Microalgae produce large quantities of lipids that can be used for biofuel feedstock. The goal of this project was to determine the effect of several engineering and management parameters on the productivity of microalgae cultivated in large, outdoor facilities. The specific objectives were focused on the effects of inoculation ratios; the effects of light, temperature, and culture depth on growth; and the minimum circulation velocity necessary to maintain growth and minimize settling in open ponds. Microalgae must first be cultured in smaller quantities before the raceway is inoculated for optimized growth. Concentration ratios are defined as the ratio of the volume of microalgae inoculum to the volume of new growth media. The microalgae species used was Nannochloris oculata (UTEX #LB 1998). Inoculation ratios studied varied from 1:1 to 1:32 and were grown in 500 mL Erlenmeyer flasks. The study found that 1:16 and 1:32 were too dilute, while the 1:8 concentration had the largest growth rate. Determination of the effects of temperature, light intensity, and cultivation depth is critical to maintaining healthy cultures. Excess light intensity can result in photoinhibition and temperatures above the maximum growing tolerance can have detrimental effects. These factors can affect growth and evidence suggests an interaction that exacerbates these effects. In an outdoor culture there are few practical control variables other than pond depth. As cultivation depth increases, the algae undergo "selfshading" and the increased cultivation volume hinders temperature changes. Scaled raceway ponds were maintained at 10.16 cm (4 inch) and 13.97 cm (5.5 inch) depths. The species used was Nannochloris oculata and it was found to grow best at 785 micromol m⁻² s⁻¹m^-2 s^-1, 20°C, and 10.16 cm. Diatoms are attractive because of high growth rates, faster lipid production, and greater cell density. The latter promotes rapid settling once mixing has stopped. Because of the silica cell wall composition, diatoms are believed to be more susceptible to shear forces which can result in lysis. Determining the natural settling rate to the minimum channel velocity relationship in cultivation ponds was the objective. No flocculants/coagulants were added which created a case of "natural" settling. Four pennate Nitzschia sp. and one centric diatom were tested in a jar tester. There was no significant difference in settling times between the species. The mean settling time was 4.55 cm min⁻¹ and the minimum channel velocity was determined to be 10.12 cm min⁻¹.

America's reliance on foreign oil has raised economic and national security issues, and in turn the U.S. has been active in reducing its dependence on foreign oil to mitigate these issues. Also, the U.S. Navy has been instrumental in driving bio-fuel research and production by setting an ambitious goal to purchase 336M gallons of bio-fuel by 2020. The production of microalgae biomass is a promising field which may be able to meet these demands. The utilization of microalgae for the production of bio-fuel requires the implementation of efficient culturing processes to maximize production and reduce costs. Therefore, three discrete rate event simulation models were developed to analyze different scaling scenarios and determine total costs associated with each scenario. Three scaling scenarios were identified by this analysis and included a stepwise, volume batching and intense culturing process. A base case and potential best case were considered in which the culturing duration, lipid content and lipid induction period were adjusted. A what-if analysis was conducted which identified and reduced capital and operational costs contributing greatly to total costs. An NPV analysis was performed for each scenario to identify the risk associated with future cash flows. The research findings indicate that the intense culturing scaling scenario yielded the greatest model throughput and least total cost for both the base case and potential best case. However, this increased productivity and cost reduction were not significantly greater than the productivity generated by the stepwise scaling scenario, suggesting that the implementation of flat plate bio-reactors in the intense culturing process may be non-advantageous given the increased operational costs of these devices. The volume batching scenario yielded the greatest total cost L^-1 of microalgae bio-oil for both, indicating an inefficient process. The scaling scenarios of the base case and potential best case yielded negative NPV's while the stepwise and intense culturing scenarios of the what-if analysis generated positive NPV's. The base case is based on current technological advances, biological limitations and costs of microalgae production therefore, a negative NPV suggests that utilizing microalgae for bio-fuel production is not an economically feasible project at this time.

