The potential impact of second‐generation biofuel landscapes on at‐risk species in the US

The recent increase in corn ethanol production has drawn attention to the environmental sustainability of biofuel production. Environmental assessments of second‐generation biofuel crops (SGBC) have focused primarily on greenhouse gas emissions and water quality. However, expanding the production of cellulosic biomass resources, especially those that require dedicated agricultural land, is also likely to have impacts on biodiversity. We developed an optimization framework for projecting the spatial pattern of SGBC expansion in the United States and intersected these predictions with occurrence data for at‐risk species. In particular, we focused on two candidate perennial grass feedstocks, Panicum virgatum (switchgrass), and Miscanthus × giganteus (Miscanthus). Tradeoffs between biodiversity and economic profitability are assessed using county level data sets of SGBC yield, agricultural land availability, land rents, and at‐risk species occurrences. Results show that future SGBC expansion is likely to occur outside of the Corn Belt, where conventional biofuel feedstocks are currently grown. The set of at‐risk species that could potentially be impacted is therefore likely to be different from the at‐risk species prevalent in the agroecological landscapes of the Upper Midwest that are dominated by corn and soy production. The total number and type of potentially impacted taxa is influenced by several factors, including the total demand for cellulosic biomass, the type of agricultural land used for production, and the method for defining at‐risk species. SGBC production is also concentrated in fewer counties when a national species conservation constraint is combined with a biofuel production mandate. This analysis provides a foundation for future research on species conservation in bioenergy production landscapes and highlights the importance of incorporating biodiversity into broader environmental assessments of biofuel sustainability.     

Engineering microbial biofuel tolerance and export using efflux pumps

Many compounds being considered as candidates for advanced biofuels are toxic to microorganisms. This introduces an undesirable trade‐off when engineering metabolic pathways for biofuel production because the engineered microbes must balance production against survival. Cellular export systems, such as efflux pumps, provide a direct mechanism for reducing biofuel toxicity. To identify novel biofuel pumps, we used bioinformatics to generate a list of all efflux pumps from sequenced bacterial genomes and prioritized a subset of targets for cloning. The resulting library of 43 pumps was heterologously expressed in Escherichia coli, where we tested it against seven representative biofuels. By using a competitive growth assay, we efficiently distinguished pumps that improved survival. For two of the fuels (n‐butanol and isopentanol), none of the pumps improved tolerance. For all other fuels, we identified pumps that restored growth in the presence of biofuel. We then tested a beneficial pump directly in a production strain and demonstrated that it improved biofuel yields. Our findings introduce new tools for engineering production strains and utilize the increasingly large database of sequenced genomes.     

An integrative modeling framework to evaluate the productivity and sustainability of biofuel crop production systems

The potential expansion of biofuel production raises food, energy, and environmental challenges that require careful assessment of the impact of biofuel production on greenhouse gas (GHG) emissions, soil erosion, nutrient loading, and water quality. In this study, we describe a spatially explicit integrative modeling framework (SEIMF) to understand and quantify the environmental impacts of different biomass cropping systems. This SEIMF consists of three major components: (1) a geographic information system (GIS)‐based data analysis system to define spatial modeling units with resolution of 56 m to address spatial variability, (2) the biophysical and biogeochemical model Environmental Policy Integrated Climate (EPIC) applied in a spatially‐explicit way to predict biomass yield, GHG emissions, and other environmental impacts of different biofuel crops production systems, and (3) an evolutionary multiobjective optimization algorithm for exploring the trade‐offs between biofuel energy production and unintended ecosystem‐service responses. Simple examples illustrate the major functions of the SEIMF when applied to a nine‐county Regional Intensive Modeling Area (RIMA) in SW Michigan to (1) simulate biofuel crop production, (2) compare impacts of management practices and local ecosystem settings, and (3) optimize the spatial configuration of different biofuel production systems by balancing energy production and other ecosystem‐service variables. Potential applications of the SEIMF to support life cycle analysis and provide information on biodiversity evaluation and marginal‐land identification are also discussed. The SEIMF developed in this study is expected to provide a useful tool for scientists and decision makers to understand sustainability issues associated with the production of biofuels at local, regional, and national scales.     

