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 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.     

Development and status of dedicated energy crops in the United States

The biofuel industry is rapidly growing because of increasing energy demand and diminishing petroleum reserves on a global scale. A multitude of biomass resources have been investigated, with high-yielding, perennial feedstocks showing the greatest potential for utilization as advanced biofuels. Government policy and economic drivers have promoted the development and commercialization of biofuel feedstocks, conversion technologies, and supply chain logistics. Research and regulations have focused on the environmental consequences of biofuels, greatly promoting systems that reduce greenhouse gas emissions and life-cycle impacts. Numerous biofuel refineries using lignocellulosic feedstocks and biomass-based triglycerides are either in production or pre-commercial development phases. Leading candidate energy crops have been identified, yet require additional efforts to realize their full potential. Advanced biofuels, complementing conventional biofuels and other renewable energy sources such as wind and solar, provide the means to substantially displace humanity’s reliance on petroleum-based energy.     

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.     

Consolidated conversion of protein waste into biofuels and ammonia using Bacillus subtilis

The non-recyclable use of nitrogen fertilizers in microbial production of fuels and chemicals remains environmentally detrimental. Conversion of protein wastes into biofuels and ammonia by engineering nitrogen flux in Escherichia coli has been demonstrated as a method to reclaim reduced-nitrogen and curb its environmental deposition. However, protein biomass requires a proteolysis process before it can be taken up and converted by any microbe. Here, we metabolically engineered Bacillus subtilis to hydrolyze polypeptides through its secreted proteases and to convert amino acids into advanced biofuels and ammonia fertilizer. Redirection of B. subtilis metabolism for amino-acid conversion required inactivation of the branched-chain amino-acid (BCAA) global regulator CodY. Additionally, the lipoamide acyltransferase (bkdB) was deleted to prevent conversion of branched-chain 2-keto acids into their acyl-CoA derivatives. With these deletions and heterologous expression of a keto-acid decarboxylase and an alcohol dehydrogenase, the final strain produced biofuels and ammonia from an amino-acid media with 18.9% and 46.6% of the maximum theoretical yield. The process was also demonstrated on several waste proteins. The results demonstrate the feasibility of direct microbial conversion of polypeptides into sustainable products.     

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.     

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.     

Advanced Biofuels from Thermochemical Processing of Sustainable Biomass in Europe

Biofuels can play an important role in decreasing the use of fossil fuels, in particular in the transport sector, which absorbs about 30 % of the EU energy requirements. This review illustrates the motivations behind biofuel development, the government incentives and regulations and the current approaches on sustainable biomass conversion in Europe, and provides an overview on the major steps involved in thermochemical processes and on the issues challenging their deployment at large scale, with particular emphasis on the pyrolysis of biomass and bio-oil upgrading using conventional oil refinery settings. Distribution of sustainable biofuels in Europe and future prospects towards achieving success of transport biofuels were also addressed. The literature suggests that importing biofuels and increasing the cost of CO₂ to at least €60/t CO₂ will be necessary to meet the renewable obligation targets in the EU. Algae represent the future feedstock for biofuels but currently are limited by their high production costs and high N content. Pyrolysis is cost competitive compared to other technologies such as fermentation and gasification, but the quality of bio-oils requires upgrading mainly to lower their oxygen content and enhance their thermal stability. The recent advances in bio-oil upgrading using catalytic cracking and hydro-treating are very promising for the future deployment of advanced biofuels in the coming decades. However, significant investments in applied research and demonstration are still required to meet the 2020/2030 biofuel targets.     

Metabolic engineering for valorization of macroalgae biomass

Marine macroalgae have huge potential as feedstocks for production of a wide spectrum of chemicals used in biofuels, biomaterials, and bioactive compounds. Harnessing macroalgae in these ways could promote wellbeing for people while mitigating climate change and environmental destruction linked to use of fossil fuels. Microorganisms play pivotal roles in converting macroalgae into valuable products, and metabolic engineering technologies have been developed to extend their native capabilities. This review showcases current achievements in engineering the metabolisms of various microbial chassis to convert red, green, and brown macroalgae into bioproducts. Unique features of macroalgae, such as seasonal variation in carbohydrate content and salinity, provide the next challenges to advancing macroalgae-based biorefineries. Three emerging engineering strategies are discussed here: (1) designing dynamic control of metabolic pathways, (2) engineering strains of halophilic (salt-tolerant) microbes, and (3) developing microbial consortia for conversion. This review illuminates opportunities for future research communities by elucidating current approaches to engineering microbes so they can become cell factories for the utilization of macroalgae feedstocks.     

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.     

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.     

Aquatic phototrophs: efficient alternatives to land-based crops for biofuels

To mitigate some of the potentially deleterious environmental and agricultural consequences associated with current land-based-biofuel feedstocks, we propose the use of biofuels derived from aquatic microbial oxygenic photoautotrophs (AMOPs), more commonly known as cyanobacteria, algae, and diatoms. Herein we review their demonstrated productivity in mass culturing and aspects of their physiology that are particularly attractive for integration into renewable biofuel applications. Compared with terrestrial crops, AMOPs are inherently more efficient solar collectors, use less or no land, can be converted to liquid fuels using simpler technologies than cellulose, and offer secondary uses that fossil fuels do not provide. AMOPs pose a new set of technological challenges if they are to contribute as biofuel feedstocks.     

