Engineering of microorganisms for the production of biofuels and perspectives based on systems metabolic engineering approaches

The increasing oil price and environmental concerns caused by the use of fossil fuel have renewed our interest in utilizing biomass as a sustainable resource for the production of biofuel. It is however essential to develop high performance microbes that are capable of producing biofuels with very high efficiency in order to compete with the fossil fuel. Recently, the strategies for developing microbial strains by systems metabolic engineering, which can be considered as metabolic engineering integrated with systems biology and synthetic biology, have been developed. Systems metabolic engineering allows successful development of microbes that are capable of producing several different biofuels including bioethanol, biobutanol, alkane, and biodiesel, and even hydrogen. In this review, the approaches employed to develop efficient biofuel producers by metabolic engineering and systems metabolic engineering approaches are reviewed with relevant example cases. It is expected that systems metabolic engineering will be employed as an essential strategy for the development of microbial strains for industrial applications.     

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.     

Protein-based biorefining: metabolic engineering for production of chemicals and fuel with regeneration of nitrogen fertilizers

Threats to stable oil supplies and concerns over environmental emissions have pushed for renewable biofuel developments to minimize dependence on fossil resources. Recent biofuel progress has moved towards fossil resource-independent carbon cycles, but environmental issues regarding use of nitrogen fertilizers have not been addressed on a global scale. The recently demonstrated conversion of waste protein biomass into advanced biofuels and renewable chemicals, while recycling nitrogen fertilizers, offers a glimpse of the efforts needed to balance the nitrogen cycle at scale. In general, the catabolism of protein into biofuels is challenging because of physiological regulation and thermodynamic limitations. This conversion became possible with metabolic engineering around ammonia assimilation, intracellular nitrogen flux, and quorum sensing. This review highlights the metabolic engineering solutions in transforming those cellular processes into driving forces for the high yield of chemical products from protein.     

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.     

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.     

Fermentative production of butanol--the academic perspective

As mobility is a major pillar of World's economic system and burning fuels from fossil resources leads to a dramatic increase in greenhouse gas emissions, the production and use of appropriate biofuels offer at least a partial solution to this problem. Butanol represents a biofuel extender or replacement with properties clearly superior to ethanol (higher mileage, not hygroscopic, usable without engine modifications, not corrosive). In addition, it is a valuable feedstock for the chemical industry. Scientific challenges for an economically competitive fermentation process include employment of cheap carbon sources, not competing with nutrition, a detailed understanding of the metabolic reactions of the biological process, development of appropriately engineered construction strains, and adaption of process technology to modern standards.     

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.     

From flavors and pharmaceuticals to advanced biofuels: Production of isoprenoids in Saccharomyces cerevisiae

Isoprenoids denote the largest group of chemicals in the plant kingdom and are employed for a wide range of applications in the food and pharmaceutical industry. In recent years, isoprenoids have additionally been recognized as suitable replacements for petroleum-derived fuels and could thus promote the transition towards a more sustainable society. To realize the biofuel potential of isoprenoids, a very efficient production system is required. While complex chemical structures as well as the low abundance in nature demonstrate the shortcomings of chemical synthesis and plant extraction, isoprenoids can be produced by genetically engineered microorganisms from renewable carbon sources. In this article, we summarize the development of isoprenoid applications from flavors and pharmaceuticals to advanced biofuels and review the strategies to design microbial cell factories, focusing on Saccharomyces cerevisiae for the production of these compounds. While the high complexity of biosynthetic pathways and the toxicity of certain isoprenoids still denote challenges that need to be addressed, metabolic engineering has enabled large-scale production of several terpenoids and thus, the utilization of these compounds is likely to expand in the future.     

Anaerobic fermentation of glycerol: a path to economic viability for the biofuels industry

Although biofuels such as biodiesel and bioethanol represent a secure, renewable and environmentally safe alternative to fossil fuels, their economic viability is a major concern. The implementation of biorefineries that co-produce higher value products along with biofuels has been proposed as a solution to this problem. The biorefinery model would be especially advantageous if the conversion of byproducts or waste streams generated during biofuel production were considered. Glycerol-rich streams generated in large amounts by the biofuels industry, especially during the production of biodiesel, present an excellent opportunity to establish biorefineries. Once considered a valuable 'co-product', crude glycerol is rapidly becoming a 'waste product' with a disposal cost attributed to it. Given the highly reduced nature of carbon in glycerol and the cost advantage of anaerobic processes, fermentative metabolism of glycerol is of special interest. This review covers the anaerobic fermentation of glycerol in microbes and the harnessing of this metabolic process to convert abundant and low-priced glycerol streams into higher value products, thus creating a path to viability for the biofuels industry. Special attention is given to products whose synthesis from glycerol would be advantageous when compared with their production from common sugars.     

Addressing the energy crisis: using microbes to make biofuels

Much of the energy being used to power our lives comes from fossil fuels such as coal, natural gas and petroleum. These energy sources are non-renewable, are being exhausted and also pollute the air, water and soil with toxic chemicals. Their mining, transportation, refining and use are associated with a large carbon footprint that contributes significantly to global warming. In addition, the geopolitical complexities surrounding the main fossil fuel producers create risks and uncertainties around the world. Replacing fossil fuels with clean, renewable forms of energy is paramount to creating a sustainable and healthy future, and for laying the foundations for global political stability and prosperity. Using biomass from plants, microbes can produce biofuels that are identical to or perform as well as fossil fuels. In addition of creating sustainable energy, advancing the biofuel industry will create new, high-quality rural jobs whilst improving energy security.     