Using species distribution models to identify suitable areas for biofuel feedstock production

The 2007 Energy Independence and Security Act mandates a five‐fold increase in US biofuel production by 2022. Given this ambitious policy target, there is a need for spatially explicit estimates of landscape suitability for growing biofuel feedstocks. We developed a suitability modeling approach for two major US biofuel crops, corn (Zea mays) and switchgrass (Panicum virgatum), based upon the use of two presence‐only species distribution models (SDMs): maximum entropy (Maxent) and support vector machines (SVM). SDMs are commonly used for modeling animal and plant distributions in natural environments, but have rarely been used to develop landscape models for cultivated crops. AUC, Kappa, and correlation measures derived from test data indicate that SVM slightly outperformed Maxent in modeling US corn production, although both models produced significantly accurate results. When compared with results from a mechanistic switchgrass model recently developed by Oak Ridge National Laboratory (ORNL), SVM results showed higher correlation than Maxent results with models fit using county‐scale point inputs of switchgrass production derived from expert opinion estimates. However, Maxent results for an alternative switchgrass model developed with point inputs from research trial sites showed higher correlation to the ORNL model than the corresponding results obtained from SVM. Further analysis indicates that both modeling approaches were effective in predicting county‐scale increases in corn production from 2006 to 2007, a time period in which US corn production increased by 24%. We conclude that presence‐only methods are a powerful first‐cut tool for estimating relative land suitability across geographic regions in which candidate biofuel feedstocks can be grown, and may also provide important insight into potential land‐use change patterns likely to be associated with increased biofuel demand.     

Toward cell‐free biofuel production: Stable immobilization of oligomeric enzymes

To overcome the main challenges facing alcohol‐based biofuel production, we propose an alternate simplified biofuel production scheme based on a cell‐free immobilized enzyme system. In this paper, we measured the activity of two tetrameric enzymes, a control enzyme with a colorimetric assay, β‐galactosidase, and an alcohol‐producing enzyme, alcohol dehydrogenase, immobilized on multiple surface curvatures and chemistries. Several solid supports including silica nanoparticles (convex), mesopourous silica (concave), diatomaceous earth (concave), and methacrylate (concave) were examined. High conversion rates and low protein leaching was achieved by covalent immobilization of both enzymes on methacrylate resin. Alcohol dehydrogenase (ADH) exhibited long‐term stability and over 80% conversion of aldehyde to alcohol over 16 days of batch cycles. The complete reaction scheme for the conversion of acid to aldehyde to alcohol was demonstrated in vitro by immobilizing ADH with keto‐acid decarboxylase free in solution. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:324–331, 2014     

How much heat can we grow in our cities? Modelling UK urban biofuel production potential

Biofuel provides a globally significant opportunity to reduce fossil fuel dependence; however, its sustainability can only be meaningfully explored for individual cases. It depends on multiple considerations including: life cycle greenhouse gas emissions, air quality impacts, food versus fuel trade‐offs, biodiversity impacts of land use change and socio‐economic impacts of energy transitions. One solution that may address many of these issues is local production of biofuel on non‐agricultural land. Urban areas drive global change, for example, they are responsible for 70% of global energy use, but are largely ignored in their resource production potential; however, underused urban greenspaces could be utilized for biofuel production near the point of consumption. This could avoid food versus fuel land conflicts in agricultural land and long‐distance transport costs, provide ecosystem service benefits to urban dwellers and increase the sustainability and resilience of cities and towns. Here, we use a Geographic Information System to identify urban greenspaces suitable for biofuel production, using exclusion criteria, in 10 UK cities. We then model production potential of three different biofuels: Miscanthus grass, short rotation coppice (SRC) willow and SRC poplar, within the greenspaces identified and extrapolate up to a UK‐scale. We demonstrate that approximately 10% of urban greenspace (3% of built‐up land) is potentially suitable for biofuel production. We estimate the potential of this to meet energy demand through heat generation, electricity and combined heat and power (CHP) operations. Our findings show that, if fully utilized, urban biofuel production could meet nearly a fifth of demand for biomass in CHP systems in the United Kingdom's climate compatible energy scenarios by 2030, with potentially similar implications for other comparable countries and regions.     

