L. Stouder, Erin G. Webb, Oak Ridge National Laboratory, Biosciences and Environmental Sciences Divisions, Oak Ridge, TN, USA

Received October 30, 2014; revised February 13, 2015; accepted February 20, 2015 View online at Wiley Online Library (wileyonlinelibrary.com); DOI: 10.1002/bbb.1549; Biofuels, Bioprod. Bioref. (2015)

Abstract: Modern biotechnology has the potential to substantially advance the feasibility, structure, and effi ciency of future biofuel supply chains. Advances might be direct or indirect. A direct advance would be improving the effi ciency of biochemical conversion processes and feedstock production. Direct advances in processing may involve developing improved enzymes and bacteria to convert lignocellulosic feedstocks to ethanol. Progress in feedstock production could include enhancing crop yields via genetic modifi cation or the selection of specifi c natural variants and breeds. Other direct results of biotechnology might increase the production of fungible biofuels and bioproducts, which would impact the supply chain. Indirect advances might include modifi cations to dedicated bioenergy crops that enable them to grow on marginal lands rather than land needed for food production. This study assesses the feasibility and advantages of near-future (10-year) biotechnological developments for a US biomass-based supply chain for bioenergy production. We assume a simplifi ed supply chain of feedstock, logistics and land use, conversion, and products and utilization. The primary focus is how likely developments in feedstock production and conversion technologies will impact bioenergy and biofuels in the USA; a secondary focus is other innovative uses of biotechnologies in the energy arenas. The assessment addresses near-term biofuels based on starch, sugar, and cellulosic feedstocks and considers some longer-term options, such as oil-crop and algal technologies. © 2015. This article is a U.S. Government work and is in the public domain in the USA.

Supporting information may be found in the online version of this article.

Keywords: biotechnology; bioenergy; bioconversion; biofeedstocks

Correspondence to: Brian H. Davison, Oak Ridge National Laboratory, Biosciences Division, P.O. Box 2008, MS 6037, Oak Ridge, TN 37831-6037, USA. E-mail: [email protected]

†This article is a US Government work and is in the public domain in the USA.

© 2015. This article is a U.S. Government work and is in the public domain in the USA. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. BH Davison et al. Perspective: The impact of biotechnological advances on the future of U.S. bioenergy

Introduction and key ﬁ ndings Also critical to bioenergy, but only indirectly infl uenced by biotechnology, are policy and economics issues. For dvanced biotechnology will be crucial to improving example, the Renewable Fuel Standard (RFS) and other biofuel supply chains. Over the next 10 years, most government policies directly support the biofuels industry, A of the progress based on biotechnology research and and uncertainty about their stability directly infl uences development will be in conversion processes. Improvements bioenergy investment. Consistent policies are essential for in feedstocks will follow over a slightly longer period. near-term deployment because they aff ect whether devel- Potential biotechnology game changers for bioenergy opers can obtain funding for fi rst-generation biorefi neries. include: Policies regarding uses of genetically modifi ed organisms (GMOs) also will have a direct bearing on biotechnol- • parallel yield and convertibility improvements in ogy implementation. Use of GMOs within the confi nes biofeedstocks and residues of a biorefi nery is generally accepted if biocontainment is • robust, easily convertible lignocellulosic feedstocks and practiced; however, the use of GMO plants and algae is a residues with minimal pre-treatment matter of debate worldwide. Public acceptance of GMO • predictable agronomic feedstock improvements for plants in the USA depends on their being regulated and yield and sustainability (e.g., low nitrogen and water thoroughly studied. Th ese restrictions can add years to use and increased soil organic carbon sequestration) fi eld testing and eventual deployment; however, biotechno- • ability to control rhizosphere (the soil microbial) com- logical advances can speed the regulatory processes. Algal munities to improve biofeedstock traits feedstock developers presume their GMOs will be closely • economically stable bioconversions able to handle bio- contained in closed photobioreactors. feedstock variability • new tools to rapidly and rationally genetically engineer Background new microbial isolates with unique complex capabilities (e.g., new enzymes and fuels or capability to Th e global biofuels industry is growing.1,2 A cellulosic thrive in harsh conditions such as pH or temperature biofuels industry is emerging with a total projected capac- extremes) ity worldwide of about 195 M gal/year (a modest amount • rational reproducible control of energy fl uxes and car- compared with the 20 to 30 B gal/year for fi rst-generation bon balance in microbes (e.g., decoupling growth from starch- and sugar-based ethanol production). Cellulosic metabolism) biorefi neries have opened in Italy, the USA, Brazil, • cellular redesign to overcome fermentation product China, and Spain; and others are scheduled to launch in inhibition while maintaining yield and rate 2014–2015. Most employ biological processes. Second- • stable, high-rate microalga lipid production in open generation biofuel refi neries have been announced for 2017 systems and beyond with a projected capacity of about 5 to 6 B gal/ • expanded compatible biotechnology processes to pro- year. Global biofeedstocks could produce about 914 mil- duce co-products(e.g., from lignin) along with fuels lion tons of residues by 2030, which could replace half of Th e developing bioenergy industry faces several broad the gasoline needed by then. Th e USA remains the deploy- issues that are critical to accelerating bioenergy deployment and technology leader in biofuels – both conven- ment but that will be infl uenced only indirectly by tional (grain-based) and advanced (e.g., cellulosic ethanol, biotechnology: algal biodiesel, drop-in biofuels).However, other nations are gaining (e.g., Brazil, China, the European Union, and • Scaling up an industry capable of achieving US and Southeast Asia). In 2011, Brazil produced just over half as global bioenergy goals requires building a huge supply much ethanol as the US total of ~53 B L/year.3 chain. Scaling up the bioenergy industry to meet US and global • Given the large scale of biofuel production needed, bioenergy goals will require a supply chain with a volume massive market demand for co-products is necessary. rivaling the current agricultural and energy industries • Th e low density and decomposition of biomass is chal- combined.4 Although biofuel production has similar key lenging for feedstock storage and logistics. requirements to other energy supply chain networks, it • Land-use and sustainability issues must be addressed. has several unique aspects related primarily to biofeed- Biotechnological and agronomic approaches can stocks and the potential use of biology for conversion. increase sustainability and manage water use. Consideration must be given to the feedstocks employed

