Physiology and Cultivation of Algae and Cyanobacteria 6. Culture methods • Batch cultures – common, simple, low cost, closed system, volume-limited – any flow of nutritions & products – Erlenmeyer flasks, tubes, Petri dishes – growth curve phases – lag, acceleration, exponential, retardation, stationary, decline Culture methods • Continuous cultures – resources are potentially infinite – cultures are maintained at chosen point on the growth curve by regulated addition of fresh medium – air pump – CO2 source, mixing – categories of contin. cult.: • turbidostat cultures • chemostat cultures • Semi-continuous cultures – periodic fresh medium addition & harvesting Culture methods • Commercial-scale cultures – volume of cca. 102 – 109 l – large open ponds, circular ponds with rotating arm, raceway ponds, large bags, tube system – factors to be considered: • biology of alga; the cost of land; labor; energy; water; nutrients; climate (if outdoors); type of product • light utilization efficiency (PBR & open ponds, surface-to-volume ratio 20-200 vs 5-10m- 1, orientation, inclination); ability to control temp.; hydrodynamic stress (mixing); oxygen accumulation; ability to maintain culture unialgal or axenic (photobioreactors vs. open ponds) -scale up ability • Harvesting (20-30%cost) – species specific – Flocculation -↑pH, cationic polymers (Chitosan, Zetag) – Centrifugation & filtration • Dehydrating – sun-drying, spray-drying, drum-drying, freeze-drying • Cell disruption – mechanical (homogenizers, bead mills, ultrasound), chemical • Product isolation and purification – Chlorella, Spirulina, Dunaliella, Nannochloropsis Methods used for algal culture growth evaluation • direct – fresh/dry mass determination – counting – number of cells (colonies) – cell volume, PCV – protein content – calorific value – flow-cytometry & epifluorescence microscopy • indirect – turbidity; optical density – chlorophyll content Algae & Men • macroalgae (commerce - 42 countries) • food – Laminaria (China, N.,S.Korea, Japan, Philipines, Chile, Norway, Indonesia, U.S., India) – Porphyra, Kappaphycus, Undaria (Wakame), Euchema, Gracilaria, Caulerpa lentillifera (green caviar) – Nori (Porphyra yezzoensis) – 13mil. t/y • microalgae • carotenoids, pigmenst, proteins, vitamins, … – Dunaliella, Haematococcus, Arthrospira, Chlorella – nutraceuticals, pharmaceuticals, animal feed additives, cosmetics, fertilizers – N2-fixing cyano.-biofertilizers in rice fields – Wastewater oxidation, bioremediation • Microalgae – platform for recombinant proteins (e.g. hGH in Chlorela) Algae & energy • Biodiesel – Algal fatty esters or vegetable oils or animal fats – "B" factor (biodiesel in fuel) • Oil Extraction – 1. Expeller/Press; 2. Hexane solvent oil extraction; 3. Supercritical Fluid extraction; Enzymatic extraction; Osmotic shock; Ultrasonicassisted Extraction • Ethanol from algae – high carbohydrate content (Sargassum, Glacilaria, Prymnesium parvum, Euglena gracilis) • Cultivation of algae for CO2 capture – can absorb over 2 million tons of Co2 a year per acre Endogenous rhythms • circadian rhythms • molecular feedback loop ~ 24h~ environmental light-dark cycles ~ positive/negative phase shifts according to phaseresponse curve of particular response • necessity to transfer sunlight-sensitive processes into the night (cell division in night) • lost under certain conditions (e.g. constatnt light, bright light, growth of Euglena on organic medium) – phototaxis – timing of cell division – photosynthetic capacity – bioluminiscence – gene expression – sensitivity to UV Endogenous rhythms • biweekly (circa-semilunar) rhythms • wave action (sea may reduce gamete concentration of marine org. with external fertilization) • e.g.:brown alga Dictyota dichotoma releases eggs twice a month in the field; synchronization signal every second full-moon light • day-length effect (photoperiodism) • circadian timer measures length of night and triggers photoperiodic response • LD (12:12~light:dark) – induce upright thali formation • SD (8:16)– induce reproductive organs formation • circannual rhythmicity • sequence of short and long days over the years results cyclic reproductive stages formation (e.g. Ascophyllum, Laminaria) • signal probably temporal sequence of different physiological stages Documetation • Microscopy (light & fluorescence) – drawings – photography – classic & digital • algal culture collections • Web pages of culture collections in the world: http://wdcm.nig.ac.jp/hpcc.html • macroalgae ~ herbarium