Current Biotechnology - Volume 5, Issue 2, 2016

Background: As interest in the use of eukaryotic microalgae as production platforms for biofuels and bioproducts increases, it is important that species-specific tools are made available. Species-specific molecular toolboxes will allow the engineering of improved performance to drive down manufacturing costs to overcome the economic thresholds for production of commercially competitive products. Historically, most genetic manipulation of eukaryotic microalgae focused on model algal strains such as Chlamydomonas reinhardtii, Chlorella sp., and Phaeodactylum tricornutum. The strong foundation built using model algal species now allows more rapid development of species-specific molecular tool boxes as novel microalgae-based products are identified in new algal strains. Customized tools for engineering improved production in strains that hold promise for fuels, foods, and high value chemicals will be a focus of future research. A snapshot of the current status of eukaryotic algal molecular engineering tools is provided plus data generated in our laboratory as we developed tools for manipulation of Auxenochlorella protothecoides. Methods: Current methods for manipulation of eukaryotic microalgae are reviewed and manipulation of Auxenochlorella protothecoides done in our laboratory is used to provide examples of some useful approaches. This paper broadly reviews all the methods currently in use or being developed to generate transgenic eukaryotic microalgae. Results: Provide original data on Auxenochlorella protothecoides for manipulation of the chloroplastic and nuclear genomes. Design of vectors and different approaches are discussed. Conclusion: Engineering improved strains of eukaryotic microalgae for economical production of biofuels and bioproducts is becoming a reality. Traditional insertional random mutagenesis is being augmented with new tools for directed strain improvement. The development and application of state of the art genome editing approaches to algal production strains has a real potential to help overcome economic thresholds and to enable microalgae as biorefineries to enter the marketplace alongside fossil fuels. The increased availability of sequenced genomes in combination with the reduced cost of generating proprietary genome sequences positions genetic engineering of eukaryotic microalgae at the threshold for rapid commercial application.

Background: Microfluidics allows manipulation of small volumes of fluids through channels with dimensions of tens to hundreds of micrometres. Microdroplet technology is a form of microfluidics in which small (10-200 μm diameter) monodispersed aqueous droplets are generated, manipulated and analysed in various ways. This multidisciplinary field provides an exciting new platform for single-cell studies of both eukaryotic microalgae and cyanobacteria, with considerable potential for enhancing algal biotechnology. Methods: Growth of several species of microalgae has been studied in detail using microfluidics and microdroplets, and individual cells have been screened and sorted according to lipid content or ethanol production. Here we provide an overview of the devices, and the range of technological advances that are being pursued. Conclusion: Microdroplet technology is an emerging technology platform that can be used in a variety of applications, including monitoring of growth characteristics at the single-cell level and high-throughput screening of algal populations. Microdroplet platforms are being developed that will allow determination of individual cell characteristics to allow screening across a population, and thus to identify and select candidate cells for biotechnological feedstocks. As the potential of this emerging technical platform is recognized, the technology will become more accessible, so that it can soon be adopted and used by researchers, without the need for specialized prior knowledge of microfluidics or expensive equipment. The platform is amenable for use with species of both microalgae and cyanobacteria.

Background: The consideration of ecological principles has brought new ideas that can be implemented to achieve sustainable production of algal biomass at the commercialscale. In particular, the key ideas of top-down control of algal pests and the potential advantages of diversifying algal crops have encouraged researchers to explore foodweb interactions in algal biomass cultivation platforms, and to investigate the characteristics of algal strains that could be used to assemble designed, multi-species consortia that outperform algal monocultures. Objective/Methods: To explore the practical applications of top-down control of algal pests and algal crop diversification, this paper reviews literature on agricultural and aquatic systems with consideration of the implementations of these ecological principles in managing commercial-scale algal cultivation. Results: Our review suggests that careful management of food-web structure in algal cultivation platforms will be needed to maximize crop protection, and that temporal and spatial diversification of algal crops may also benefit industrial algal biofuels production. Extensive domestication and genetic improvements of algal strains are currently underway worldwide, and we suggest that careful selection of endogenous algal community which proliferates under selective environmental condition has the potential to engineer algal communities of high commercial interest. Conclusion: Overall, our review suggests that the careful management of food-web structure and algal crop diversity, as well as experiences and insights from modern agriculture can be used to guide the design and operation of industrial-scale algal biomass production systems. We urge thorough experimental tests of these ideas in both laboratory and field settings.