© 2015. This article is a U.S. Government work and is in the public domain in the USA. | Biofuels, Bioprod. Bioref. (2015); DOI: 10.1002/bbb Perspective: The impact of biotechnological advances on the future of U.S. bioenergy BH Davison et al.

(e.g., agricultural residues, dedicated energy crops), logistics Feedstocks have great mid-term potential and receive less and land-use decisions associated with the selected feed- attention.) A main current driver for continued improve- stocks, conversion technologies used to convert the feed- ment in bioenergy is that liquid biofuels remain the only stocks (e.g., thermochemical conversion, fermentation) to renewable alternative for the diesel and jet markets, which products (e.g., ethanol),and utilization of the products pro- are benefi ting from biotechnological improvements. duced (Fig. 1). See Supplemental S1 for longer defi nitions. Table 1 summarizes factors in the biofuel supply chain Th is assessment examines the infl uence of biotechnol- and highlights how advanced biotechnology might signifi - ogy on US biofuel production through the lens of the sup- cantly impact them. Th e following sections consider these ply chain. Currently, ethanol from corn provides close to factors in sequence – feedstocks, logistics and land use, 10% of US liquid ‘gasoline’ fuel supply. Mandates of the conversion, and products – and present analyses of the US Energy Independence and Security Act of 2007 will feasibility of advances, risks, barriers to major advances require signifi cant increases in feedstock amounts and and crosscutting impacts on the supply chain. sources. Th us, future supply chains may include cellulosic Drawing on improved understanding of mechanisms, feedstocks for ethanol production (currently in deploy- advanced biotechnologies lead to targeted manipulations ment) and advanced fungible biofuels based on cellulosic using genetic engineering, synthetic biology, directed evo- or other biomass feedstocks (e.g., algal oils). lution, or genetically assisted breeding. Th e biotechnological approaches most likely to have the greatest impact on Discussion the biofuel supply chain include synthetic biology, protein design, and associated tools. Synthetic biology can be Advanced biotechnology can benefi t the bioenergy sup- defi ned as the rational design of biological systems on the ply chain with near-term developments in conversion and basis of engineering principles. Central to this approach is co-products. (Most of our discussion covers this area. the capability to regulate genes in native and introduced

Figure 1. Schematic of major components of the biofuels supply chain.

© 2015. This article is a U.S. Government work and is in the public domain in the USA. | Biofuels, Bioprod. Bioref. (2015); DOI: 10.1002/bbb BH Davison et al. Perspective: The impact of biotechnological advances on the future of U.S. bioenergy

Table 1. Critical factors affecting deployment and scale-up of US bioenergy industries Feedstocks Logistics and land-water use Conversion technologies Products and utilization Insuffi cient yield Spatially dispersed feedstock Insuffi cient yield, rate, and titer Certifi cation of new fuels sources Tolerance to environmental Low bulk density and high Convert all variable feedstock Biofuel demand –ethanol blend stresses (e.g., drought) moisture content of feedstock compositions (e.g., recalcitrance) wall, development of new biofuels (e.g., biojet fuel) Amendment requirements Biomass stability during storage Marketable Policy stability (e.g., fertilizer) Co-products (e.g., Renewable Fuel Standard) Adoption of genetically Feedstock displacement of High capital costs and investment Compatibility with existing modifi ed crops cropland risk infrastructure Halophytes, saline agriculture Indirect land use change Scalability tradeoffs Algae production Scalability tradeoffs Direct production of drop-in fuels Water requirements Factors for which advanced biotechnologies are expected to make a signifi cant impact are indicated in italic type.

