The growth of algae, irrespective of cultivation system, requires large volumes of water. Almost all areas with high solar energy also have a high evaporation rate. Therefore, it is logical to use sea water for large-scale algae biomass production. As highlighted previously, it is also critical to recycle the culture medium to reduce the nutrient use. Sea water must also be used to replace evaporative loss. This means that the salt concentration in the pond will gradually increase over the time. For instance, in conditions with evaporations rate of 2 m year-1, productivity of 20 g m 2 day 1 and 80 % medium recycling, the medium salinity will rise from

3.5 % NaCl to 25 % NaCl in 490 days. Salinity is usually growth-limiting at the extremes of salt tolerance in some microalgal species, and every microalga has an optimum salinity range (Borowitzka and Moheimani 2013a). The effect of salinity on microalgal growth relates to osmoregulation, which in microalgae is achieved through diverse strategies. Osmoregulatory metabolites are organic substances produced by microalgae that, when the latter are exposed to water stress conditions, respond appropriately to the changes in extracellular water activity. Microalgae main osmoregulators (function as intracellular osmotic regulators) are as follows: (a) polyhydric alcohols (i. e. glycerol, mannitol or sorbitol), (b) variety of glycosides (i. e. galactosyl glycerides, floridoside and isofloridoside) and (c) amino acids (i. e. glutamic acid and proline).

Freshwater algae grow between 0 and 1-2 % NaCl; hypotonic algae grow between 3.0 and 5-5.5 % NaCl; halotolerant algae grow between 6-7 and 14-15 % NaCl; and halophylic algae can grow above 15-16 % NaCl. The majority of microalgae can grow in freshwater and hypotonic conditions. Some microalgae (i. e. diatom, chlorophyta and cyanobacteria) are capable of growth under halotol — erant conditions. However, only a few species of microalgae are hypersaline (i. e. D. salina). There is no single strain of algae capable of optimal growth in the whole range of salinity from sea water to saturation. Interestingly, almost all companies interested in large-scale algae production focus on growing either freshwater or hypotonic algae which will not be sustainable (Moheimani et al. 2013c). Halotolerant algae can normally grow under optimal condition in a wider range of salinities (Fon Sing 2010).

An alternative method of cultivation is to mix microalgae while salinity is being increased. In such a method, a new species can be introduced to the culture of a mono-species while the salinity is rising. That is, a halotolerant microalga species will be introduced to the culture of hypotonic algae when salinity is above the optimum growth condition of the hypotonic algae. If the halotolerant alga can flourish while salinity is increased, the medium use can be maximised. The same technique can be applied for mixed cultures of halotolerant and halophylic mic­roalgae. Such a mixed cultivation technique is yet to be tested at the laboratory or outdoor conditions. One very important advantage of mixed microalgae cultivation is avoiding unnecessary water and nutrient discharge. Considering the species change throughout the cultivation, less negative effect of autoinhibitors is also expected. When the salinity of the culture becomes very high, one option is to have large evaporation ponds for the hypersaline wastewater. Alternatively, this hyper­saline water can also be used in salt gradient solar ponds for generating additional energy.