Background: Algaculture, like any form of agriculture, is highly sensitive to fertilizer or nutrient costs. A major roadblock to commercial algaculture is efficiently utilizing volatile nutrients, specifically carbon dioxide and ammonia, to feed microalgal cultures. These nutrients are typically plentiful in multiple agricultural and industrial waste streams and can potentially provide fertilizer or nutrients for large-scale microalgal cultivation. However, individual methodologies need to be developed for each type of waste stream to remove uncertainty in the assumption of yield and cost of production in microalgae cultivation systems. Methods: This review focused on the use of waste nutrients used for cultivation of microalgae, specifically nutrients that are currently being underutilized or wasted. Those nutrients, carbon dioxide and ammonia, require special capture and/or handling steps in order for them to be used for microalgae cultivation. A variety of techniques, using carbon dioxide for pH control, mixing sumps, sparging, controlled dosing techniques and pond covers represent approaches used for handling and maximizing the use of available carbon dioxide and ammonia for microalgae cultivation. Results: Efficient nutrient recycling has been shown to reduce fertilizer input costs, although this is highly dependent on the specific processes being employed, and the nutrient utilization efficiency. This review summarizes efforts to quantify and improve carbon dioxide and ammonia utilization in microalgal cultivation and to reduce overall volatilization and loss of these valuable nutrients. Practical methods of preventing volatilization and increasing gas transfer to the microalgae crop included: pH control, nutrient dosing and improving gas transfer efficiency. Conclusion: Finding an economical nutrient source to drive microalgal biofuel production remains a key challenge. Wastewaters represent a relatively untapped source of nutrients for microalgae cultivation. Key nutrients in wastewaters are volatile (carbon dioxide and ammonia), potentially toxic, and require special handling approaches such as balancing pH, temperature, contact time, and contact surface area. Preliminary results suggest that these volatile nutrients can be effectively utilized for microalgal cultivation.

Background: Previous studies have shown the positive effect of genetically induced antenna truncation on the photosynthetic performance of mutant strains (tla). From the increase of maximum photosynthesis rates per unit chlorophyll under saturating irradiance, it has been concluded that photobioreactors with tla mutants could be operated at higher cell numbers and should show increased biomass production under high irradiance. Objective: The potential of three wild type (WT) strains and one tla mutant of Chlamydomonas reinhardtii to adjust cellular Chlorophyll content (Chlcell) in response to different light conditions and its consequences for the cell physiology was investigated. Results: Under high growth irradiance, some WT strains can achieve a comparable level of antenna reduction as observed in the tla mutant. Respiration and maximum photosynthesis rate showed an inverse correlation with Chlcell independently of whether antenna size reduction was achieved by light adaptation or by genetic transformation. Thus, under diurnal light conditions increased respiratory losses compensate for increased photosynthesis rates and exert negative impact on biomass production. The reduction of Chlcell inevitably resulted in an increase of the absorption efficiency of Chl. This implies that cell number and transparency in a bioreactor cannot be increased to a similar extent as expected from Chlcell reduction. Conclusion: Under natural light conditions, the negative impact of increased respiration and absorptivity of antenna truncated cells has to be taken into account to judge the biomass production potential of antenna truncated alga strains. A further reduction of antenna size beyond the current degree could even decrease biomass productivity. Alternative approaches to increase biomass productivity of photobioreactors are suggested. Keywords: antenna truncation, tla mutants, biomass production, Chlamydomonas reinhardtii

Background: This study was completed as part of a larger effort to develop emulsions that have high stability for long-term algal cell storage and that release cells rapidly when applied to a pond surface as inoculum. Past research has shown that including silica nanoparticles in the oil phase yields stable emulsions but that cell release upon application takes place on the order of days. Methods: Water-in-oil (W/O) emulsions were prepared with Chlorella sorokiniana and brilliant sulfoflavine (BSF) in the internal phase to investigate the effects of oil phase thickeners on BSF and cell release upon spray application of the emulsion to a water surface. Three oil phase thickeners: Aerosil R974, Bentone 38, and Bentone 150, were tested at concentrations ranging from 0.25-1% (w/w) in the oil phase. Results: The oil phase thickeners had a modest impact on the release of BSF but no significant effect on cell release. In particular, emulsions with Bentone 150 had significantly faster release of BSF than other thickeners. Spray application of the emulsion onto a water surface, however, had a dramatic effect on cell release rate without negatively affecting cell viability. Average spray droplet size was found to be ~45 μm and nearly 100% release of both cells and BSF could be achieved within ~30 minutes. Conclusion: Spray application resulted in rapid release of algae and dye even for emulsions with a high concentration of silica nanoparticles. The fact that thickeners affected the release rate of soluble BSF but not cells suggests the potential to design emulsions with differential release rates of cells and compounds upon spray application.

Background: Microalgae have been identified as potential biodiesel feedstock because of their high lipid and biomass productivity. The present work was aimed to the mass culture of microalgae in a shallow pond and circular tank under open-air condition and to determine the productivity of biomass, lipid, and fatty acid methyl esters. Methods: Chlorella vulgaris (GenBank: KF937218.1) isolated from the offshore water of Visakhapatnam along the East coast of India and Platymonas convolutae obtained from CMFRI, India, were mass cultured in a shallow pond and circular tank under open-air conditions. Outdoor mass cultivation was carried out for both the species in shallow pond and circular tank. The daily productivity and growth kinetics were calculated. Results: C. vulgaris had the highest biomass concentration (0.98 g l-1) with 21% of the lipid content in the circular tank culture. The maximum aerial productivities of 42 g m2 d-1 and 32.1 g m2 d-1 were recorded for C. vulgaris and P. convolutae, respectively. Fatty acid profiles showed the presence of C14:0, C16:0, C16:1, C18:0, C18:1, C18:2, and C18:3. FAME yield determined via different extraction methods was also reported. Conclusion: The results suggest that the coastal area of Visakhapatnam is a feasible location for the production of biomass via large-scale mass culture in an open-air shallow pond. Fatty acid profile showed the feasibility production of biodiesel.