metabolic pathways in a controlled fashion. Examples of and directed evolution. ‘Rational design’ is defi ned as the bioengineering options include knowledge-based design of new proteins; ‘directed evolution’ refers to the generation of new protein functions • varying promoter activity and effi cacy. through functional screening of randomly generated vari- • using diff erently inducible promoters. ant copies of the protein. DNA writing and site-specifi c • modifying ribosome binding strength. genome engineering capabilities form the basis of the • exploiting messenger ribonucleic acid (mRNA) rational design approach. Th e directed evolution approach secondary structures to diff erentially. stabilizing requires high-throughput, automated design, construction, specifi c mRNA segments. engineering, and evaluation of tens of thousands of combi- • amplifying genes on chromosomes. nations using robotics and computational techniques. • using nonnative RNA polymerase. Other important biotechnological tools include: Site-specifi c genome bioengineering tools that facilitate • molecular techniques relevant to targeted genome editing and transcription modulation – robust, reliable, and rapid single-cell genomics. are essential for elucidating the function ofdeoxyribonu- – single-molecule detection. cleic acid (DNA) elements. Such tools include homologous – protein profi ling from very small samples. gene targeting, transposases, site-specifi c recombinases, – DNA writing. meganucleases, integration of viral vectors, and artifi cial – gene stacking. zinc-fi nger technology. When coupled to eff ector domains, – genome engineering. an emerging technique – customizable transcription – transient and stable transformation. activator-like eff ectors – provides a promising platform • phenotyping techniques for in situ measurements. for achieving a wide variety of targeted genome manipu- • standards-adhered reconstructions of transcriptional 5 lations. Th ese site-specifi c genome bioengineering tools networks and nodes. may achieve greater precision and predictability in modi- • annotated genome sequences from hundreds of multi- fying biological traits in feedstocks and potentially in ple relevant species and genotypes. microbial-base conversion technologies. Variations in enzymatic function and protein features Th e creation of transgenics or GMOs can prove the func- ultimately have functional relevance at the organism level tion of key genes or pathways leading to the desired phe- and thus have been tested over evolutionary time via natu- notypes. Armed with this knowledge, genetically assisted ral selection. Th e nascent fi eld of protein engineering has breeding can greatly accelerate the development of specifi c been most used for sector design in microbial sciences.6 phenotypes that are nontransgenic and thus not regulated Th ere are two main approaches to improving or creat- as GMOs. Th is will be especially useful for improved non- ing new protein functions: rational design of proteins GMO plant biofeedstocks, especially where the traits are

either cisgenic or already within the natural variation in duction by advanced breeding and biotechnology if the the population.7 markets make such eff orts worthwhile.11 Similar changes might also be seen in residues from other food crops. Feedstocks Biotechnology will be used to improve robustness and decrease water/nutrient requirements for energy crops. Biotechnology can improve the feedstock component of Two active avenues are in control of the plant (such as the supply chain in multiple ways: osmotic capacities, transpiration, nutrient transporters) • increasing feedstock yields and manipulation and control of the rhizosphere, where • improving robustness and reducing amendment (e.g., certain microbes have been shown to enhance drought fertilizer) requirements resistance in switchgrass.13 • increasing tolerance for environmental and biological Leading candidate crops that may supply second- stresses such as drought and pest attacks generation feedstocks for lignocellulosic ethanol include • modifying feedstock composition (e.g., to reduce bio- perennial grasses such as switchgrass, Miscanthus, energy mass recalcitrance to conversion). cane, and short-rotation woody crops like poplar and wil- Improvements will likely occur through genetic under- low. Perennial oilseed species such as Jatropha may serve standing that aff ects the phenotypic characteristics of as biodiesel feedstocks,14 and algae may eventually become interest. Potential useful manipulations include breeding a major feedstock. eff orts that select for traits of interest and direct genetic Th e attractiveness of microalgae stems from their rapid modifi cations. Traditional breeding will transform into growth under a broad variety of conditions, including in genetically assisted breeding using biotechnological tools saline or nutrient-poor water or waste water. Th ey can be to rapidly identify desired traits in plants at earlier stages. grown in open ponds, closed chambers, or vertical stacks, Plant biotechnology will have a major impact (although eliminating the use of arable land. However, there are bar- fi eld trials slow the impact of new discoveries). Some con- riers to making algal biofuels economically viable, includ- cepts in yield and robustness are already in fi eld trials, and ing high infrastructure and energy costs for growth and research into composition will follow. harvest; the necessity of year-round warm weather; and a high possibility of contamination, evaporation, or incon- Yield, robustness, and planting requirements sistent mixing.15 Additional barriers are outlined in Cheng 16 17 Agricultural and forest residues are critical feedstocks for and Timilsina and in the DOE algal fuels roadmap. the fi rst biofuel conversion facilities.8 Corn stover, a par- Traditional biology or advanced biotechn ology may be able ticularly important agricultural residue, is the feedstock to improve the salt tolerance of algae, allowing the use of source for cellulosic ethanol facilities currently under con- brackish water for growth. Most research targets improve- struction by POET9 and DuPont.10 But because corn grain ments in eukaryotic algae, especially those that naturally probably will continue to be a higher-value commodity accumulate large quantities of lipids under stress. Recent than stover for bioenergy, most biotechnology eff orts to advances suggest a combination of metabolic engineering improve corn focus on optimizing grain yield and qual- and process control can alleviate algal challenges. Further ity. Average grain yields have been projected to approach discussion of how biotechnology could advance algal feasi- 250 bu/ac in 2030,11 up from an average 160 bu/ac in bility is in supplemental material section S2. 2013.12 Th is increase could have the paradoxical eff ect of actually reducing corn stover feedstocks. Th e total amount Feedstock composition of stover produced depends on the harvest index (ratio of Lignocellulosic-biomass-based biofuels would use dedi- grain mass to total aboveground crop mass). Currently, the cated energy crops such as switchgrass, along with green harvest index averages 0.5 (half of the above ground plant waste or residue from food or nonfood crops (e.g., corn, mass is grain); but genetic selection to increase grain yields wheat, rice, sorghum). Th e lignin and cellulose, their could increase the harvest index to as much as 0.7,11 greatly organization and quality, structural features of the plant reducing the amount of corn stover available. For example, cell wall, and patterns of cell wall development directly if the corn grain yield were 250 bu/ac with a harvest index aff ect biomass properties and in turn the sugar yield and of 0.5, the pre-harvest stover yield would be 15.7 Mg/ha the potential for producing liquid transportation fuels. (7 ton/ac). If the harvest index increased to 0.7, the stover A major goal of feedstock biotechnology eff orts is mak- yield would drop to 6.7 Mg/ha (3 ton/ac). However, the ing more easily convertible plants, for instance by reduc- harvest index can be modifi ed to increase biomass pro- ing lignin content. Early work dispelled the intuitive idea