Prospects for Cellulosic Biofuel Production in the Northeastern United States: A Scenario Analysis

Secure access to energy and food are two of the challenges facing the Northeast region of the United States. Traditional biofuel feedstocks, such as corn and oil seed, are able to satisfy energy requirements. However, they compete with food production for desirable land and water resources and, in any case, are not likely to exploit the region's current comparative advantages. This study investigates a potential solution to the energy security problem in the Northeast: biofuel from advanced feedstock in the form of net forest growth and woody wastes, of which the region has abundant endowments. The federal government has committed to requiring 79.5 billion liters (BL) of advanced biofuel production annually by 2022. We evaluate both the physical capacity for its production and its cost competitiveness using an input‐output model of consumption, production, and trade in the 13‐state region. The model minimizes resource use required to satisfy given consumer demand using alternative technological options and subject to resource constraints. We compile data from the technical literature quantifying state‐level biofuel feedstock endowments and the technological requirements for cellulosic ethanol production. We find that exploiting the region's endowment of cellulosic feedstock requires either making the price of biofuels competitive with gasoline through subsidies or restricting imports of gasoline. Based on this initial investigation, we conclude that the region can produce significant amounts of advanced biofuel, up to 20.28 BL of cellulosic ethanol per year, which could displace nearly 12.5% of the gasoline that is now devoted to motorized transport in the region.     

Plant biotechnology for lignocellulosic biofuel production

Lignocelluloses from plant cell walls are attractive resources for sustainable biofuel production. However, conversion of lignocellulose to biofuel is more expensive than other current technologies, due to the costs of chemical pretreatment and enzyme hydrolysis for cell wall deconstruction. Recalcitrance of cell walls to deconstruction has been reduced in many plant species by modifying plant cell walls through biotechnology. These results have been achieved by reducing lignin content and altering its composition and structure. Reduction of recalcitrance has also been achieved by manipulating hemicellulose biosynthesis and by overexpression of bacterial enzymes in plants to disrupt linkages in the lignin–carbohydrate complexes. These modified plants often have improved saccharification yield and higher ethanol production. Cell wall‐degrading (CWD) enzymes from bacteria and fungi have been expressed at high levels in plants to increase the efficiency of saccharification compared with exogenous addition of cellulolytic enzymes. In planta expression of heat‐stable CWD enzymes from bacterial thermophiles has made autohydrolysis possible. Transgenic plants can be engineered to reduce recalcitrance without any yield penalty, indicating that successful cell wall modification can be achieved without impacting cell wall integrity or plant development. A more complete understanding of cell wall formation and structure should greatly improve lignocellulosic feedstocks and reduce the cost of biofuel production.     

Industrial‐strength ecology: trade‐offs and opportunities in algal biofuel production

Microalgae represent one of the most promising groups of candidate organisms for replacing fossil fuels with contemporary primary production as a renewable source of energy. Algae can produce many times more biomass per unit area than terrestrial crop plants, easing the competing demands for land with food crops and native ecosystems. However, several aspects of algal biology present unique challenges to the industrial‐scale aquaculture of photosynthetic microorganisms. These include high susceptibility to invading aquatic consumers and weeds, as well as prodigious requirements for nutrients that may compete with the fertiliser demands of other crops. Most research on algal biofuel technologies approaches these problems from a cellular or genetic perspective, attempting either to engineer or select algal strains with particular traits. However, inherent functional trade‐offs may limit the capacity of genetic selection or synthetic biology to simultaneously optimise multiple functional traits for biofuel productivity and resilience. We argue that a community engineering approach that manages microalgal diversity, species composition and environmental conditions may lead to more robust and productive biofuel ecosystems. We review evidence for trade‐offs, challenges and opportunities in algal biofuel cultivation with a goal of guiding research towards intensifying bioenergy production using established principles of community and ecosystem ecology.     