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.     

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.     

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.     

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.     

Metabolic systems analysis to advance algal biotechnology

Algal fuel sources promise unsurpassed yields in a carbon neutral manner that minimizes resource competition between agriculture and fuel crops. Many challenges must be addressed before algal biofuels can be accepted as a component of the fossil fuel replacement strategy. One significant challenge is that the cost of algal fuel production must become competitive with existing fuel alternatives. Algal biofuel production presents the opportunity to fine-tune microbial metabolic machinery for an optimal blend of biomass constituents and desired fuel molecules. Genome-scale model-driven algal metabolic design promises to facilitate both goals by directing the utilization of metabolites in the complex, interconnected metabolic networks to optimize production of the compounds of interest. Network analysis can direct microbial development efforts towards successful strategies and enable quantitative fine-tuning of the network for optimal product yields while maintaining the robustness of the production microbe. Metabolic modeling yields insights into microbial function, guides experiments by generating testable hypotheses, and enables the refinement of knowledge on the specific organism. While the application of such analytical approaches to algal systems is limited to date, metabolic network analysis can improve understanding of algal metabolic systems and play an important role in expediting the adoption of new biofuel technologies.     

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.     

Photobioreactor cultivation strategies for microalgae and cyanobacteria

The current burden on fossil‐derived chemicals and fuels combined with the rapidly increasing global population has led to a crucial need to develop renewable and sustainable sources of chemicals and biofuels. Photoautotrophic microorganisms, including cyanobacteria and microalgae, have garnered a great deal of attention for their capability to produce these chemicals from carbon dioxide, mineralized water, and solar energy. While there have been substantial amounts of research directed at scaling‐up production from these microorganisms, several factors have proven difficult to overcome, including high costs associated with cultivation, photobioreactor construction, and artificial lighting. Decreasing these costs will substantially increase the economic feasibility of these production processes. Thus, the purpose of this review is to describe various photobioreactor designs, and then provide an overview on lighting systems, mixing, gas transfer, and the hydrodynamics of bubbles. These factors must be considered when the goal of a production process is economic feasibility. Targets for improving microalgae and cyanobacteria cultivation media, including water reduction strategies will also be described. As fossil fuel reserves continue to be depleted and the world population continues to increase, it is imperative that renewable chemical and biofuel production processes be developed toward becoming economically feasible. Thus, it is essential that future research is directed toward improving these processes. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:811–827, 2018     

Greenhouse gas emissions and abatement costs of biofuel production in South Africa

Transport accounts for about one quarter of South Africa's final energy consumption. Most of the energy used is based on fossil fuels causing significant environmental burdens. This threat becomes even more dominant as a significant growth in transport demand is forecasted, especially in South Africa's economic hub, Gauteng province. The South African government has realized the potential of biofuel usage for reducing oil import dependency and greenhouse gas (GHG) and has hence developed a National Biofuels Industrial Strategy to enforce their use. However, there is limited experience in the country in commercial biofuel production and some of the proposed crops (i.e. rapeseed and sugar beet) have not been yet cultivated on a larger scale. Furthermore, there is only limited research available, looking at the feasibility of commercial scale biofuel production or abatement costs of GHG emissions. To assess the opportunities of biofuel production in South Africa, the production costs and consumer price levels of the fuels recommended by the national strategy are analysed in this article. Moreover, the lifecycle GHG emissions and mitigation costs are calculated compared to the calculated fossil fuel reference including coal to liquid (CTL) and gas to liquid (GTL) fuels. The results show that the cost for biofuel production in South Africa are currently significantly higher (between 30% and 80%) than for the reference fossil fuels. The lifecycle GHG emissions of biofuels (especially for sugar cane) are considerably lower (up to 45%) than the reference fossil GHG emissions. The resulting GHG abatement costs are between 1000 and 2500 ZAR₂₀₀₇ per saved ton of carbon dioxide equivalent, which is high compared to the current European CO₂ market prices of ca. 143 ZAR₂₀₀₇ t^?1. The analysis has shown that biofuel production and utilization in South Africa offers a significant GHG‐mitigation potential but at relatively high cost.     

The path to next generation biofuels: successes and challenges in the era of synthetic biology

Volatility of oil prices along with major concerns about climate change, oil supply security and depleting reserves have sparked renewed interest in the production of fuels from renewable resources. Recent advances in synthetic biology provide new tools for metabolic engineers to direct their strategies and construct optimal biocatalysts for the sustainable production of biofuels. Metabolic engineering and synthetic biology efforts entailing the engineering of native and de novo pathways for conversion of biomass constituents to short-chain alcohols and advanced biofuels are herewith reviewed. In the foreseeable future, formal integration of functional genomics and systems biology with synthetic biology and metabolic engineering will undoubtedly support the discovery, characterization, and engineering of new metabolic routes and more efficient microbial systems for the production of biofuels.     

Renewable fuels from algae: An answer to debatable land based fuels

This article reviews the utilization of first and second-generation biofuels as the suitable alternatives to depleting fossil fuels. Then the concern has been presented over a debate on most serious problem arising from the production of these biofuels; which is the increase of food market prices because of the increased use of arable land for the cultivation of biomass used for the production of first and second-generation biofuels. The solution to this debate has been suggested with the use of non-arable land for the cultivation of algal biomass for the generation of third generation biofuels. The recent research and developments in the cultivation of algal biomass and their use for biofuel production have been discussed.