that reducing lignin content would reduce the strength manipulated at a time. Th e manipulation of one gene oft en of plants and lead to lodging (i.e., plants falling over in has both expected and unexpected pleiotropic and unde- the fi elds, making them diffi cult to harvest).18 Instead, sirable eff ects on the plants. Th e paucity of publications increased lignin was found to be more likely to cause lodg- reporting dramatic improvements may be due to the failure ing, possibly by making plants more brittle. As researchers to target the right combination of gene(s) and promoter ele- continue to develop plants with lower lignin content, there ments, and to the lack of understanding of the correlation may be a point at which improvements in digestibility are between protein structure and the most eff ective enzyme not worth the reductions in strength, water movement or biochemistry. Th erefore, a whole pathway or systems view pest resistance. A review by Pedersen et al.19 found that, is needed. Th ese bottlenecks to realizing biotechnological although results were mixed, reduced lignin tends to crop advancements can be addressed with decrease the agricultural fi tness of plants. However, they noted the signifi cance of reduced lignin content is strongly • robust statistical analysis methods to handle complex aff ected by interactions with the environment and genetic and diverse datasets. background. Lignin has been shown to play a role in plant • plant models with shorter life cycles. shear strength;20 since grinding equipment designed to • fi eld trials coordinated tightly with greenhouse exert shear forces on biomass is more energy-effi cient, assessments. reducing lignin could in turn reduce the energy required • parallel and concerted investment by industry. for grinding. As adequate quantities of modifi ed bioenergy crop material become available in fi eld trials, experiments Logistics and land and water use are needed to test the relationship between lignin co ntent Eff orts to genetically enhance energy crop production and and grinding energy. conversion will aff ect supply-chain logistics (i.e., harvest, Th ere has been a rapid rise in reports on genetic and storage, transport, and size reduction) and resource uti- molecular underpinnings of biomass properties in both lization. Speculative biotechnological improvements that woody and herbaceous plants. Several excellent recent would improve logistics include increased biomass density, reviews are available on genomics and biotechnological improved storability, decreased variability, and reduced approaches to improving plant cell wall characteristics and ash content (especially for thermochemical processes). 21–27 saccharifi cation effi ciency. Th ese reviews report that Higher yields for biomass crops – a primary goal of feed- changes in cellulose, xylan, and pectin can all have benefi - stock biotechnology research – typically help reduce crop cial impacts. production and logistics costs. Sokhansanj et al.31 show Another goal of feedstock-related biotechnology is that increasing switchgrass yields from 4.5 to 13.4 ton/ac increasing plant-oil production in natural oil-seed crops, dropped production costs by more than 50% (from $37.66 algae, or new plant species. Th ese plant oils can be directly to $17.37 ton/ac). made into biodiesel or upgraded into a biojet fuel, as Although many biotechnological advances, such as 28–30 several recent reviews report. increasing yields, will improve logistical effi ciency and reduce costs, others may increase the diffi culty of building Barriers to improvement of feedstocks supply chains capable of delivering high-quality year-round A nearly universal goal of plant biotechnology research is feedstocks. For example, how will genetic improvements increased yields, in terms of overall plant growth (height or that make plants more easily convertible impact size- mass) and/or the plant component needed for the feed, food, reduction and storage operations? More discussion is in the or fi ber market. It remains a colossal challenge to precisely supplemental material, sections S3 and S4. defi ne the genetic basis of a plant trait. Th e more complex the Competition between bioenergy crops and food/feed trait, the more factors there are that control the trait, making production (real and perceived) for land and water is it harder to pin down targets for transformation. a challenge. Measures to reduce land competition will Recent biotechnological approaches include studying include increasing biomass yield (dry mass produced pathways and genome-wide-omics*, but when it comes to per unit of land per unit of time), without harming soil 32 validating a hypothesis for a role or function, one gene is health, to minimize the land area required for bioenergy feedstocks,33 and designing plants that tolerate environ-