Cellulase production from palm oil mill effluent in Malaysia: Economical and technical perspectives

The demand for cellulases has increased tremendously over the last few decades. This is due to its numerous applications in industry and also because it can be used to hydrolyze cellulosic materials into sugars that can be fermented into bioethanol and bio‐based products. This does not only open up a big and significant market for cellulases, but also provides another source of biofuel and bioenergy in the future. Nevertheless, the cost of the existing substrates for cellulase fermentation is very high if required for large‐scale production. Sustainable supplies and an economically feasible biomass are needed to reduce the cost of cellulase production. Palm oil mill effluent (POME) is rich in carbohydrates, proteins, nitrogenous compounds, lipids, minerals, cellulose, hemicelluloses and lignin. It can be used naturally as a fermentation medium, either for cellulase or other value‐added product fermentation. In Malaysia, a large and continually increasing amount of POME is produced every year because of the high global demand for palm oil. Hence, the development of cellulase production from POME is reviewed, covering the POME production, cellulase production and the major challenges together with the future prospects of these processes.     

Simulating the effects of harvest and biofuel production on the forest system carbon balance of the Midwest,USA

Forests of the Midwestern United States are an important source of fiber for the wood and paper products industries. Scientists, land managers, and policy makers are interested in using woody biomass and/or harvest residue for biofuel feedstocks. However, the effects of increased biomass removal for biofuel production on forest production and forest system carbon balance remain uncertain. We modeled the carbon (C) cycle of the forest system by dividing it into two distinct components: (1) biological (net ecosystem production, net primary production, autotrophic and heterotrophic respiration, vegetation, and soil C content) and (2) industrial (harvest operations and transportation, production, use, and disposal of major wood products including biofuel and associated C emissions). We modeled available woody biomass feedstock and whole‐system carbon balance of 220 000 km² of temperate forests in the Upper Midwest, USA by coupling an ecosystem process model to a collection of greenhouse gas life‐cycle inventory models and simulating seven forest harvest scenarios in the biological ecosystem and three biofuel production scenarios in the industrial system for 50 years. The forest system was a carbon sink (118 g C m^?2 yr^?1) under current management practices and forest product production rates. However, the system became a C source when harvest area was doubled and biofuel production replaced traditional forest products. Total carbon stores in the vegetation and soil increased by 5–10% under low‐intensity management scenarios and current management, but decreased up to 3% under high‐intensity harvest regimes. Increasing harvest residue removal during harvest had more modest effects on forest system C balance and total biomass removal than increasing the rate of clear‐cut harvests or area harvested. Net forest system C balance was significantly, and negatively correlated (R² = 0.67) with biomass harvested, illustrating the trade‐offs between increased C uptake by forests and utilization of woody biomass for biofuel feedstock.     

Algal swimming velocities signal fatty acid accumulation

The use of microalgae for biofuel production will be beneficial to society if we can produce biofuels at large scales with minimal mechanical energy input in the production process. Understanding micro‐algal physiological responses under variable environmental conditions in bioreactors is essential for the optimization of biofuel production. We demonstrate that measuring micro‐algal swimming speed provides information on culture health and total fatty acid accumulation. Three strains of Chlamydomonas reinhardtii were grown heterotrophically on acetate and subjected to various levels of nitrogen starvation. Other nutrient levels were explored to determine their effect on micro‐algal kinetics. Swimming velocities were measured with two‐dimensional micro‐particle tracking velocimetry. The results show an inverse linear relationship between normalized total fatty acid mass versus swimming speed of micro‐algal cells. Analysis of RNA sequencing data confirms these results by demonstrating that the biological processes of cell motion and the generation of energy precursors are significantly down‐regulated. Experiments demonstrate that changes in nutrient concentration in the surrounding media also affect swimming speed. The findings have the potential for the in situ and indirect assessment of lipid content by measuring micro‐algal swimming kinetics. Biotechnol. Bioeng. 2013; 110: 143–152. © 2012 Wiley Periodicals, Inc.     

