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.     

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.     

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.     

Methods of energy extraction from microalgal biomass: a review

The potential of algal biomass as a source of liquid and gaseous biofuels is a highly topical theme, The process operations for algal biofuel production can be grouped into three areas: growth, harvesting and energy extraction, with a wide range of combinations of unit operations that can form a microalgal biofuel production system, but as yet there is no successful economically viable commercial system producing biofuel. This suggests that there are major technical and engineering difficulties to be resolved before economic algal biofuel production can be achieved. This article briefly reviews the methods by which useful energy may be extracted from microalgae biomass: (a) direct combustion, (b) pyrolysis, (c) gasification, (d) liquefaction, (e) hydrogen production by biochemical processes in certain algae, (f) fuel cells, (g) fermentation to bioethanol, (h) trans-esterification to biodiesel, (i) anaerobic digestion.     

Seasonal productivity of a periphytic algal community for biofuel feedstock generation and nutrient treatment

Algal biomass is a promising feedstock for biofuel production. With a high lipid content and high rate of production, algae can produce more oil on less land than traditional bioenergy crops. Algal communities can also be used to remove nutrients from impacted waters. The purpose of this study was to demonstrate the ability of an algal turf scrubber (ATS)™ to facilitate the growth of periphytic algal communities for the production of biomass feedstock and the removal of nutrients from a local stream. A pilot-scale ATS was implemented in Springdale, AR, and operated over the course of a nine-month sampling period. System productivity over the nine-month operating time averaged 26 g m⁻² d⁻¹. Total phosphorus and total nitrogen removal averaged 48% and 13%, respectively. The system showed potential for biomass generation and nutrient removal across three seasons.     

Accumulation of lipid production in Chlorella minutissima by triacylglycerol biosynthesis-related genes cloned from Saccharomyces cerevisiae and Yarrowia lipolytica

Discovery of an alternative fuel is now an urgent matter because of the impending issue of oil depletion. Lipids synthesized in algal cells called triacylglycerols (TAGs) are thought to be of the most value as a potential biofuel source because they can use transesterification to manufacture biodiesel. Biodiesel is deemed as a good solution to overcoming the problem of oil depletion since it is capable of providing good performance similar to that of petroleum. Expression of several genomic sequences, including glycerol-3-phosphate dehydrogenase, glycerol-3-phosphate acyltransferase, lysophosphatidic acid acyltransferase, phosphatidic acid phosphatase, diacylglycerol acyltransferase, and phospholipid:diacylglycerol acyltransferase, can be useful for manipulating metabolic pathways for biofuel production. In this study, we found this approach indeed increased the storage lipid content of C. minutissima UTEX 2219 up to 2-fold over that of wild type. Thus, we conclude this approach can be used with the biodiesel production platform of C. minutissima UTEX 2219 for high lipid production that will, in turn, enhance productivity.     

Predaceous insects may limit algal grazers in high-nutrient environments: implications for wastewater remediation in open bioreactors

Algal biofuel has potential as a source of renewable fuel and a tool for wastewater remediation. Open algal bioreactors fertilized with wastewater can have net energy gain but are vulnerable to colonization by algal grazers. However, colonizing predaceous insects may limit grazer impacts on algae. Here, we investigate the effects of grazers, predators, and invading algae species on algal production and community structure in high-nutrient environments. First, we grew diverse algal assemblages in treated municipal wastewater in a greenhouse with Daphnia grazers and different insect predators that were added experimentally. When Daphnia were present without predators, they eliminated suspended algae. But, dragonfly larvae [Odonata: Libellulidae] and backswimmers [Hemiptera: Notonectidae], but not larval diving beetles [Coloeoptera: Dytiscidae], suppressed Daphnia allowing suspended algae to persist. Second, we grew Chlorella algae in field tanks that were open or protected from natural invertebrate colonization and half the tanks received wild-collected plankton in a factorial design. Mosquito larvae [Culex sp.] readily colonized open tanks and reduced algal mass and dissolved phosphorus concentrations. Colonist addition to open tanks shifted algal functional and taxonomic composition but did not impact suspended algal production. Our study indicates that large numbers of grazer individuals can rapidly colonize open bioreactors. Experimentally added and naturally colonizing grazers altered algal community structure and reduced algal standing crops but may also aid in nutrient removal from wastewater-fed bioreactors. Effective operation of open algal bioreactors must consider cultivated algae species’ vulnerability to competition and local grazers as well as the ability of potential predators to both naturally disperse into bioreactors and to control grazers.     

