A model for improving microbial biofuel production using a synthetic feedback loop

Cells use feedback to implement a diverse range of regulatory functions. Building synthetic feedback control systems may yield insight into the roles that feedback can play in regulation since it can be introduced independently of native regulation, and alternative control architectures can be compared. We propose a model for microbial biofuel production where a synthetic control system is used to increase cell viability and biofuel yields. Although microbes can be engineered to produce biofuels, the fuels are often toxic to cell growth, creating a negative feedback loop that limits biofuel production. These toxic effects may be mitigated by expressing efflux pumps that export biofuel from the cell. We developed a model for cell growth and biofuel production and used it to compare several genetic control strategies for their ability to improve biofuel yields. We show that controlling efflux pump expression directly with a biofuel-responsive promoter is a straightforward way of improving biofuel production. In addition, a feed forward loop controller is shown to be versatile at dealing with uncertainty in biofuel production rates.     

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.     

Metabolic engineering strategies toward production of biofuels

Exacerbation of climate change and air pollution around the world have emphasized the necessity of replacing fossil fuels with clean and sustainable energy. Metabolic engineering has provided strategies to engineer diverse organisms for the production of biofuels from renewable carbon sources. Although some of the processes are commercialized, there has been continued effort to produce advanced biofuels with higher efficiencies. In this article, metabolic engineering strategies recently exploited to enhance biofuel production and facilitate utilization of non-edible low-value carbon sources are reviewed. The strategies include engineering enzymes, exploiting new pathways, and systematically optimizing metabolism and fermentation processes, among others. In addition, metabolic and bioprocess engineering strategies to achieve competitiveness of current biofuel production systems compared with fossil fuels are discussed.     

Engineering biofuel tolerance in non-native producing microorganisms

Large-scale production of renewable biofuels through microbiological processes has drawn significant attention in recent years, mostly due to the increasing concerns on the petroleum fuel shortages and the environmental consequences of the over-utilization of petroleum-based fuels. In addition to native biofuel-producing microbes that have been employed for biofuel production for decades, recent advances in metabolic engineering and synthetic biology have made it possible to produce biofuels in several non-native biofuel-producing microorganisms. Compared to native producers, these non-native systems carry the advantages of fast growth, simple nutrient requirements, readiness for genetic modifications, and even the capability to assimilate CO₂ and solar energy, making them competitive alternative systems to further decrease the biofuel production cost. However, the tolerance of these non-native microorganisms to toxic biofuels is naturally low, which has restricted the potentials of their application for high-efficiency biofuel production. To address the issues, researches have been recently conducted to explore the biofuel tolerance mechanisms and to construct robust high-tolerance strains for non-native biofuel-producing microorganisms. In this review, we critically summarize the recent progress in this area, focusing on three popular non-native biofuel-producing systems, i.e. Escherichia coli, Lactobacillus and photosynthetic cyanobacteria.     

Triple bottom line study of a lignocellulosic biofuel industry

Growing concerns about energy security and climate change have prompted interest in Australia and worldwide to look for alternatives of fossil fuels. Among the renewable fuel sources, biofuels are one such alternative that have received unprecedented attention in the past decade. Cellulosic biofuels, derived from agricultural and wood biomass, could potentially increase Australia's oil self‐sufficiency. In this study, we carry out a hybrid life‐cycle assessment (LCA) of a future cellulose‐refining industry located in the Green Triangle region of South Australia. We assess both the upstream and downstream refining stages, and consider as well the life‐cycle effects occurring in conventional industries displaced by the proposed biofuel supply chains. We improve on conventional LCA method by utilising multi‐region input–output (IO) analysis that allows a comprehensive appraisal of the industry's supply chains. Using IO‐based hybrid LCA, we evaluate the social, economic and environmental impacts of lignocellulosic biofuel production. In particular, we evaluate the employment, economic stimulus, energy consumption and greenhouse gas impacts of the biofuel supply chain and also quantify the loss in economic activity and employment in the paper, pulp and paperboard industry resulting from the diversion of forestry biomass to biofuel production. Our results reveal that the loss in economic activity and employment will only account for 10% of the new jobs and additional stimulus generated in the economy. Lignocellulosic biofuel production will create significant new jobs and enhance productivity and economic growth by initiating the growth of new industries in the economy. The energy return on investment for cellulosic biofuel production lies between 2.7 and 5.2, depending on the type of forestry feedstock and the travel distance between the feedstock industry and the cellulose refinery. Furthermore, the biofuel industry will be a net carbon sequester.     

