Combinatorial optimization of cyanobacterial 2,3-butanediol production

A vital goal of renewable technology is the capture and re-energizing of exhausted CO₂ into usable carbon products. Cyanobacteria fix CO₂ more efficiently than plants, and can be engineered to produce carbon feedstocks useful for making plastics, solvents, and medicines. However, fitness of this technology in the economy is threatened by low yields in engineered strains. Robust engineering of photosynthetic microorganisms is lagging behind model microorganisms that rely on energetic carbon, such as Escherichia coli, due in part to slower growth rates and increased metabolic complexity. In this work we show that protein expression from characterized parts is unpredictable in Synechococcus elongatus sp. strain PCC 7942, and may contribute to slow development. To overcome this, we apply a combinatorial approach and show that modulation of the 5'-untranslated region (UTR) can produce a range of protein expression sufficient to optimize chemical feedstock production from CO₂.     

A comparison of lipid storage in Phaeodactylum tricornutum and Tetraselmis suecica using laser scanning confocal microscopy

Microalgae contain lipid bodies (LBs) composed of triacylglycerols, which can be converted to biodiesel. Here we demonstrate a method to study the accumulation patterns of LBs in different microalgae strains and culture conditions utilizing laser scanning confocal microscopy (LSCM) with BODIPY 505/515 (4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene) staining, in parallel with Nile Red (9-diethylamino-5H-benzo-a-phenoxazine-5-one) fluorescence analysis of intracellular lipids in microplates. Phaeodactylum tricornutum and Tetraselmis suecica were selected as model organisms and monitored throughout the growth phases in standard and nitrogen-deficient growth conditions. Utilizing image quantification techniques, the number and morphology of LBs suggest that P. tricornutum accumulates lipids by merging with existing LBs, while T. suecica synthesizes new LBs. We observed that T. suecica accumulates a higher number of LBs and total volume of lipids per cell, while P. tricornutum accumulates only 1–2 LBs with a larger volume per LB. LSCM analysis complements Nile Red (NR) methods because LSCM provides three-dimensional images of lipid accumulation at a cellular level, while NR analysis can quickly monitor the total levels of intracellular lipids for phenotypic screening. Using NR analysis, we have observed that the optimal harvest date for P. tricornutum and T. suecica in standard cultivation conditions is 24 and 42 days, respectively. Comparison with nitrogen-deficient growth conditions is utilized as a model to confirm that LSCM and NR analysis can be used to study lipid storage and productivity for diverse growth conditions and various strains of microalgae.     

Size-modulated synergy of cellulase clustering for enhanced cellulose hydrolysis

Immobilization of enzymes onto nanoparticles for enhanced biocatalytic activity via enzyme clustering is a growing field. In this paper, the effect of nanoparticle size on the hydrolytic activity of artificial cellulosomes was investigated. A simple method based on metal affinity coordination was employed to directly conjugate two enzymes, an endoglucanase CelA and an exoglucanase CelE, onto CdSe–ZnS core–shell quantum dots (QDs) without the use of any chemical modification or linker molecules such as streptavidin. Artificial cellulosomes were created by clustering the enzymes onto two different QDs (5 and 10 nm) to systematically study the influence of particle size and QD to enzyme ratio on the enhancement in cellulose hydrolysis. Our results indicate that enzyme proximity is the most important factor for activity enhancement while the influence of particle size is relatively modest. This detailed understanding will provide insights for the design of other artificial cellulosomes based on nanoclustering of multiple catalytic domains with significantly enhanced activities, and may be applicable for designing improved nanobiocatalysts for biofuel production, bioremediation, and drug design.     

Laying the foundations for a bio-economy

Biological technologies are becoming an important part of the economy. Biotechnology already contributes at least 1% of US GDP, with revenues growing as much as 20% annually. The introduction of composable biological parts will enable an engineering discipline similar to the ones that resulted in modern aviation and information technology. As the sophistication of biological engineering increases, it will provide new goods and services at lower costs and higher efficiencies. Broad access to foundational engineering technologies is seen by some as a threat to physical and economic security. However, regulation of access will serve to suppress the innovation required to produce new vaccines and other countermeasures as well as limiting general economic growth.

Evaluation of halophilic microalgae isolated from Rabigh Red Sea coastal area for biodiesel production: Screening and biochemical studies

In the present study, different water samples from Red Sea coastal area at Rabigh city, Saudi Arabia were studied for their dominant algal species. Microalgal isolation was carried out based on dilution method and morphologically examined using F/2 as a growth medium. Dry weight and main biochemical composition (protein, carbohydrates, lipids) of all species were performed at the end of the growth, and biodiesel characteristics were estimated. Nannochloropsis sp., Dunaliella sp., Tetraselmis sp., Prorocentrum sp., Chlorella sp., Nitzschia sp., Coscinodiscus sp., and Navicula sp. were the most dominant species in the collected water samples and were used for further evaluation. Nannochloropsis sp. surpassed all other isolates in concern of biomass production with the maximum recorded dry weight of 0.89 g L^?1, followed by Dunaliella sp. (0.69 g L^?1). The highest crude protein content was observed in Nitzschia sp. (38.21%) and Dunaliella sp. (18.01%), while Nannochloropsis sp. showed 13.38%, with the lowest recorded lipid content in Coscinodiscus sp. (10.09%). Based on the growth, lipid content, and biodiesel characteristics, the present study suggested Dunaliella sp. and Nitzschia sp. as promising candidates for further large-scale biodiesel production.     

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.     