A thermophilic ionic liquid-tolerant cellulase cocktail for the production of cellulosic biofuels

Generation of biofuels from sugars in lignocellulosic biomass is a promising alternative to liquid fossil fuels, but efficient and inexpensive bioprocessing configurations must be developed to make this technology commercially viable. One of the major barriers to commercialization is the recalcitrance of plant cell wall polysaccharides to enzymatic hydrolysis. Biomass pretreatment with ionic liquids (ILs) enables efficient saccharification of biomass, but residual ILs inhibit both saccharification and microbial fuel production, requiring extensive washing after IL pretreatment. Pretreatment itself can also produce biomass-derived inhibitory compounds that reduce microbial fuel production. Therefore, there are multiple points in the process from biomass to biofuel production that must be interrogated and optimized to maximize fuel production. Here, we report the development of an IL-tolerant cellulase cocktail by combining thermophilic bacterial glycoside hydrolases produced by a mixed consortia with recombinant glycoside hydrolases. This enzymatic cocktail saccharifies IL-pretreated biomass at higher temperatures and in the presence of much higher IL concentrations than commercial fungal cocktails. Sugars obtained from saccharification of IL-pretreated switchgrass using this cocktail can be converted into biodiesel (fatty acid ethyl-esters or FAEEs) by a metabolically engineered strain of E. coli. During these studies, we found that this biodiesel-producing E. coli strain was sensitive to ILs and inhibitors released by saccharification. This cocktail will enable the development of novel biomass to biofuel bioprocessing configurations that may overcome some of the barriers to production of inexpensive cellulosic biofuels.     

Biofuel alternatives to ethanol: pumping the microbial well

Engineered microorganisms are currently used for the production of food products, pharmaceuticals, ethanol fuel and more. Even so, the enormous potential of this technology has yet to be fully exploited. The need for sustainable sources of transportation fuels has generated a tremendous interest in technologies that enable biofuel production. Decades of work have produced a considerable knowledge-base for the physiology and pathway engineering of microbes, making microbial engineering an ideal strategy for producing biofuel. Although ethanol currently dominates the biofuel market, some of its inherent physical properties make it a less than ideal product. To highlight additional options, we review advances in microbial engineering for the production of other potential fuel molecules, using a variety of biosynthetic pathways.     

A state of the art of metabolic networks of unicellular microalgae and cyanobacteria for biofuel production

The most promising and yet challenging application of microalgae and cyanobacteria is the production of renewable energy: biodiesel from microalgae triacylglycerols and bioethanol from cyanobacteria carbohydrates. A thorough understanding of microalgal and cyanobacterial metabolism is necessary to master and optimize biofuel production yields. To this end, systems biology and metabolic modeling have proven to be very efficient tools if supported by an accurate knowledge of the metabolic network. However, unlike heterotrophic microorganisms that utilize the same substrate for energy and as carbon source, microalgae and cyanobacteria require light for energy and inorganic carbon (CO₂ or bicarbonate) as carbon source. This double specificity, together with the complex mechanisms of light capture, makes the representation of metabolic network nonstandard. Here, we review the existing metabolic networks of photoautotrophic microalgae and cyanobacteria. We highlight how these networks have been useful for gaining insight on photoautotrophic metabolism.     

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.     

Bioconversion of natural gas to liquid fuel: Opportunities and challenges

Natural gas is a mixture of low molecular weight hydrocarbon gases that can be generated from either fossil or anthropogenic resources. Although natural gas is used as a transportation fuel, constraints in storage, relatively low energy content (MJ/L), and delivery have limited widespread adoption. Advanced utilization of natural gas has been explored for biofuel production by microorganisms. In recent years, the aerobic bioconversion of natural gas (or primarily the methane content of natural gas) into liquid fuels (Bio-GTL) by biocatalysts (methanotrophs) has gained increasing attention as a promising alternative for drop-in biofuel production. Methanotrophic bacteria are capable of converting methane into microbial lipids, which can in turn be converted into renewable diesel via a hydrotreating process. In this paper, biodiversity, catalytic properties and key enzymes and pathways of these microbes are summarized. Bioprocess technologies are discussed based upon existing literature, including cultivation conditions, fermentation modes, bioreactor design, and lipid extraction and upgrading. This review also outlines the potential of Bio-GTL using methane as an alternative carbon source as well as the major challenges and future research needs of microbial lipid accumulation derived from methane, key performance index, and techno-economic analysis. An analysis of raw material costs suggests that methane-derived diesel fuel has the potential to be competitive with petroleum-derived diesel.     