Algal biofuels

The world is facing energy crisis and environmental issues due to the depletion of fossil fuels and increasing CO₂ concentration in the atmosphere. Growing microalgae can contribute to practical solutions for these global problems because they can harvest solar energy and capture CO₂ by converting it into biofuel using photosynthesis. Microalgae are robust organisms capable of rapid growth under a variety of conditions including in open ponds or closed photobioreactors. Their reduced biomass compounds can be used as the feedstock for mass production of a variety of biofuels. As another advantage, their ability to accumulate or secrete biofuels can be controlled by changing their growth conditions or metabolic engineering. This review is aimed to highlight different forms of biofuels produced by microalgae and the approaches taken to improve their biofuel productivity. The costs for industrial-scale production of algal biofuels in open ponds or closed photobioreactors are analyzed. Different strategies for photoproduction of hydrogen by the hydrogenase enzyme of green algae are discussed. Algae are also good sources of biodiesel since some species can make large quantities of lipids as their biomass. The lipid contents for some of the best oil-producing strains of algae in optimized growth conditions are reviewed. The potential of microalgae for producing petroleum related chemicals or ready-make fuels such as bioethanol, triterpenic hydrocarbons, isobutyraldehyde, isobutanol, and isoprene from their biomass are also presented.     

Selection and optimization of microbial hosts for biofuels production

Currently, the predominant microbially produced biofuel is starch- or sugar-derived ethanol. However, ethanol is not an ideal fuel molecule, and lignocellulosic feedstocks are considerably more abundant than both starch and sugar. Thus, many improvements in both the feedstock and the fuel have been proposed. In this paper, we examine the prospects for bioproduction of four second-generation biofuels (n-butanol, 2-butanol, terpenoids, or higher lipids) from four feedstocks (sugars and starches, lignocellulosics, syngas, and atmospheric carbon dioxide). The principal obstacle to commercial production of these fuels is that microbial catalysts of robust yields, productivities, and titers have yet to be developed. Suitable microbial hosts for biofuel production must tolerate process stresses such as end-product toxicity and tolerance to fermentation inhibitors in order to achieve high yields and titers. We tested seven fast-growing host organisms for tolerance to production stresses, and discuss several metabolic engineering strategies for the improvement of biofuels production.     

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.     

Sources of variability in greenhouse gas and energy balances for biofuel production: a systematic review

Across the energy sector, alternatives to fossil fuels are being developed, in response to the dual drivers of climate change and energy security. For transport, biofuels have the greatest potential to replace fossil fuels in the short‐to medium term. However, the ecological benefits of biofuels and the role that their deployment can play in mitigating climate change are being called into question. Life Cycle Assessment (LCA) is a widely used approach that enables the energy and greenhouse gas (GHG) balance of biofuel production to be calculated. Concerns have nevertheless been raised that published data show widely varying and sometimes contradictory results. This review describes a systematic review of GHG emissions and energy balance data from 44 LCA studies of first‐ and second‐generation biofuels. The information collated was used to identify the dominant sources of GHG emissions and energy requirements in biofuel production and the key sources of variability in published LCA data. Our analysis revealed three distinct sources of variation: (1) ‘real’ variability in parameters e.g. cultivation; (2) ‘methodological’ variability due to the implementation of the LCA method; and (3) ‘uncertainty’ due to parameters rarely included and poorly quantified. There is global interest in developing a sustainability assessment protocol for biofuels. Confidence in the results of such an assessment can only be assured if these areas of uncertainty and variability are addressed. A more defined methodology is necessary in order to allow effective and accurate comparison of results. It is also essential that areas of uncertainty such as impacts on soil carbon stocks and fluxes are included in LCA assessments, and that further research is conducted to enable a robust calculation of impacts under different land‐use change scenarios. Without the inclusion of these parameters, we cannot be certain that biofuels are really delivering GHG savings compared with fossil fuels.     

Improving olefin tolerance and production in E. coli using native and evolved AcrB

Microorganisms can be engineered for the production of chemicals utilized in the polymer industry. However many such target compounds inhibit microbial growth and might correspondingly limit production levels. Here, we focus on compounds that are precursors to bioplastics, specifically styrene and representative alpha‐olefins; 1‐hexene, 1‐octene, and 1‐nonene. We evaluated the role of the Escherichia coli efflux pump, AcrAB‐TolC, in enhancing tolerance towards these olefin compounds. AcrAB‐TolC is involved in the tolerance towards all four compounds in E. coli. Both styrene and 1‐hexene are highly toxic to E. coli. Styrene is a model plastics precursor with an established route for production in E. coli (McKenna and Nielsen, 2011). Though our data indicates that AcrAB‐TolC is important for its optimal production, we observed a strong negative selection against the production of styrene in E. coli. Thus we used 1‐hexene as a model compound to implement a directed evolution strategy to further improve the tolerance phenotype towards this alpha‐olefin. We focused on optimization of AcrB, the inner membrane domain known to be responsible for substrate binding, and found several mutations (A279T, Q584R, F617L, L822P, F927S, and F1033Y) that resulted in improved tolerance. Several of these mutations could also be combined in a synergistic manner. Our study shows efflux pumps to be an important mechanism in host engineering for olefins, and one that can be further improved using strategies such as directed evolution, to increase tolerance and potentially production. Biotechnol. Bioeng. 2015;112: 879–888. © 2015 Wiley Periodicals, Inc.     