Transgenic switchgrass (Panicum virgatum L.) targeted for reduced recalcitrance to bioconversion: a 2‐year comparative analysis of field‐grown lines modified for target gene or genetic element expression

Transgenic Panicum virgatum L. silencing (KD) or overexpressing (OE) specific genes or a small RNA (GAUT4‐KD, miRNA156‐OE, MYB4‐OE, COMT‐KD and FPGS‐KD) was grown in the field and aerial tissue analysed for biofuel production traits. Clones representing independent transgenic lines were established and senesced tissue was sampled after year 1 and 2 growth cycles. Biomass was analysed for wall sugars, recalcitrance to enzymatic digestibility and biofuel production using separate hydrolysis and fermentation. No correlation was found between plant carbohydrate content and biofuel production pointing to overriding structural and compositional elements that influence recalcitrance. Biomass yields were greater for all lines in the second year as plants establish in the field and standard amounts of biomass analysed from each line had more glucan, xylan and less ethanol (g/g basis) in the second‐ versus the first‐year samples, pointing to a broad increase in tissue recalcitrance after regrowth from the perennial root. However, biomass from second‐year growth of transgenics targeted for wall modification, GAUT4‐KD, MYB4‐OE, COMT‐KD and FPGS‐KD, had increased carbohydrate and ethanol yields (up to 12% and 21%, respectively) compared with control samples. The parental plant lines were found to have a significant impact on recalcitrance which can be exploited in future strategies. This summarizes progress towards generating next‐generation bio‐feedstocks with improved properties for microbial and enzymatic deconstruction, while providing a comprehensive quantitative analysis for the bioconversion of multiple plant lines in five transgenic strategies.     

Macroalgae in biofuel production

The conversion processes of macroalgae for biofuels can be divided into thermochemical (dry) and microbiological (wet) processes. The chemical composition of macroalgae together with the pre‐treatment method, conversion conditions, and the characteristics of the microbes involved (wet processes) determine the yield and the properties of the biofuel produced. Macroalgae are often rich in carbohydrates, and therefore well suited for biogas, biobutanol and bioethanol productions. The content of triacylglycerols (TAGs) is the best indicator for the suitability of the alga for biodiesel production. TAGs have a high conversion rate to biodiesel, high percentage of fatty acids, and they lack phosphorus, sulfur and nitrogen. Macroalgae can have high metal concentrations, which can have an impact on conversion processes: metals may inhibit or catalyse the processes. High sulfur (especially in green algae) and nitrogen contents are also characteristic to macroalgae, and may be problematic in the production of biogas (NH₃‐toxicity) and the use of the oil and biodiesel (high concentrations of H₂S and NO_x‐compounds). Macroalgae have proven to be suitable material for conversion processes, but further optimization of the processes is needed. At present, macroalgae are not economically, or in many cases not even environmentally, sustainable material when the whole production chain is considered. In this review we summarize information on the chemical composition of macroalgae in a prospect of biofuel production, and the current situation in the field of macroalgal‐based biofuel production.     