*The term ‘-omics’ refers to the growing suite of large-data biologi- mental stresses such as drought and salt and could be 33,34 cal analyses, including genomics, transcriptomics, proteomics, and grown on currently unproductive or underused lands. metabolomics. Strategies such as double-cropping, either seasonal

( planting biomass as a winter cover crop) or spatial (plant- the past two decades and probably will continue to do so, ing grasses between rows of a tree plantation) can help but major SSF breakthroughs are unlikely. energy crops coexist with food crops. Some energy crops, Consolidated bioprocessing (CBP) uses microorganisms particularly trees, appear to be able to tolerate saline soils that produce their own hydrolytic enzymes and complete that are not suitable for food crop production. It has been fermentation into the product in one unit operation. CBP estimated that trees selected or designed for ability to probably will be realized in some form within the next 10 grow on salt-aff ected land could supply up to 8% of global years; hybrid CBP/SSF approaches also will be deployed primary energy consumption.35,36 Miscanthus, which can that combine cellulase-expressing industrial microbes substitute for corn in ethanol production, requires less that are incapable or poorly capable of converting plant land and water than corn. Carefully selecting and trans- biomass alone, but that require less added cellulase than forming crops for higher yields and lower water use will current strains. Th ere has been progress both in engi- be necessary to achieve sustainability in the use of basic neering cellulolytic microbes to make fuels38,39 and engi- resources such as land and water. neering fuel-making microbes to degrade cellulose.40–42 Expanded research to improve the productivity of bioen- Synthetic biology and metabolic engineering tools have ergy crops with low or no chemical inputs will achieve the been brought to bear on issues such as co-utilization dual goals of expanding the production and use of biomass of glucose (the sole component of cellulose) and xylose and improving sustainability.32 (the primary component of hemicellulose) in a variety of organisms.43–46 Further near-term progress toward effi - Conversion technologies cient co-utilization is certain. Non-xylose hemicellulosic sugars such as rhamnose, arabinose, and galactose are Advanced biotechnology is critical to developing and underutilized; advanced synthetic biology tools for meta- deploying solutions for biomass conversion. Th ere are two bolic control are being developed47 that will enable them aspects of this issue: (1) modifi cation of the microbes used to be used more effi ciently, thus increasing the fuel/chemi- in conversion processes, via metabolic engineering or syn- cal yield per ton of biomass without decreasing the fi tness thetic biology, to produce new or improved products and or robustness of the CBP micro-organism. (2) improvement in the key factors of bioconversion: yield, Preliminary reports suggest un-pretreated biomass could titer, and rate. be a feasible substrate for bioconversion.48 If biotechnol- Biotechnology has the potential to enhance three pri- ogy can improve the yield and rate of product formation mary aspects of conversion: from un-pretreated biomass, it would be a game-changing • additional feasible feedstock streams technology because it would eliminate the need for costly • product diversifi cation chemical pretreatment. • process technologies and effi ciencies Planned waste streams from bioprocessing – including lignin, acetic acid, and glycerol – need to be utilized to add Metabolic engineering, synthetic biology, and other bio- value for biorefi neries. Biotechnology could help remedy technological advances will allow more rapid optimization the underutilization of lignin,49 a highly amorphous and of conversion processes by rational enzyme engineering, hydrophobic polymeric network of substituted aromatic by introduction of new pathways and regulation, and by compounds that accounts for approximately 25% of plant control of the fl ux. biomass.50 It is typically burned for process heat and elec- Conversion of additional feedstocks and tricity, b ut it could be used to make fi ne and/or bulk chemicals to increase the economic viability of biorefi neries. components One challenging potential solution would be engineering Th e most critical bioenergy feedstocks will be cellulosic a microbe to depolymerize lignin and convert it to a fuel and hemicellulosic sugars from plant biomass. Several or a bulk chemical. Given the vast quantities of lignin that companies (Abengoa, Beta Renewables, and DuPont37) are would be produced by a mature biofuels industry, the target using conversion processes driven by separately produced products would need pre-existing large markets to prevent cellulolytic enzymes [e.g., simultaneous saccharifi cation market saturation and enable rapid commercialization. and fermentation (SSF)]. Enzyme engineering will lead to Glycerol is a by-product of both biodiesel and bioethanol advances in rational improvement of hydrolytic cellulo- production. Both native and engineered organisms have lytic enzyme activity and will help lower cellulase produc- been demonstrated to convert crude glycerol to value- tion costs. Enzyme cocktails have steadily improved over added products such as succinic acid, propanediol, and