Metabolic engineering for advanced biofuels production from Escherichia coli

Global energy and environmental problems have stimulated increasing efforts toward synthesizing liquid biofuels as transportation energy. Compared to the traditional biofuel, ethanol, advanced biofuels should offer advantages such as higher energy density, lower hygroscopicity, lower vapor pressure, and compatibility with existing transportation infrastructure. However, these fuels are not synthesized economically using native organisms. Metabolic engineering offers an alternative approach in which synthetic pathways are engineered into user-friendly hosts for the production of these fuel molecules. These hosts could be readily manipulated to improve the production efficiency. This review summarizes recent progress in the engineering of Escherichia coli to produce advanced biofuels.     

Coupling Input‐Output Tables with Macro‐Life Cycle Assessment to Assess Worldwide Impacts of Biofuels Transport Policies

Many countries see biofuels as a replacement to fossil fuels to mitigate climate change. Nevertheless, some concerns remain about the overall benefits of biofuels policies. More comprehensive tools seem required to evaluate indirect effects of biofuel policies. This article proposes a method to evaluate large‐scale biofuel policies that is based on life cycle assessment (LCA), environmental extensions of input‐output (I‐O) tables, and a general equilibrium model. The method enables the assessment of indirect environmental effects of biofuels policies, including land‐use changes (LUCs), in the context of economic and demographic growth. The method is illustrated with a case study involving two scenarios. The first one describes the evolution of the world economy from 2006 to 2020 under business as usual (BAU) conditions (including demographic and dietary preferences changes), and the second integrates biofuel policies in the United States and the European Union (EU). Results show that the biofuel scenario, originally designed to mitigate climate change, results in more greenhouse gas emissions when compared to the BAU scenario. This is mainly due to emissions associated with global LUCs. The case study shows that the method enables a broader consideration for environmental effects of biofuel policies than usual LCA: Global economic variations calculated by a general equilibrium economic model and LUC emissions can be evaluated. More work is needed, however, to include new biofuel production technologies and reduce the uncertainty of the method.     

Biofuel production from microalgae as feedstock: current status and potential

Algal biofuel has become an attractive alternative of petroleum-based fuels in the past decade. Microalgae have been proposed as a feedstock to produce biodiesel, since they are capable of mitigating CO₂ emission and accumulating lipids with high productivity. This article is an overview of the updated status of biofuels, especially biodiesel production from microalgae including fundamental research, culture selection and engineering process development; it summarizes research on mathematical and life cycle modeling on algae growth and biomass production; and it updates global efforts of research and development and commercialization attempts. The major challenges are also discussed.     

Systems biology of yeast: enabling technology for development of cell factories for production of advanced biofuels

Transportation fuels will gradually shift from oil based fuels towards alternative fuel resources like biofuels. Current bioethanol and biodiesel can, however, not cover the increasing demand for biofuels and there is therefore a need for advanced biofuels with superior fuel properties. Novel cell factories will provide a production platform for advanced biofuels. However, deep cellular understanding is required for improvement of current biofuel cell factories. Fast screening and analysis (-omics) methods and metabolome-wide mathematical models are promising techniques. An integrated systems approach of these techniques drives diversity and quantity of several new biofuel compounds. This review will cover the recent technological developments that support improvement of the advanced biofuels 1-butanol, biodiesels and jetfuels.     

Life cycle assessment of biofuels from Jatropha curcas in West Africa: a field study

In recent years, liquid biofuels for transport have benefited from significant political support due to their potential role in curbing climate change and reducing our dependence on fossil fuels. They may also participate to rural development by providing new markets for agricultural production. However, the growth of energy crops has raised concerns due to their high consumption of conventional fuels, fertilizers and pesticides, their impacts on ecosystems and their competition for arable land with food crops. Low-input species such as Jatropha curcas , a perennial, inedible crop well adapted to semiarid regions, has received much interest as a new alternative for biofuel production, minimizing adverse effects on the environment and food supply. Here, we used life-cycle assessment to quantify the benefits of J. curcas biofuel production in West Africa in terms of greenhouse gas emissions and fossil energy use, compared with fossil diesel fuel and other biofuels. Biodiesel from J. curcas has a much higher performance than current biofuels, relative to oil-derived diesel fuels. Under West Africa conditions, J. curcas biodiesel allows a 72% saving in greenhouse gas emissions compared with conventional diesel fuel, and its energy yield (the ratio of biodiesel energy output to fossil energy input) is 4.7. J. curcas production studied is eco-compatible for the impacts under consideration and fits into the context of sustainable development.     