Unicellular microalgae are a renewable bioresource that can meet the challenge forfood and energy in a growing world population. Using sunlight, CO2, nutrients,and water, algal cells produce biomass in the form of sugars, proteins and oils, allof which carry commercial value as food, feed and bioenergy. Flue gas CO2 andwastewater nutrients are inexpensive sources of carbon and fertilizers. Microalgaecan mitigate CO2 emissions and reduce nutrients from waste streams whileproducing valuable biomass.My focus was on some of the challenging aspects of cultivating microalgae ascrop: the response of biomass production and quality to seasonality, nutrients andbiological interactions. Approach spans from laboratory experiments to large-scaleoutdoor cultivation, using single microalgal strains and natural communities insouthern (Portugal) and northern (Sweden) Europe.Half of the seasonal variation in algal oil content was due to changes in light andtemperature in outdoor large-scale cultures of a commercial strain (Nannochloropsisoculata). Seasonal changes also influence algal oil composition with more neutrallipids stored in cells during high light and temperature. Nitrogen (N) stress usuallyenhances lipid storage but suppresses biomass production. Our manipulationshowed that N stress produced more lipids while retaining biomass. Thus,projecting annual biomass and oil yields requires accounting for both seasonalchanges and N stress to optimize lipid production in commercial applications.Baltic Sea microalgae proved to be a potential biological solution to reduce CO2emissions from cement flue gas with valuable biomass production. A multi-speciescultivation approach rather than single-species revealed that natural or constructedcommunities of microalgae can produce equivalent biomass quality. Diversecommunities of microalgae can offer resilience and stability due to more efficientresource utilization with less risk of contamination, less work and cost for culturemaintenance.Stable algal biomass production (annual basis) was achieved in outdoor pilot-scale(1600 L) cultivation of Baltic Sea natural communities using cement flue gas as aCO2 source. Results indicate favorable algal oil content at northern Europeanlatitudes compared to southern European latitudes.My thesis establishes the potential of cultivating microalgae as a bioresource inScandinavia, and using a community approach may be one step towardssustainable algal technology.

Continuous culture fermentation (CCF) was used to test the hypotheses that: marine microalgae (MA) and macroalgae (seaweeds) alter rumen microbial metabolism; MA types differ in abilities to provide rumen escape n-3 polyunsaturated fatty acids (PUFA); and algae have the potential to reduce enteric methane emission. The CCF system of Teather and Sauer (1988) was modified to reduce clogging, refrigerate effluent, and allow for determination of gas production. The CCF systems were inoculated with pooled rumen fluid from 4 cows. Total mixed ration was fed at the rate of 30 g DM d-1. Temperature was maintained at 39 oC, and buffered with artificial saliva to maintain pH 6.2. Response variables were measured from effluent digesta (fatty acids, NH4+-N, digestibility), fermentor contents (CCF density, volatile fatty acids), and the gas phase (CO2, CH4). The experimental design for MA testing was a 3**3 factorial. Treatments consisted of heterotrophic and photoautotrophic MA as well as a 1:1 blend with protection levels of zero, 33 and 50 % of encapsulation (w/w), and fluid turnover rates of 5, 7.5, and 10 % h-1. The seaweed treatments consisted of a PEI shoreweed mix containing Laminaria longicruris and Fucus vesiculosus tested as a component of the mix, and Chondrus crispus and Furcellaria fastigiata tested individually. The design for seaweed testing was an unbalanced 5*5 Latin square. The heterotrophic MA destabilized the digesta mat while the autotroph improved stability. Biohydrogenation was extensive for C18 FA in the basal ration (> 90 %) and less for C22:6n3 (75 %) from the heterotroph and C20:5n3 (60 %) from the photoautotroph. The recovery of PUFA was improved by encapsulation, however PUFA in the MA were not greatly affected and digestibility was improved by turnover rate. Seaweeds had no effect on CCF stability, however they reduced CH4 production without reduction in OM digestibility. The heterotroph reduced overall fermentation resulting in diminished density and volatile fatty acids and NH4+-N concentrations. Seaweed supplementation decreased NH4+-N, CO2 and CH4 production, and increased density.

