Cultures
Three strains of benthic cyanobacteria were used in this study; all are now present in and are publically from the Cawthron Institute Culture Collection of Microalgaea. Phormidium autumnale, CAWBG26, was isolated from the Hutt River, Wellington, New Zealand and is a common species found throughout New Zealand rivers. During stable flow conditions, Ph. autumnale mats proliferate, at times forming expansive black/brown leathery mats across large expanses of river bed (Heath et al. 2010). Phormidium murrayi, CAWBG22, was isolated from a small tarn in the Red Hills, Nelson, New Zealand, where it formed extensive mats. Prior to this identification, Ph. murrayi was known only from Antarctica (Heath et al. 2010). The final strain, CAWBG59, was isolated from a benthic mat in the Waitaki River, Christchurch, New Zealand, and has tentatively been identified as a Planktothrix sp. (Wood et al. 2010).
Growth experiments
Aliquots (4.2-4.8 mg wet mass) of each strain were added to 50 mL clear polystyrene bottles (pottles) (Biolab, New Zealand) containing 30 mL of MLA medium (Bolch and Blackburn 1996). For each strain 15 pottles were set up, enabling collection in triplicate at five time points. The samples were grown at 18°C ± 1°C under 36.8 μE.m2.s-1 of light from cool white fluorescent tubes arranged above the cultures on a 12:12 h light:dark regime. Light levels were assessed by averaging ten measurements (LI_185B, LiCor Inc., USA). Cultures were static. After five days, triplicate samples were harvested. Media was aspirated off and pottles were dried at 50°C for 26.5 h. Each pottle was weighed, thoroughly cleaned and dried and re-weighed to calculate the increase in dry mass. Samples were harvested approximately every four days. The growth was noticeably slower in CAWBG22 and harvesting was extended to eight days.
Media optimisation experiment
Phormidium autumnale (CAWBG26) was selected for an experiment to optimize MLA media under conditions described above. MLA is comprised of NaNO3 (2.00 mM), NaHCO3 (2.019 mM), MgSO4 7H2O (200.43 μM), CaCl2 2H2O (200 μM), K2HPO4 (199.77 μM), NaEDTA (11.7 μM), H2SeO3 (10.00 μM), H3BO3 (39.95 μM), MnCl2 4H2O (18.19 μM), FeCl3 6H2O (5.85 μM), CuSO4 5H2O (40.1 pM), ZnSO4 7H2O (76.5 pM), CoCl2 6H2O (79.86 pM), Na2MoO4 2H2O (24.8 pM), Biotin (0.05 μg/L), Vitamin B12 (0.05 μg/L) and Thiamine HCl (100 μg/L) (Bolch and Blackburn 1996). The concentrations of the following elements were modified: nitrogen, iron, calcium, and selenium. Nitrogen amounts were varied due to the element's role as a major macronutrient, the abundance of which is often responsible for algal blooms (Oliver and Ganf 2000). Iron was chosen as it plays an important role in cellular functions, especially redox reactions, and carbon and nitrogen reduction (Rueter and Petersen 1987). Iron has also been shown to stimulate growth in many species (Paerl et al. 1994; Hyenstrand et al. 2001; Li et al. 2009; Swingley et al. 2005). Calcium was selected because in most biological systems it's heavily involved in cellular signaling and following this may be important in regulating responses to environmental variables (Norris et al. 1996; Smith 1995; Giraldez-Ruiz et al. 1997). The selenium concentration was altered in the attempt to replicate the natural environment of the cyanobacteria in New Zealand as soils in this country are selenium deficient.
Nitrogen in standard MLA medium is at 2.00 mM concentration (representing a 10:1:1 ratio of nitrogen: phosphorus: potassium). In the media manipulation experiments nitrogen was increased to 4.00 mM, providing an experimental 20:1:1 NPK ratio. Iron was increased from 5.85 μM to 11.7 μM (2× original concentration) and 58.5 μM (10× original concentration). Calcium was increased from 199.98 μM to 400 μM (2× original concentration) and 4.0 mM (20× original concentration). Selenium, in standard MLA media at a concentration of 10 μM, was experimentally removed and also increased to 20 μM (2× original concentration) and 100 μM (10× original concentration). Each experiment was set up in triplicate and harvested at five time points, approximately four days apart, as described above.
Geometry and surface area optimisation experiment
Phormidium autumnale (CAWBG26) was used in further experiments to explore bPBR geometry and configurations that might be suitable for large-scale growth. Aliquots (39.7-46.6 mg wet mass) of CAWBG26 were used to inoculate plastic bags with the dimensions of 15 cm × 24 cm made of heat-sealed 250 μm thick crystalline polyethylene (Aperio Plastic Ltd, Christchurch, New Zealand) with approximately 600 cm2 of usable surface area. The bPBR bags were filled with 1 L optimized media (10 × iron MLA, as described above) and bubbled through with air, with no additional carbon dioxide (CO2), from the base at a rate of 200 mL min-1. Light and temperature were as for previous experiments. Previously when growing benthic species the above bags had been positioned horizontally in a 'lay flat' position (Figure 1a). We compared this configuration with the same bags hanging vertically (vertical, Figure 1b) and also to vertical bags that had additional growth surface areas by adding coiled silicone tubing (coiled spiral, Figure 1c), a PVC bottle brush (Silva-Aciares and Riquelme 2008), Figure 1d), or loops of silicone tubing (Figure 1e). The coiled spiral silicone supplied ~220 cm2 extra surface area, the bottle brush an additional ~250 cm2 surface area, and the silicone loops ~264 cm2 extra surface area. Each experiment was set up in triplicate and harvested 36 days after inoculation. Date of detachment was recorded.
Statistical analysis was performed with GraphPad Prism 5.0 software for Mac OS-X.