Food and bioenergy: reviewing the potential of dual‐purpose wheat crops

Within the bioenergy debate, the ‘food vs. fuel’ controversy quickly replaced enthusiasm for biofuels derived from first‐generation feedstocks. Second‐generation biofuels offer an opportunity to produce fuels from dedicated energy crops, waste materials or coproducts such as cereal straw. Wheat represents one of the most widely grown arable crops around the world, with wheat straw, a potential source of biofuel feedstock. Wheat straw currently has limited economic value; hence, wheat cultivars have been bred for increased grain yield; however, with the development of second‐generation biofuel production, utilization of straw biomass provides the potential for ‘food and fuel’. Reviewing the evidence for the development of dual‐purpose wheat cultivars optimized for food grain and straw biomass production, we present a holistic assessment of a potential ideotype for a dual‐purpose cultivar (DPC). An ideal DPC would be characterized by high grain and straw yields, high straw digestibility (i.e. biofuel yield potential) and good lodging resistance. Considerable variation in these traits exists among current wheat cultivars, facilitating the selection of improved individual traits; however, increasing straw yield and digestibility could potentially have negative trade‐off impacts on grain yield and lodging resistance, reducing the feasibility of a single ideotype. Adoption of alternative management practices could potentially increase straw yield and digestibility, albeit these practices are also associated with potential trade‐offs among cultivar traits. Benefits from using DPCs include reduced logistics costs along the biofuel feedstock supply chain, but practical barriers to differential pricing for straw digestibility traits are likely to reduce the financial incentive to farmers for growing higher ‘biofuel‐quality’ straw cultivars. Further research is required to explore the relationships among the ideotype traits to quantify potential DPC benefits; this will help to determine whether stakeholders along the bioenergy feedstock supply chain will invest in the development of DPCs that provide food and fuel potential.     

Environmental impacts and limitations of third‐generation biobutanol: Life cycle assessment of n‐butanol produced by genetically engineered cyanobacteria

Photosynthetic cyanobacteria have attracted interest as production organisms for third‐generation biofuels, where sunlight and CO₂ are used by microbes directly to synthesize fuel molecules. A particularly suitable biofuel is n‐butanol, and there have been several laboratory reports of genetically engineered photosynthetic cyanobacteria capable of synthesizing and secreting n‐butanol. This work evaluates the environmental impacts and cumulative energy demand (CED) of cyanobacteria‐produced n‐butanol through a cradle‐to‐grave consequential life cycle assessment (LCA). A hypothetical production plant in northern Sweden (area 1 ha, producing 5–85 m³ n‐butanol per year) was considered, and a range of cultivation formats and cellular productivity scenarios assessed. Depending on the scenario, greenhouse gas emissions (GHGe) ranged from 16.9 to 58.6 gCO₂eq/MJ_BuOH and the CED from 3.8 to 13 MJ/MJ_BuOH. Only with the assumption of a nearby paper mill to supply waste sources for heat and CO₂ was the sustainability requirement of at least 60% GHGe savings compared to fossil fuels reached, though placement in northern Sweden reduced energy needed for reactor cooling. A high CED in all scenarios shows that significant metabolic engineering is necessary, such as a carbon partitioning of >90% to n‐butanol, as well as improved light utilization, to begin to displace fossil fuels or even first‐ and second‐generation bioethanol.     

Biofuel production in Escherichia coli: the role of metabolic engineering and synthetic biology

The microbial production of biofuels is a promising avenue for the development of viable processes for the generation of fuels from sustainable resources. In order to become cost and energy effective, these processes must utilize organisms that can be optimized to efficiently produce candidate fuels from a variety of feedstocks. Escherichia coli has become a promising host organism for the microbial production of biofuels in part due to the ease at which this organism can be manipulated. Advancements in metabolic engineering and synthetic biology have led to the ability to efficiently engineer E. coli as a biocatalyst for the production of a wide variety of potential biofuels from several biomass constituents. This review focuses on recent efforts devoted to engineering E. coli for the production of biofuels, with emphasis on the key aspects of both the utilization of a variety of substrates as well as the synthesis of several promising biofuels. Strategies for the efficient utilization of carbohydrates, carbohydrate mixtures, and noncarbohydrate carbon sources will be discussed along with engineering efforts for the exploitation of both fermentative and nonfermentative pathways for the production of candidate biofuels such as alcohols and higher carbon biofuels derived from fatty acid and isoprenoid pathways. Continued advancements in metabolic engineering and synthetic biology will help improve not only the titers, yields, and productivities of biofuels discussed herein, but also increase the potential range of compounds that can be produced.