polyhydroxyalkanoates (PHAs).51 Acetic acid, a low-value for either expanded feedstock streams or new products to or waste substance present in plant biomass in the form be economically viable. Additional barriers are outlined in of acetylated xylan, is also a product of microbial metabo- Cheng and Timilsina.16 lism. Microbes will be engineered to convert acetic acid Th e fi nal titer of a desired product (e.g., ethanol) is to value-added products rather than allowing the carbon oft en controlled by product inhibition or tolerance. to go to waste. Recently, Saccharomyces cerevisiaewas Much research in a variety of organisms has targeted engineered to consume acetic acid.52 It is unclear whether understanding and mitigating the toxicity of process substantial value will be added to these waste streams inhibitors, including alcohols, hydrocarbons, phenolic within 10 years, but the rapid progress of synthetic biology compounds, and organic acids.54–57 Most eff orts to makes these high-risk, high-reward projects more feasible. increase product tolerance involve adaptation and evolution; learning how to truly engineer tolerance would be a Increased product diversiﬁ cation major advance. High-titer (10% to 20%) soluble products are usually separated via distillation. Direct production of New biomass-derived fuels, chemicals, and other products insoluble hydrocarbons provides a distinct biotechnology- are likely to become more prominent over the next dec- driven advantage for processing because the initial process ade. Cellulose-derived short-chain alcohols such as etha- can be a liquid/liquid phase separation. However, insolu- nol will be the fi rst to be commercially successful; other ble solvents can cause cellular disruption and inhibition in products will follow. Th ere are substantial cost barriers to many microorganisms. Moderate but important advances the commercialization of microbially produced hydrocar- are likely in the realm of tolerance of products and other bons (biogasoline, biojet fuel) and medium-to long-chain inhibitors, which will allow higher titer production. fatty acids (for esterifi cation to biodiesel) as fuels, but an Rational decoupling of growth from metabolic fl ux has intense research eff ort over the next decade would have the potential to increase yield by eliminating the fl ux of a moderate chance of reducing the cost to a competitive substrate to production of new cells while also increasing range. Even if costs remain too high to allow their use as titer by reducing product inhibition.58 Previous technolo- fuels, many of these compounds are potential high-value gies include microbial or biocatalyst retention processes co-products. For example, farnesene can be sold as a high- such as cell recycling or immobilization to maintain a value precursor for cosmetics and other applications, and high rate. Future developments in synthetic biology to Amyris’s business model targets doing so in the near term. control cellular physiology have a moderate chance of Even bio-compounds such as n-butanol and isobutanol enabling the rational decoupling of growth and metabo- are current feedstocks for the chemical industry and may lism. However, economical decoupling with a concomitant begin displacing petroleum-derived compounds. Other increase in yield and titer would result in substantial proc- compounds with high potential for deployment within the ess improvements. next decade include organic acids (e.g. succinic acid, malic Challenges associated with growing and processing algae acid, lactic acid, adipic acid), diols (e.g. propanediols, – including concerns over water use, pond contamination, butanediols), and PHAs, which add to the current biocom- use of GMOs in open ponds, photobioreactor scale-up, modities of acetic acid and 1,3-propanediol. product harvest, and product dewatering – will continue A long-range, high-risk application of synthetic biology to be barriers17 and will be diffi cult to address using would be to combine the metabolic engineering approaches biotechnology only. In this case, issues associated with used to convert sugars to useful products with the engineer- phototrophic growth are combined with the challenge of ing of photosynthetic microbes to provide direct conversion genetic manipulation of non-model bacteria. of sunlight into chemical products, fuels, or electricity.53 Current bioprocesses tend to rely on mesophilic micro- However, this combines the challenges of effi cient bioprod- organisms (20 to 45°C), but the ability to operate under uct formation with the major challenges of algal bioprocess- extreme conditions could simplify process operations ing and is seen as unlikely in the next decade. and lower costs. Extremophiles can tolerate high levels of temperature (up to 100°C), pH, or salts, and thermophilic Improved process technologies and microbes oft en operate at increased rates.59 Th ermophilic efﬁ ciencies processes are speculated to improve separations (i.e., in Th e key factors in a bioconversion process are yield, titer, situ vapor extraction) and lower heating and cooling costs. and rate. Synthetic biology and metabolic engineering are Partial analyses show generally small advantages because the main drivers of increased yield and will be required some heating, cooling or product separations will still be

