## 2 Results and Discussion
### Algae Are Harvested and Concentrated onto Amberlite Anion Exchange Resin
After washing the column with distilled water, the operation was repeated for three more cycles, eluting with either fresh methanol sulfuric acid reagent (fresh) or reusing the previously used reagent (used). The binding capacity of the resin was determined for each cycle of algal loading onto the resin. **Figure 2.** FAME yield and characterization by resin-bound transesterification. **(a)** FAMEs from normal or stressed (nitrogen-starved) _Neochloris_ and KAS 603 were generated either by elution of resin-bound algae with 5% sulfuric acid/methanol reagent or by subjecting dry algal pellets subjected to sequential base hydrolysis followed by treatment with sulfuric acid in methanol to esterify free fatty acids. The FAME yields were expressed as percent of dry cell weight. The weight of a crude lipid extract from parallel batches of algae and the TAG content those extracts (determined by HPLC) are given for comparison. The FAME prepared from resin-bound algae were analyzed by HPLC/MS to determine the abundance of **(b)** saturated C16:0, C18:0, and C20:0 and **(c)** unsaturated C18:3 and C21:4 acyl constituents relative to total FAME derived. Figure 3: Efficiency of FAME production using recycled reagent, and FAME removal. **(a)** Algal were loaded onto Amberlite and eluted with 5% sulfuric acid/methanol four times in succession using either fresh reagent or reusing the old reagent after the initial elution. Twelve h after each elution the FAME was extracted with hexane, solvent was removed and the amount of FAME determined as a percentage of algal dry cell weight; **(b)** The sulfuric acid/methanol reagent containing algal FAME was either extracted with hexane or, for comparison, passed over a hydrophobic resin column composed of 80% EGDMA and 20% HMA; **(c)** FAME relative to total dry algal weight obtained by hexane extraction or after eluting FAME bound to the hydrophobic column. The results showed that the binding capacity for _Neochloris_ was 37.0 mg/g resin whereas that for KAS 603 was 12.8 mg/g resin [Figure 1(b)]. In terms of efficiency of removal of algae from 1 L of 0.4 g/L suspension, 10 g of Amberlite will sequester 93% of the _Neochloris_, whereas 30 g of Amberlite will sequester 96% of the KAS603 during a single pass through the column. Although the difference in binding capacity could possibly be attributed to differences in surface charge density, studies to be detailed elsewhere show that the nature of the resin backbone, especially its hydrophobicity has a large impact on binding capacity [25]. In resins based on a methacrylate backbone, both _Neochloris_ and KAS 603 showed similar binding capacities. Furthermore, binding capacity was greatly increased with the inclusion of glycol groups, which do not alter the charge characteristics of the resin but make it more hydrophilic. Figure 4: Functionalized resins for improved binding of KAS603. The relative binding capacity of Amberlite for KAS 603 is compared to anion exchange resins composed of either (**a**) EGDMA-IM-DEG (60:30:10) or (**b**) DVB-DMA (60:40); (**c**) The results show that the binding capacity of Amberlite is low compared to the other resins; (**d**) In addition to binding more algae, subsequent elution of either Amberlite or DVB-DMA shows that greater amounts of FAME are produced from the DVB:DMA resin. After binding, algae were eluted with 100 mL of 5% sulfuric acid in methanol. This treatment visibly removed algae from the resin and regenerated the resin. It appears that most of the algae was dissolved by the sulfuric acid/methanol reagent as shown by filtering an eluate of _Neochloris_ through a pre-weighed PTFE filter (Millipore Omnipore, pore size 0.1 \(\upmu\)m). Only 6.1% of the original DCW was recovered on the filter. In order to determine how completely the resin was regenerated, resin columns were loaded with _Neochloris_ [Figure 1(c) \"fresh\"] or KAS 603 [Figure 1(d) \"fresh\"] and eluted with fresh portions of sulfuric acid/methanol four times in succession.