Influence of biofuel crops on mosquito production and oviposition site selection

The proposed expansion of biofuels production may cause unintended land‐use changes and potentially alter ecosystem services. This study evaluated the impact of first‐generation (corn) and second‐generation (switchgrass and Miscanthus) biofuel crops on production and oviposition site selection by two vector mosquitoes, the yellow fever mosquito Aedes aegypti and the Asian tiger mosquito Aedes albopictus. Larvae of the two species were reared at varying conspecific and heterospecific densities in senescent leaf infusions prepared from one of the three biofuel crops and their survival and development time to adulthood determined. The effects of the three leaf infusions on water chemistry and oviposition site selection by the two mosquito species were also determined. Ae. albopictus females deposited significantly fewer eggs in Miscanthus than in corn infusion while Ae. aegypti females deposited significantly fewer eggs in Miscanthus than in both corn and switchgrass infusion. Survival to adulthood for both mosquito species was significantly lower in corn than in switchgrass and Miscanthus infusions; was consistently lower at high‐ (0:40 and 20:20) than at low density treatments in both switchgrass and Miscanthus infusions; and significantly lower at high intraspecific density (40:0 and 0: 40) than at high interspecific density (20:20) in Miscanthus infusion. Development time to adulthood was positively related to larval density, but was not influenced by biofuel leaf treatment. Corn infusion had lower pH values and higher salinity, conductivity, total dissolved solids (TDS), and temperature values than switchgrass and Miscanthus infusions. These findings demonstrate the potential for biofuel crops to modify the chemistry of aquatic habitats in ways that may influence mosquito production and thereby the risk of exposure to mosquito‐borne diseases.     

Synechocystis sp. PCC6803 metabolic models for the enhanced production of hydrogen

In the present economy, difficulties to access energy sources are real drawbacks to maintain our current lifestyle. In fact, increasing interests have been gathered around efficient strategies to use energy sources that do not generate high CO₂ titers. Thus, science-funding agencies have invested more resources into research on hydrogen among other biofuels as interesting energy vectors. This article reviews present energy challenges and frames it into the present fuel usage landscape. Different strategies for hydrogen production are explained and evaluated. Focus is on biological hydrogen production; fermentation and photon-fuelled hydrogen production are compared. Mathematical models in biology can be used to assess, explore and design production strategies for industrially relevant metabolites, such as biofuels. We assess the diverse construction and uses of genome-scale metabolic models of cyanobacterium Synechocystis sp. PCC6803 to efficiently obtain biofuels. This organism has been studied as a potential photon-fuelled production platform for its ability to grow from carbon dioxide, water and photons, on simple culture media. Finally, we review studies that propose production strategies to weigh this organism’s viability as a biofuel production platform. Overall, the work presented in this review unveils the industrial capabilities of cyanobacterium Synechocystis sp. PCC6803 to evolve interesting metabolites as a clean biofuel production platform.     

Modification of native grasses for biofuel production may increase virus susceptibility

Bioenergy production is driving modifications to native plant species for use as novel biofuel crops. Key aims are to increase crop growth rates and to enhance conversion efficiency by reducing biomass recalcitrance to digestion. However, selection for these biofuel‐valuable traits has potential to compromise plant defenses and alter interactions with pests and pathogens. Insect‐vectored plant viruses are of particular concern because perennial crops have potential to serve as virus reservoirs that influence regional disease dynamics. In this study, we examined relationships between growth rates and biomass recalcitrance in five switchgrass (Panicum virgatum) populations, ranging from near‐wildtype to highly selected cultivars, in a common garden trial. We measured biomass accumulation rates and assayed foliage for acid detergent lignin, neutral detergent fiber, in vitro neutral detergent fiber digestibility and in vitro true dry matter digestibility. We then evaluated relationships between these traits and susceptibility to a widely distributed group of aphid‐transmitted Poaceae viruses (Luteoviridae: Barley and cereal yellow dwarf viruses, B/CYDVs). Virus infection rates and prevalence were assayed with RT‐PCR in the common garden, in greenhouse inoculation trials, and in previously established switchgrass stands across a 300‐km transect in Michigan, USA. Aphid host preferences were quantified in a series of arena host choice tests with field‐grown foliage. Contrary to expectations, biomass accumulation rates and foliar digestibility were not strongly linked in switchgrass populations we examined, and largely represented two different trait axes. Natural B/CYDV prevalence in established switchgrass stands ranged from 0% to 28%. In experiments, susceptibility varied notably among switchgrass populations and was more strongly predicted by potential biomass accumulation rates than by foliar digestibility; highly selected, productive cultivars were most virus‐susceptible and most preferred by aphids. Evaluation and mitigation of virus susceptibility of new biofuel crops is recommended to avert possible unintended consequences of biofuel production on regional pathogen dynamics.     