required. Th e greater advantage of extremophilic processes At the same time, fundamental studies will continue to is indirect – they are likely to resist contamination by unde- provide an intellectual foundation for future work. Th is sired microbes. In addition, feed stream cleanup for these might include discovery of unique enzymatic activities or processes (e.g., for salts or low pH) would be less stringent. pathways that could be harnessed for biotechnology, along However, extensive biotechnological modifi cation will be the lines of the recently discovered enzyme fatty aldehyde required either to use most current extremophiles (likely for decarbonylase61 that produces hydrocarbons from fatty selected extremophiles) or to make current robust microbial acid derivatives. hosts into extremophiles (unlikely soon). Oft en, a microbe newly isolated from the environment Biotechnology can accelerate thermochemical conver- has desirable properties but a lack of genetic tools hinders sion of plant material and municipal waste into syngas its development as an industrial biocatalyst. Typically, followed by syngas bioconversion. Bacterial strain engi- several years of intensive trial-and-error genetic tool neering and development by companies such as LanzaTech development are needed to genetically engineer any novel have demonstrated the potential for bioconversion of microbe. More rational, reliable approaches to develop- syngas to a suite of fuels and chemicals. Although biotech- ing genetic tools and cultivation methods would allow nology is unlikely to signifi cantly overcome all limitations the use of many unique features of novel microbes (such of syngas as a feedstock, such as mass transfer of CO, the as extremophiles or microbes that can consume complex product diversifi cation barrier is ripe for biotechnological substrates or produce novel products). Th is development innovation. is only moderately likely over the next decade but could be In addition to using microbes alone for bioconversion, a game-changer if accomplished. Genetic tools for these an alternate approach is in vitro product formation with non-model organisms are improving.62 enzymes alone. It removes the desired enzyme pathway from the living microorganism and uses a mixture of spe- Products cifi c enzymes. Pathways of more than ten enzymes have been devised and tested.60 Breakthroughs are needed in Potential applications of biotechnology to by-product pro- enzyme stability and cofactors, as well as in lowering the duction are primarily in cost of enzyme production. Improved in vitro enzymatic • altering feedstock characteristics so by-products are processes are likely but will be cost prohibitive for most more suitable for the intended use. commodities. • altering enzymes, bacteria, or yeast used in the fermen- Microbial communities may be assembled to perform tation process to yield higher-value or more consistent desired functions. However, nearly all industrial product co-products. formation uses single isolated microbes or enzymes – pure microbial cultures – as opposed to mixed cultures. Given the scale of biofuel production necessary to make Exceptions are in food production or waste treatment (e.g., an impact on the market, co-products must be useful in biomethane production) and are driven by consumption massive quantities to avoid collapsing their market prices. for growth. A major breakthrough would be the ability to For example, a major by-product of bioenergy produc- design, assemble, and control a mixed culture to produce a tion, particularly biodiesel, is glycerin. Glycerin is valuable specifi c desired product (such as a biofuel). However, many because it is useful in producing hundreds of products; challenges in pure culture apply also to mixed popula- however, because of increased biofuel production, the tions, making this approach unlikely in the next decade. glycerin supply has exceeded demand and the price has col- lapsed.63 Th is example illustrates the problem of targeting specialty chemicals as co-products of biofuel production. Barriers to improving conversion processes At least two by-products, distillers’ grain and lignin, Th e major barriers to biotechnological advancements could supply a large market and thus improve the econom- in conversion of additional feedstock streams and new ics of biofuel production. Th e use of distillers’ grain in products are limited knowledge of microbial metabolism animal feed is a proven contributor to the economics of and diffi culty in controlling metabolic fl ux. Advances in biofuel production from corn. Lignin has potential uses as synthetic biology, particularly, will begin to enable more a precursor in carbon fi ber production or as an additive to dynamic control of metabolic fl ux via coupling sensing plastics to improve the qualities of plastic products.49 of the environment with gene expression, translational US exports of distillers’ grain as an animal feed have and posttranslational control, and allosteric regulation. risen sevenfold since 2005/200612 with the rise in ethanol