Revival of the biological sunlight‐to‐biogas energy conversion system

In the quest for renewable resources, algae are increasingly receiving attention. Their high growth rate, high CO₂ fixation and their lack of requirement for fertile soil surface represent several advantages as compared to conventional (energy) crops. Through their ability to store large amounts of oils, they qualify as a source for biodiesel. Algal biomass, however, can also be used as such, namely as a substrate for anaerobic digestion. In the present research, we investigated the use of algae for energy generation in a stand‐alone, closed‐loop system. The system encompasses an algal growth unit for biomass production, an anaerobic digestion unit to convert the biomass to biogas and a microbial fuel cell to polish the effluent of the digester. Nutrients set free during digestion can accordingly be returned to the algal growth unit for a sustained algal growth. Hence, a system is presented that continuously transforms solar energy into energy‐rich biogas and electricity. Algal productivities of 24–30 ton VS ha^?1 year^?1 were reached, while 0.5 N m³ biogas could be produced kg^?1 algal VS. The system described resulted in a power plant with a potential capacity of about 9 kW ha^?1 of solar algal panel, with prospects of 23 kW ha^?1. Biotechnol. Bioeng. 2009;103: 296–304. © 2009 Wiley Periodicals, Inc.     

Applications of systems biology towards microbial fuel production

Harnessing the immense natural diversity of biological functions for economical production of fuel has enormous potential benefits. Inevitably, however, the native capabilities for any given organism must be modified to increase the productivity or efficiency of a biofuel bioprocess. From a broad perspective, the challenge is to sufficiently understand the details of cellular functionality to be able to prospectively predict and modify the cellular function of a microorganism. Recent advances in experimental and computational systems biology approaches can be used to better understand cellular level function and guide future experiments. With pressure to quickly develop viable, renewable biofuel processes a balance must be maintained between obtaining depth of biological knowledge and applying that knowledge.     

Merging Metabolism and Power: Development of a Novel Photobioelectric Device Driven by Photosynthesis and Respiration

Generation of renewable energy is one of the grand challenges facing our society. We present a new bio-electric technology driven by chemical gradients generated by photosynthesis and respiration. The system does not require pure cultures nor particular species as it works with the core metabolic principles that define phototrophs and heterotrophs. The biology is interfaced with electrochemistry with an alkaline aluminum oxide cell design. In field trials we show the system is robust and can work with an undefined natural microbial community. Power generated is light and photosynthesis dependent. It achieved a peak power output of 33 watts/m² electrode. The design is simple, low cost and works with the biological processes driving the system by removing waste products that can impede growth. This system is a new class of bio-electric device and may have practical implications for algal biofuel production and powering remote sensing devices.     

Strategies to enhance production of microalgal biomass and lipids for biofuel feedstock

ABSTRACT

Microalgae have enormous potential as feedstock for biofuel production compared with other sources, due to their high areal productivity, relatively low environmental impact, and low impact on food security. However, high production costs are the major limitation for commercialization of algal biofuels. Strategies to maximize biomass and lipid production are crucial for improving the economics of using microalgae for biofuels. Selection of suitable algal strains, preferably from indigenous habitats, and further improvement of those ‘platform strains’ using mutagenesis and genetic engineering approaches are desirable. Conventional approaches to improve biomass and lipid productivity of microalgae mainly involve manipulation of nutritional (e.g. nitrogen and phosphorus) and environmental (e.g. temperature, light and salinity) factors. Approaches such as the addition of phytohormones, genetic and metabolic engineering, and co-cultivation of microalgae with yeasts and bacteria are more recent strategies to enhance biomass and lipid productivity of microalgae. Improvement in culture systems and the use of a hybrid system (i.e. a combination of open ponds and photobioreactors) is another strategy to optimize algal biomass and lipid production. In addition, the use of low-cost substrates such as agri-industrial wastewater for the cultivation of microalgae will be a smart strategy to reduce production costs. Such systems not only generate high algal biomass and lipid productivity, but are also useful for bioremediation of wastewater and bioremoval of waste CO₂. The aim of this review is to highlight the advances in the use of various strategies to enhance production of algal biomass and lipids for biofuel feedstock.     