Elevated acetyl‐CoA by amino acid recycling fuels microalgal neutral lipid accumulation in exponential growth phase for biofuel production

Microalgal neutral lipids [mainly in the form of triacylglycerols (TAGs)], feasible substrates for biofuel, are typically accumulated during the stationary growth phase. To make microalgal biofuels economically competitive with fossil fuels, generating strains that trigger TAG accumulation from the exponential growth phase is a promising biological approach. The regulatory mechanisms to trigger TAG accumulation from the exponential growth phase (TAEP) are important to be uncovered for advancing economic feasibility. Through the inhibition of pyruvate dehydrogenase kinase by sodium dichloroacetate, acetyl‐CoA level increased, resulting in TAEP in microalga Dunaliella tertiolecta. We further reported refilling of acetyl‐CoA pool through branched‐chain amino acid catabolism contributed to an overall sixfold TAEP with marginal compromise (4%) on growth in a TAG‐rich D. tertiolecta mutant from targeted screening. Herein, a three‐step α loop‐integrated metabolic model is introduced to shed lights on the neutral lipid regulatory mechanism. This article provides novel approaches to compress lipid production phase and heightens lipid productivity and photosynthetic carbon capture via enhancing acetyl‐CoA level, which would optimize renewable microalgal biofuel to fulfil the demanding fuel market.     

Advanced biofuel production in microbes

The cost-effective production of biofuels from renewable materials will begin to address energy security and climate change concerns. Ethanol, naturally produced by microorganisms, is currently the major biofuel in the transportation sector. However, its low energy content and incompatibility with existing fuel distribution and storage infrastructure limits its economic use in the future. Advanced biofuels, such as long chain alcohols and isoprenoid- and fatty acid-based biofuels, have physical properties that more closely resemble petroleum-derived fuels, and as such are an attractive alternative for the future supplementation or replacement of petroleum-derived fuels. Here, we review recent developments in the engineering of metabolic pathways for the production of known and potential advanced biofuels by microorganisms. We concentrate on the metabolic engineering of genetically tractable organisms such as Escherichia coli and Saccharomyces cerevisiae for the production of these advanced biofuels.     

Metabolite labelling reveals hierarchies in Clostridium acetobutylicum that selectively channel carbons from sugar mixtures towards biofuel precursors

Clostridial fermentation of cellulose and hemicellulose relies on the cellular physiology controlling the metabolism of the cellulosic hexose sugar (glucose) with respect to the hemicellulosic pentose sugars (xylose and arabinose) and the hemicellulosic hexose sugars (galactose and mannose). Here, liquid chromatography–mass spectrometry and stable isotope tracers in Clostridium acetobutylicum were applied to investigate the metabolic hierarchy of glucose relative to the different hemicellulosic sugars towards two important biofuel precursors, acetyl‐coenzyme A and butyryl‐coenzyme A. The findings revealed constitutive metabolic hierarchies in C. acetobutylicum that facilitate (i) selective investment of hemicellulosic pentoses towards ribonucleotide biosynthesis without substantial investment into biofuel production and (ii) selective contribution of hemicellulosic hexoses through the glycolytic pathway towards biofuel precursors. Long‐term isotopic enrichment demonstrated incorporation of both pentose sugars into pentose‐phosphates and ribonucleotides in the presence of glucose. Kinetic labelling data, however, showed that xylose was not routed towards the biofuel precursors but there was minor contribution from arabinose. Glucose hierarchy over the hemicellulosic hexoses was substrate‐dependent. Kinetic labelling of hexose‐phosphates and triose‐phosphates indicated that mannose was assimilated but not galactose. Labelling of both biofuel precursors confirmed this metabolic preference. These results highlight important metabolic considerations in the accounting of clostridial mixed‐sugar utilization.