Ultrahigh bioproductivity from algae

The potential for dramatic increases in bioproductivity in algal photobioreactors relative to current biomass approaches, e.g., for converting sunlight into biofuels, by an unorthodox integration of photonics and biotechnologies is described. The key to greater biomass yields—projected as high as 100 g dry weight m⁻² h⁻¹—is a pronounced heightening of algal flux tolerance, achieved by tailoring the photonic temporal, spectral and intensity characteristics with pulsed light-emitting diodes. Such tailored photonic input is applied in concert with thin-channel ultradense culture photobioreactors with flow patterns that produce rapid light/dark algae exposure cycles. The artificial-light scheme is globally feasible only with electricity generated from renewables. Recent advances in ultra-efficient concentrator photovoltaics, as well as high-performance light-emitting diodes, create a practical reality for converting sunlight into pulsed red light and delivering it to indoor photobioreactors, with characteristic pulse times and intensities optimally suited to the rate-limiting dark reactions of photosynthesis. Cellular engineering built upon recent progress in modifying algal chlorophyll antenna size, in combination with metabolic engineering, could further enhance bioproductivity. The proposed strategy requires no major advances for implementation and adopts existing technologies. Revision submitted to Applied Microbiology and Biotechnology on 25 June 2007.     

Life cycle assessment of biodiesel production from microalgae in ponds

This paper analyses the potential environmental impacts and economic viability of producing biodiesel from microalgae grown in ponds. A comparative Life Cycle Assessment (LCA) study of a notional production system designed for Australian conditions was conducted to compare biodiesel production from algae (with three different scenarios for carbon dioxide supplementation and two different production rates) with canola and ULS (ultra-low sulfur) diesel. Comparisons of GHG (greenhouse gas) emissions (g CO₂-e/t km) and costs (¢/t km) are given. Algae GHG emissions (−27.6 to 18.2) compare very favourably with canola (35.9) and ULS diesel (81.2). Costs are not so favourable, with algae ranging from 2.2 to 4.8, compared with canola (4.2) and ULS diesel (3.8). This highlights the need for a high production rate to make algal biodiesel economically attractive.     

Microalgae biofuels: A critical review of issues, problems and the way forward

Culturing of microalgae as an alternative feedstock for biofuel production has received a lot of attention in recent years due to their fast growth rate and ability to accumulate high quantity of lipid and carbohydrate inside their cells for biodiesel and bioethanol production, respectively. In addition, this superior feedstock offers several environmental benefits, such as effective land utilization, CO(2) sequestration, self-purification if coupled with wastewater treatment and does not trigger food versus fuel feud. Despite having all these 'theoretical' advantages, review on problems and issues related to energy balance in microalgae biofuel are not clearly addressed until now. Base on the maturity of current technology, the true potential of microalgae biofuel towards energy security and its feasibility for commercialization are still questionable. Thus, this review is aimed to depict the practical problems that are facing the microalgae biofuel industry, covering upstream to downstream activities by accessing the latest research reports and critical data analysis. Apart from that, several interlink solutions to the problems will be suggested with the purpose to bring current microalgae biofuel research into a new dimension and consequently, to revolutionize the entire microalgae biofuel industry towards long-term sustainability.     

Dual purpose microalgae-bacteria-based systems that treat wastewater and produce biodiesel and chemical products within a Biorefinery

Excess greenhouse gas emissions and the concomitant effect on global warming have become significant environmental, social and economic threats. In this context, the development of renewable, carbon-neutral and economically feasible biofuels is a driving force for innovation worldwide. A lot of effort has been put into developing biodiesel from microalgae. However, there are still a number of technological, market and policy barriers that are serious obstacles to the economic feasibility and competitiveness of such biofuels. Conversely, there are also a number of business opportunities if the production of such alternative biofuel becomes part of a larger integrated system following the Biorefinery strategy. In this case, other biofuels and chemical products of high added value are produced, contributing to an overall enhancement of the economic viability of the whole integrated system. Additionally, dual purpose microalgae-bacteria-based systems for treating wastewater and production of biofuels and chemical products significantly contribute to a substantial saving in the overall cost of microalgae biomass production. These types of systems could help to improve the competitiveness of biodiesel production from microalgae, according to some recent Life Cycle Analysis studies. Furthermore, they do not compete for fresh water resources for agricultural purposes and add value to treating the wastewater itself. This work reviews the most recent and relevant information about these types of dual purpose systems. Several aspects related to the treatment of municipal and animal wastewater with simultaneous recovery of microalgae with potential for biodiesel production are discussed. The use of pre-treated waste or anaerobic effluents from digested waste as nutrient additives for weak wastewater is reviewed. Isolation and screening of microalgae/cyanobacteria or their consortia from various wastewater streams, and studies related to population dynamics in mixed cultures, are highlighted as very relevant fields of research. The species selection may depend on various factors, such as the biomass and lipid productivity of each strain, the characteristics of the wastewater, the original habitat of the strain and the climatic conditions in the treatment plant, among others. Some alternative technologies aimed at harvesting biomass at a low cost, such as cell immobilization, biofilm formation, flocculation and bio-flocculation, are also reviewed. Finally, a Biorefinery design is presented that integrates the treatment of municipal wastewater with the recovery of oleaginous microalgae, together with the use of seawater supplemented with anaerobically digested piggery waste for cultivating Arthrospira (Spirulina) and producing biogas, biodiesel, hydrogen and other high added value products. Such strategies offer new opportunities for the cost-effective and competitive production of biofuels along with valuable non-fuel products.