production from corn. Distillers’ grain is a good source altered composition of soil symbionts). Biotechnological of animal feed, and using it as such improves the green- improvements in these areas will reduce plants’ dependence house gas benefi ts of biofuel production. It is oft en blended on fertilizers, pesticides, and other agrochemicals. Th is in with other plant materials and supplemented with spe- turn is expected to result in (i) reduced pollution and (ii) cifi c amino acids for optimal nutritional quality. Because reduced production costs; however, these sustainability fac- there is little information on equivalent materials that will tors will need to be measured.67 come from cellulosic biofuel plants, research is needed on If microbes can be engineered to effi ciently convert potential issues with using them, including nutritional cellulosic material into liquid fuels and chemicals from quality, presence of toxic products, and predictability of the unmodifi ed plants, this would suggest that plant biomass content. Feedstock characteristics and the processes used yield per acre would become a primary plant engineer- in preprocessing it will aff ect the utility of the by-products ing target. However, if the former goal remains out of as an animal feed. Genetic manipulation of the feedstock reach, then an essential target for improvement would be could increase the value of the distillers’ grain by increas- to modify plants so that they are more easily converted to ing total protein content and the content of amino acids products. Another niche use to provide additional value that might be lacking (e.g., lysine). Alternatively, the fer- within the supply chain might be the use of biorefi nery mentative microorganisms could be engineered to do the wastewater for bioproduction of fuels and chemicals.68 same, improving the value of the product. However, given Conversion will also impact how fuels are used. Th e the time scale of deploying engineered plants and the cur- choice of compounds produced is highly pertinent. If rent lack of nutritional data, this has a low probability of ethanol remains the dominant product, then the blend completion within the next 10 years. wall will continue to be a barrier and to limit the size of Potential uses of lignin include use as a feedstock for the biofuels market. However, if biogasoline, biodiesel, and structural materials such as carbon fi ber64 and materials biojet drop-in fuels become economical, then the blend for energy-related applications, such as anodes for lithium wall will no longer be an issue. ion batteries.65 Th e current price of carbon fi ber limits its Developing microbes designed for mutually benefi - use to specialty applications, but a price drop would open cial interactions with plants has been identifi ed as a way new markets for it as a structural material.66 One chal- to increase crop yields, decrease nutrient applications, lenge is the variability of the structure and composition of improve resistance to pests and diseases, and improve lignin,65 and no one has demonstrated cost-eff ective pro- plant water use.69 Th ese symbiotic relationships are duction of carbon fi ber from lignin that meets the strength expected to increase grower profi t by stabilizing yields requirements.66 Ongoing research into how genetic vari- across years with varying weather conditions, and by ation in the feedstock aff ects the suitability of the lignin reducing grower costs. Th ey should help address the chal- for use as a carbon fi ber precursor could lay the founda- lenges of greenhouse gas emissions and competition for tion for future plant-engineering strategies for improving land by improving yields with reduced inputs. Th ere are lignin conversion to carbon fi ber. Lignin pathways are other important questions related to the sustainability of being altered in bioenergy crops to increase conversion. biofuel plantations: we do not understand how the biota Th ese same plants will likely also be assessed for the of ecosystems (e.g. pollinators, aphids, birds, small mam- eff ect of lignin composition on the production of useful mals) will be aff ected by the cultivation of plants improved products, including carbon fi ber. using advanced biotechnology, in nonnative or untested geographical niches, and the related land use changes. Th e impacts of improved biofuel crops on the carbon Crosscut impacts on supply chain cycle, biosequestration,70 and the promise of biofuel as a It is essential to study, understand, and improve the envi- carbon-neutral fuel option need to be demonstrated. Th e

ronmental and ecological sustainability of future bioenergy impacts of changes in atmospheric CO2 levels, precipita- crops that may be planted at the scale of millions of hectares tion regimes, and temperatures require that bioenergy of land. Th e ideotypic or most desirable bioenergy plant will plant performance and sustainability be assessed within need to be productive on marginal lands (land having poor the context of climate change models. soil structure, nutrient composition, and moisture) and in Th e ultimate success will come from combining fl uctuating weather conditions and will need to be resilient advances that result from biotechnology approaches with in the face of various abiotic stresses (e.g., water, nutrients, more traditional chemical engineering to develop cost- and temperature) and biotic stresses (e.g., pathogens or eff ective processes.

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Dr Udaya Kalluri Richard L. Stouder Dr Udaya Kalluri is a Staff Scientist in Richard L. Stouder was the Director of the Biosciences Division at the Oak Technology Development and Deploy- Ridge National Laboratory where she ment in the Global Security Directorate leads cellulose biosynthesis research at Oak Ridge National Laboratory within the US DOE BioEnergy Science (ORNL), retiring 31 March 2014. Before Center. Her research areas include joining ORNL in 2001 as a Senior Pro- plant cell wall research, genome gram Manager he served 30 years in the biology, systems biology, transcription factor and US Army, retiring as Colonel in June 2001. Mr Stouder has signaling pathway analyses, imaging, and studies of an undergraduate degree in Political Science and History bioenergy, carbon biosequestration, and plant-microbe (University of Southern Mississippi) and a graduate degree interactions. in Personnel Management (Central Michigan University).

Dr Anthony V. Palumbo Erin Webb Dr Anthony V. Palumbo is the Direc- Erin Webb, PhD, PE, is an agricultural tor of the Biosciences Division of Oak engineer in the Oak Ridge National Ridge National Laboratory. His re- Laboratory Environmental Sciences search is focused on microbial popula- Division where she serves as principal tions, geochemistry, and contaminants investigator of the Feedstock Supply in surface and subsurface environ- Modeling and Biomass Codes and ments. His most recent research has Standards projects. She also holds been on the role of bacterial communities in mercury adjunct faculty appointments at the University of Tennes- transformations in the environment. see and the University of Kentucky. Dr Webb’s current work focuses on simulation and optimization of biomass supply chains and evaluation of building and fire codes for biomass industries.