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.     

Association with an ammonium-excreting bacterium allows diazotrophic culture of oil-rich eukaryotic microalgae

Concerns regarding the depletion of the world's reserves of oil and global climate change have promoted an intensification of research and development toward the production of biofuels and other alternative sources of energy during the last years. There is currently much interest in developing the technology for third-generation biofuels from microalgal biomass mainly because of its potential for high yields and reduced land use changes in comparison with biofuels derived from plant feedstocks. Regardless of the nature of the feedstock, the use of fertilizers, especially nitrogen, entails a potential economic and environmental drawback for the sustainability of biofuel production. In this work, we have studied the possibility of nitrogen biofertilization by diazotrophic bacteria applied to cultured microalgae as a promising feedstock for next-generation biofuels. We have obtained an Azotobacter vinelandii mutant strain that accumulates several times more ammonium in culture medium than wild-type cells. The ammonium excreted by the mutant cells is bioavailable to promote the growth of nondiazotrophic microalgae. Moreover, this synthetic symbiosis was able to produce an oil-rich microalgal biomass using both carbon and nitrogen from the air. This work provides a proof of concept that artificial symbiosis may be considered an alternative strategy for the low-N-intensive cultivation of microalgae for the sustainable production of next-generation biofuels and other bioproducts.     

Selection, breeding and engineering of microalgae for bioenergy and biofuel production

Microalgal production technologies are seen as increasingly attractive for bioenergy production to improve fuel security and reduce CO(2) emissions. Photosynthetically derived fuels are a renewable, potentially carbon-neutral and scalable alternative reserve. Microalgae have particular promise because they can be produced on non-arable land and utilize saline and wastewater streams. Furthermore, emerging microalgal technologies can be used to produce a range of products such as biofuels, protein-rich animal feeds, chemical feedstocks (e.g. bioplastic precursors) and higher-value products. This review focuses on the selection, breeding and engineering of microalgae for improved biomass and biofuel conversion efficiencies.     

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.     

木质纤维素预处理及高值化技术研究进展

木质纤维素生物质是地球上最丰富的可再生生物资源.随着化石能源的消耗及环境的污染,以取代石化燃料为目标的由生物质向生物燃料的转化受到了广泛的关注.木质纤维素有很强的天然抗降解屏障,需先通过物理、化学及微生物等手段进行预处理,进而以更低的成本和更高的效率转化为生物燃料及其他高附加值产品.本文在总结酸碱等传统预处理方法优缺点...     

2013生物能源专刊序言

生物能源作为可再生能源,可以替代部分石化能源,有望缓解能源供给中对石油的依赖程度.本期专刊结合第6届国际生物能源会议,包括综述和研究报告两部分,报道了我国生物能源专家学者在燃料乙醇、生物柴油、微生物油脂、生物燃料标准、航空生物燃料等领域的最新研究进展.     

Significance and limitations of first generation biofuels

Formerly on the margins of the European agricultural landscape, liquid biofuels for transport have recently come into sharp focus with the help of three drivers: the depletion of oil resources and the political motto of energy independence, international negotiations on climate, and finally - in Europe at least - the overhaul of the common agricultural policy underpinning the need to diversify this sector. This political purpose has led to aggressive development targets in both Europe and the United States, implying a nearly ten-fold increase of biofuel production within ten years. This article introduces the current biofuel production technologies (so-called ;first generation'), whose common marker is the reliance on the storage organs of agricultural plants. This implies a relatively strong demand in arable areas, along with only moderately positive energy and environmental advantages compared to fossil fuels. 'Second generation' biofuels, which are based on generic biomass (ligno-cellulose) are expected to overcome these limitations, but will not be deployed on the market for another ten years.     

Non-sterile fermentations for the economical biochemical conversion of renewable feedstocks

Heavy reliance on petroleum-based products drives continuous exploitation of fossil fuels, and results in serious environmental and climate problems. To address such an issue, there is a shift from petroleum sources to renewable ones. Biochemical conversion via fermentation is a primary platform for converting renewable sources to biofuels and bulk chemicals. In order to provide cost-competitive alternatives, it is imperative to develop efficient, cost-saving, and robust fermentation processes. Non-sterile fermentation offers several benefits compared to sterile fermentation, including elimination of sterility, reduced maintenance requirements, relatively simple bioreactor design, and simplified operation. Thus, cost effectiveness of non-sterile fermentation makes it a practical platform for low cost, large volume production of biofuels and bulk chemicals. Many approaches have been developed to conduct non-sterile fermentation without sacrificing the yields and productivities of fermentation products. This review focuses on the strategies for conducting non-sterile fermentation. The challenges facing non-sterile fermentation are also discussed.