The biodiesel in this study is produced by conversion of dry microalgae biomass from the VIP using a one-step thermochemolysis reaction into an open pyrolysis prototype reactor. The biomass may be used partially wet, but Hatcher and Liu rec­ognized that drying increases the yield of FAMEs [15] . During this reaction, the transesterification occurs at a temperature sufficient to hydrolyze and alkylate trig­lycerides in the biomass [15] . This process is possible because the association of TMAH and methanol provides a single step high-temperature saponification and methylation of the ester functional groups of the triglycerides [11]. It is also likely that a transesterification occurs at lower temperatures (approximately 100°C) to produce the FAMEs, much like what is observed with alkali like sodium hydroxide [12] as long as there is residual methanol to act as a transesterification reagent. Using temperatures above 100°C and evaporating the methanol before insertion of the reaction mixture into the reactor created conditions that minimized the transesterification product at low temperatures. The produced FAMEs become vola­tile at reaction temperatures above 250°C and can be condensed at the exit of the reactor. To establish the optimum reaction temperature, multiple assays at tempera­tures ranging from 250 to 550°C were used. The volatile products trapped in an ice bath were collected and analyzed by GC-MS in order to identify and quantify the individual FAMEs and other products for each assayed reactor temperature.

The total ion chromatograms (TIC) for VIP algae assayed at 250 and 450°C are shown and peak identifications and their concentrations are reported (Fig. 2 and Table 1). Mostly linear saturated and unsaturated FAMEs are detected in the chro­matogram. The dominant peaks are fatty acids, typical of biodiesel, containing C140, C160, C161, and C181 corresponding to peaks 1, 6, 5, and 9a, b in the table [8].

It is also worth noticing that both chromatograms of biodiesel produced at 250 and 450°C are very similar showing that the conversion of triglycerides to biodiesel using TMAH/MeOH reagent does not change its selectivity for FAMEs with respect to temperature. A similar pattern was observed at all assay temperatures. Minor amounts of other compounds are observed in the products.

All of the peaks correspond to FAMEs except peaks 4, 8, and 11 which are respectively ascribed to a C20 isoprenoid hydrocarbon, a lactone, and dodecanamide. The isoprenoid hydrocarbon probably is produced from chlorophyll and the dode- canamide from reaction of ammonia with triglycerides in the reaction chamber.

The yields of FAMEs are summed for each assay temperature along with an estimate of the standard deviation of the analysis for multiple assays at each tem­perature (Fig. 3). A yield of approximately 3% (±0.3%) is achieved at 250°C for the VIP algae. The highest yield of FAMEs, approximately 4% (±0.8 and 0.6%) of the dry biomass, is achieved at 350 and 450°C. At higher temperature, a slight decrease in yield of FAMEs is observed with a yield below 3% (±0.2%) at 550°C. Total lipid content obtained by Bligh-Dyer extraction was found to be on average 12% (±3.6%) from Scenedesmus spp., which is the dominant species from the VIP algae, and similar to reported values [3, 6, 31]. The lipid extract is determined by gravimetric

yield and includes triglyceride lipids as well as other extractable organic and inorganic materials. The TMAH procedure only converts the triglycerides and free fatty acids to methyl esters, and we can determine that approximately 30-40% of the total extract is converted to FAMEs. Samples run at each temperature range show standard

deviation overlap of percent yield. Though a slightly higher yield is observed for algae run at 350 and 450°C, they are not significantly different from those run at the lower temperature of 250 and higher temperature of 550°C. The effect of tempera­ture difference, however, is apparent in the algae residue after biodiesel collection.

The algal residue obtained following the thermochemolysis may potentially be useful as a fertilizer and even as an animal feed. Our initial goal was to examine its chemical composition for possible future evaluation as these end uses. Raw algae in coastal regions, such as seaweeds, have a long history of use as soil conditioners and fertilizers [14]. Seaweed composting, however, presents some problems such as high salinity and excessive sand content that can limit plant growth and develop­ment [10 ] . Using algal residue produced in our thermochemolysis reactor from freshwater algae would potentially eliminate these problems. The carbon and nitro­gen content of algae and algal residues are collected at different temperatures (Table 2). The algal residue collected at 250°C had an average carbon of 46.67% and nitrogen of 5.83%. Both carbon and nitrogen values decreased slightly with increasing temperatures. The C/N ratios, however, were highest at a temperature of 450°C with a value of 9.41. Further testing is being conducted to verify the use of algae and algae residue as an organic fertilizer.

The solid-state CPMAS 13C NMR spectra for whole algae and residue collected at various temperatures are displayed (Fig. 4). Whole algae samples collected both

200 50

Fig. 4 13C solids NMR (a) whole algae VIP, (b) whole algae Scenedesmus/Desmodesmus spp. from algae farm Spring Grove, VA, (c) VIP algae residue from reactor at 250°C, (d) VIP algae resi­due from reactor at 450°C from the VIP and the algal farm are shown. Dominant species in the VIP samples were microscopically identified as pennate diatoms and in the algae farm samples as Scenedesmus/Desmodesmus spp. The NMR spectra of the two algal collections are quite similar, being dominated by strong signals in the 0-60 ppm region, repre­senting lipid-like aliphatic carbons and proteins. Carbohydrates, characterized by signals between 60 and 105 ppm, are subordinate components of the spectra indicat­ing that they constitute a small fraction of the algal biomass. Peaks in the region for aromatic/olefinic carbons (105-160 ppm) are also subordinate, reflecting aromatic amino acids comprising proteinaceous components of the algae and olefinic struc­ture contained in the lipids. The large peak at 175 ppm is assigned to amide and carboxyl groups, structural components of both lipids and proteins.

When the algal samples are treated with TMAH at different temperatures, some significant changes are observed in what remains as a residue. The spectrum shown has many of the same signals as the original algae, but contains significantly more olefinic/aromatic carbon (Fig. 4c). This is most likely an indicator that the 250°C heating has transformed the algae such that increased aromatization occurs. The transformation, however, is not overly drastic, suggesting that this residue probably has sufficiently preserved structures to be used as a fertilizer. It is well known that
heating materials containing carbohydrates and proteins (algae) together produces furanosic materials often referred to as melanoidins [2, 20].

The diminution of carbohydrates (60-105 ppm) and portions of the proteina­ceous signals (NCH at 50 ppm) observed in this residue is consistent with the involvement of carbohydrates and proteins to form these aromatic substances. The presence of a peak at 160 ppm (assigned to the O-bearing aromatic carbons), along with a broad signal in the range between 105 and 150 ppm, is suggestive of furans, but also could be derived from heterocyclic N rings such as indoles or pyrroles. Usually, a signal at 160 ppm is assigned to phenolic substances that are commonly found in lignin. We know that lignin is not present within algae, so the emergence of this peak at 160 ppm can only be rationalized as that derived from furans or heterocyclic N. Heating to a temperature of 450°C causes drastic changes in the structure of the residue, which shows signals mainly like charred material. The high aromatic content and minor aliphatic content is suggestive of extensive polymeriza­tion into condensed aromatic moieties. We can speculate that the furans forming from carbohydrates might undergo cyclization and aromatization and eventual for­mation of dibenzofurans. The protein-derived heterocyclic N might probably undergo increased aromatization to structures similar to carbazoles. Interestingly, the C/N ratio for this material (9.41) coupled to the high aromatic content are suggestive of the fact that a significant amount of N-containing structures are embodied within aromatic units. Use of this material as a fertilizer where the N can be released via decomposition is unknown at this time.