This dissertation investigates harvesting of marine microalgae for bioenergy and production of two recombinant proteins for therapeutic applications in Chlamydomonas reinhardtii. The first study describes harvesting of marine microalgae by flocculation using aluminum chloride (AlCl_3), natural polymer chitosan, and synthetic cationic polymers. Harvesting and concentration process of low concentration microalgae cultures ranging from 1 to 2 g dry weight per liter was affected by algogenic organic matter (AOM), ionic strength, cell concentration, polymer charge density, and media pH. Marine microalgae flocculation was greatly affected by the presence of AOM independently of the flocculant chemistry. Presence of AOM demanded extra flocculant dosage i.e., 3-fold of AlCl3, 7-fold of highly charged synthetic cationic polymer, and 10-fold of chitosan. Flocculant dosage required for > 90 % flocculation efficiency in the presence of AOM was 160 mg/L, 50 mg/L, and 20 mg/L when using AlCl_3, chitosan, and best (more efficient) synthetic polymer respectively. The high-ionic strength of saline water did not have a significant effect on flocculation efficiency when using AlCl_3. However, to achieve efficient algal biomass removal, application of highly-charged synthetic polymers was required to overcome the presence of electrolytes. The best synthetic cationic polymer tested herein, which achieved greater than 90 % flocculation efficiency at 20 mg/L dosage, was a polymer with 99 % cationic charge density. Cell concentration also affected flocculant dosage requirement; low density cultures (10^6 cells/mL) required 6-fold greater dosages than cultures grown until early stationary phase (10^7 cells/mL). The second study addresses cultivation, extraction and purification challenges of two complex recombinant proteins, an immunotoxin molecule (MT51) and malaria vaccine antigen (Pfs25) produced in the chloroplast of C. reinhardtii. Main challenges identified were i) low transgene expression level, ii) proteolytic instability of MT51 immunotoxin, and iii) aggregation of Pfs25 antigen. Optimal expression and accumulation of Pfs25 antigen required growing C. reinhardtii cultures to late exponential phase (10^6 cells/mL) and inducing transgene expression for 24 h at a photon irradiance of 120 µmol/m^2s.

Thesis (M.Tech.:Biotechnology)-Dept. of Biotechnology, Durban University of Technology, 2007 xvi, 185 leaves / Spirulina is a blue-green, multicellular, filamentous cyanobacterium that can grow to sizes of 0.5 millimetres in length. It is an obligate photoautotroph and has a pH growth range from 8.3 to 11.0.The large-scale production of Spirulina biomass depends on many factors, the most important of which are nutrient availability, temperature and light. These factors can influence the growth of Spirulina and the composition of the biomass produced by changes in metabolism. Brine effluent from cooling towers of electricity generating plants may provide an ideal growth medium for Spirulina based on its growth requirements, i.e. high alkalinity and salinity. The aim of this research was to optimise brine effluent from cooling towers by supplementing it with salts, in order to use this optimised effluent in a small open laboratory raceway pond in an attempt to increase the biomass production of Spirulina.

Algae are primary producers in aquatic ecosystems and are thus the most important organisms in maintaining ecosystem functioning and stability. The usage of algae by humans is quite extensive; they act as an ingredient in aquaculture feed, a potential biomedical resource, as a fertiliser and as a nutritional source. Recently, algae have been identified as a third generation biofuel feedstock for fuel generation which essentially means that algae are more efficient, net carbon neutral and have less impacts on the environment. Algae as organisms are extremely sensitive to changes in the immediate environment. The interaction of parameters with each other causes minute changes in the environment which may alter the algae biomass present and the lipids that can be extracted from the biomass. The focus of this study is to model and determine which conditions maximise algal biomass and the subsequent lipids that can be extracted from the biomass. This will allow biofuel producers to understand which conditions are the best for harvesting algae in artificial conditions or harvesting algae from the wild. Furthermore, the model developed has broad application for biofuel specialists, pollution remediation specialists and biologists. This model developed is able to determine the present state of the algal bloom and uses the present state to predict the future state of bloom hence determining the optimal conditions to harvest. The model was developed under optimal ranges described by the Food and Agriculture Organisation (FAO) and designed to replicate the most common combinations of parameters present in the wild. For the purposes of this study, various combinations of parameters within their optimal ranges that is temperature (18 – 24°C), salinity (20 – 24 p.p.t.) and photoperiod (25 – 75% light exposure) were assessed. The model was run for 72 hours with sampling every 6 hours. Every six hours, algal growth was measured by the biomass present (chloro-pigments used as estimators); this was done by fluorescence. Lipids were then extracted from algal biomass using the Bligh and Dyer method (1959). Spline curves were fitted to the data and analysis performed using Mathematica 8.0. It was found that photoperiod was the most important variable in controlling algal growth. Furthermore, lipids extracted from biomass were at their highest when algae were exposed to the conditions 75% light exposure, 21°C and 22 p.p.t. These conditions would allow for the highest amount of biofuel to be produced. Generally, algae biomass trend graphs mimic lipid trend graphs over the 72 hour period that is when lipids are at their maximum, biomass concentrations are at their maximum. It can be concluded from time model that the best time to harvest biomass is 48 hours from the initial start time of algal growth to gain the highest amount of lipids for biofuel production. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2012.

