Sample Collection and Preparation: Both filtration and centrifugation were used to obtain highly concentrated samples of algae from various sources. Algae were col­lected from the effluent of the sewage treatment plant Virginia Initiative Plant (VIP), close to Old Dominion University (ODU), Norfolk, VA, from May, 2007 to February, 2008. The dominant algae at the VIP were Centric diatoms (10-30 mm), Scenedesmus/Desmodesmus spp., and Chlorella sp. Water from the wastewater facil­ity with entrained algae was passed through a tangential flow ultrafiltration system (Millipore Pellicon) with 0.2 mm filter, and the retentate, as condensed algal concen­trate, was collected. Total cell concentration and algae specification for each sample was examined under microscope [21] . To remove salts, the algal concentrate was centrifuged (6,500 RPM, 250 mL rotor, 30 min), and the supernatant was decanted and replaced with MilliQ water. This procedure was repeated twice. The final con­centrate was freeze-dried before chemical treatment. Algae were also collected from the ODU Algae Farm located in Spring Grove, VA. This farm, established in 2008, is an open pond design in which mainly Scenedesmus/Desmodesmus spp. algae are being grown and harvested. Algae at the ODU algal farm were concentrated by use of a continuous flow centrifuge (Lavin Centrifuge Model 12-413 V). The concen­trated algae, collected as a paste, were removed and lyophilized to dryness.

Conversion of Algae to Biodiesel in a Prototype Reactor: The TMAH method was adapted for the conversion of algae to biodiesel in a horizontal tube furnace reactor [16,17,26]. Approximately 1-2 g of algae sample was used in the prototype reactor (Fig. 1) . Algae samples were placed in a 30-mL glass culture tube with 1-2 mL of a TMAH in methanol solution (25 wt%). The methanol was evaporated to dryness under N2 and placed in the Thermo Scientific Lind-berg® Blue M reactor tube oven. The temperature was programmed to increase from 25°C to the desired temperature (250°C up to 550°C) over 15 min. The sample was held at final tem­perature for 45 min and the reactor was cooled to room temperature. An oxygen — free environment was created by using nitrogen gas flowing at a rate of approximately 100 mL/min. Gaseous vapors were collected and condensed in an ice bath. These condensed vapors were analyzed for FAME content by GC-MS.

Condenser

Biodiesel

Collected

Fig. 1 Schematic diagram of the prototype reactor. Reactor is designed for use with TMAH in a batch mode

I dentification by GC-MS: Samples were analyzed by an Agilent 6890 gas chromatograph interfaced to a Leco III mass spectrometer. The temperature was programmed from 50 to 300°C at a rate of 15°C/min. The mass spectrometer was repeatedly scanned in the low-resolution mode from 45 to 500 mass (u) at a rate of 20 spectra/s. Compounds were identified by their mass spectra. Most peaks were identified by comparison with the Wiley/NBS library and some were confirmed by comparison with standards. Quantitative measurements of the concentrations of individual peaks were made using an internal standard л-tetracosane as well as with external fatty acid methyl ester (FAME) standards. FAME standards were GLC-40, 50, and 90 standard mixtures (Supelco Analytical) containing fatty acid mixtures of

C16:0’ C180 C20:0, and C22:0; C16:1’ C18:1 ’ C20:T and C22:1; and C13:0, C150 C17:0, C19:0, and

C210; respectively.

Elemental Carbon (C) and Nitrogen (N) Analysis of Algae Residue: The carbon (C) and nitrogen (N) contents of algae residues were determined by a Thermo Finnigan Flash 1112 Elemental Analyzer using a nicotinamide standard for calibra­tion. Approximately 1-2 mg of each solid sample was placed in a 3.3 x 5-mm tin capsule for combustion. The method used for analysis was a furnace at 900°C, oven at 75°C, and carrier gas helium at 91 mL/min.

Solid-state 13C NMR: The NMR cross-polarization magic angle spinning (CPMAS) 13C experiments, with a recycle delay time of 1.0 s and 2,048 scans, were performed using a Bruker Avance II 400 spectrometer operating at 100 MHz for 13C and 400 MHz for 1H [18]. All the experiments were conducted using a 4-mm triple resonance probe. Dry algae and conversion residues were packed into a 5-mm Zirconia NMR rotor fitted with a Kel-F cap for analysis.