Optimization of industrial microorganisms: recent advances in synthetic dynamic regulators

Production of biochemicals by industrial fermentation using microorganisms requires maintaining cellular production capacity, because maximal productivity is economically important. High-productivity microbial strains can be developed using static engineering, but these may not maintain maximal productivity throughout the culture period as culture conditions and cell states change dynamically. Additionally, economic reasons limit heterologous protein expression using inducible promoters to prevent metabolic burden for commodity chemical and biofuel production. Recently, synthetic and systems biology has been used to design genetic circuits, precisely controlling gene expression or influencing genetic behavior toward a desired phenotype. Development of dynamic regulators can maintain cellular phenotype in a maximum production state in response to factors including cell concentration, oxygen, temperature, pH, and metabolites. Herein, we introduce dynamic regulators of industrial microorganism optimization and discuss metabolic flux fine control by dynamic regulators in response to metabolites or extracellular stimuli, robust production systems, and auto-induction systems using quorum sensing.     

A novel cold-adapted lipase, LP28, from a mesophilic Streptomyces strain

Fossil fuel is limited but its usage has been growing rapidly, thus the fuel is predicted to be completely running out and causing an unbearable global energy crisis in the near future. To solve this potential crisis, incorporating with increasing environmental concerns, significant attentions have been given to biofuel production in the recent years. With the aim of isolating a microbial biocatalyst with potential application in the field of biofuel, a lipase from Streptomyces sp. CS628, LP28, was purified using hydroxyapatite column chromatography followed by a gel filtration. Molecular weight of LP28 was estimated to be 32,400 Da by SDS-PAGE. The activity was the highest at 30 °C and pH 8.0 and was stable at pH 6.0-8.0 and below 25 °C. The enzyme preferentially hydrolyzed p-nitrophenyl decanoate (C10), a medium chain substrate. Furthermore, LP28 non-specifically hydrolyzed triolein releasing both 1,2- and 1,3-diolein. More importantly, LP28 manifestly catalyzed biodiesel production using palm oil and methanol; therefore, it can be a potential candidate in the field of biofuel.     

Socioeconomic indicators for sustainable design and commercial development of algal biofuel systems

Social and economic indicators can be used to support design of sustainable energy systems. Indicators representing categories of social well‐being, energy security, external trade, profitability, resource conservation, and social acceptability have not yet been measured in published sustainability assessments for commercial algal biofuel facilities. We review socioeconomic indicators that have been modeled at the commercial scale or measured at the pilot or laboratory scale, as well as factors that affect them, and discuss additional indicators that should be measured during commercialization to form a more complete picture of socioeconomic sustainability of algal biofuels. Indicators estimated in the scientific literature include the profitability indicators, return on investment (ROI) and net present value (NPV), and the resource conservation indicator, fossil energy return on investment (EROI). These modeled indicators have clear sustainability targets and have been used to design sustainable algal biofuel systems. Factors affecting ROI, NPV, and EROI include infrastructure, process choices, and financial assumptions. The food security indicator, percent change in food price volatility, is probably zero where agricultural lands are not used for production of algae‐based biofuels; however, food‐related coproducts from algae could enhance food security. The energy security indicators energy security premium and fuel price volatility and external trade indicators terms of trade and trade volume cannot be projected into the future with accuracy prior to commercialization. Together with environmental sustainability indicators, the use of a suite of socioeconomic sustainability indicators should contribute to progress toward sustainability of algal biofuels.     

A perspective: Photosynthetic production of fatty acid-based biofuels in genetically engineered cyanobacteria

Biofuels are expected to play a key role in the development of a sustainable, economical and environmentally safe source of energy. Microbes offer great potential for applications in technology based biofuel production. Three fundamental questions need to be addressed in order for the development of microbial synthesis of biofuels to be successful. Firstly, what energy resource platform could be used to make biofuels. Secondly, what type of biofuel is the ideal fuel molecule that should be targeted. Finally, what microbial system could be used to transform energy resources into the targeted biofuel molecules. In this perspective, the potential of using photosynthetic microbes (cyanobacteria in particular) in the solar energy driven conversion of carbon dioxide to fatty acid-based biofuels is explored.