Fast pyrolysis, the rapid thermal decomposition of organic material in the absence of oxygen, is a process that can be used to convert biomass into liquid fuels and chemicals. When performed at the micro-scale, pyrolysis is useful for characterizing biomass structure, as well as determining the pyrolysis products that can be generated from specific biomass feedstocks. Indeed, microscale pyrolysis coupled with on-line analysis of the pyrolysis vapors by GC/MS, so-called pyrolysis-GC/MS (Py-GC/MS), is a technique that can be used to characterize the structure and composition of the various components of lignocellulosic and microalgal biomass based on their pyrolysate distributions. Pyrolysates produced also provide insight into the range of products that can be expected when biomass feedstocks are subjected to thermal decomposition processes. This dissertation focuses on the Py-GC/MS analysis of lignocellulosic biomass such as sorghum and Scenedesmus sp. microalgae, in addition to high-lignin feedstocks such as walnut shells, coconut shells, olive pits and peach pits. The differences in the pyrolysate distributions among these biomass types are correlated with differences in the structure and composition of the biopolymers, mainly cellulose, hemicellulose and lignin, present in the biomass. Py-GC/MS analysis of lignin extracted from endocarp feedstocks is also emphasized. In addition to biomass and extracted lignin, sinapyl (S) and coniferyl (G) alcohol have been analyzed by Py-GC/MS in order to understand the relationship between the corresponding pyrolysates and sinapyl/coniferyl ratios of lignin present in lignocellulosic biomass.

This study was conducted in Esquimalt lagoon, located southwest of Victoria, British Columbia, Canada. Physical characteristics of the water column, e.g. circulation and stratification, changed seasonally resulting from variations in tides, temperature, precipitation and wind. Chemical characteristics, e.g. oxygen and dissolved nutrient concentrations, also differed temporally relative to those in the lagoon’s ocean source water (Juan de Fuca Strait) because of variations in local photosynthesis and nutrient use by phytoplankton. Diatom blooms occurred in the spring, and blooms of photosynthetic flagellates (Heterosigma akashiwo (2009) and Akashiwo sanguinea (2009 and 2010)) occurred in the late summer and early fall when nitrate, ammonium, and urea were depleted. Proliferation of these flagellates led to the development of harmful algal blooms (HABs) associated with oxygen depletion in the lagoon bottom waters. Increased oxygen demand from bacterial degradation of algal biomass and exudates was the likely cause for bottom water hypoxia under reduced tidal exchange. / Graduate

Immobilization of photosynthetic cultures has been used to generate biofuels and high value compounds through direct conversion of CO2 and water using sunlight. Compared with suspended cultures, immobilized bacteria can achieve much higher densities resulting in greater areal productivity. Limitations exist however, on the density that can be reached without compromising access to light and other nutrients. In this thesis an optofluidic approach to overcoming the challenge of light delivery to high density cultures of cyanobacteria is described and proof of concept experiments presented. This approach uses optical waveguides to deliver light to cells through bacterial interaction with the evanescent field and is tailored to meet each cell's need for light and nutrients. Experiments presented here demonstrate biofilm proliferation in the presence of evanescent fields. Illumination of surfaces by surface plasmon enhanced evanescent fields is also shown to be an effective and potentially useful technique to grow biofilms within optofluidic architectures.