Microalgae belong to the fastest-growing photosynthetic organisms since their cell doubling time can be as little as a few hours. Biomass production by microalgae (oxygenic photosynthetic organisms) is based on the simple scheme shown below, which determines all the necessary requirements of this biological process:
CO2 + H2O + nutrients + light energy ! biomass + O2
In biotechnology, generally, the production of biomass requires well-defined conditions. The necessary cultivation requirements for the growth of microalgal mass cultures are light, a suitable temperature and pH, and a sufficient carbon and nutrient supply in the growth medium. Since microalgal mass cultures grow in dense suspensions, some kind of turbulent mixing is necessary to expose cells to light and to allow for an efficient mass transfer.
Light is the most important factor for microalgal growth. The amount of photon energy received by each cell is a combination of several factors: photon flux density, cell density, length of optical path (thickness of culture layer), and rate of mixing. The light capture by photosynthetic pigments is roughly 10 times higher under full sunlight (2000 p,mol photons m~ s~ ) than that required to saturate growth. In other words, up to 90% of the photons captured in full sunlight by chlorophyll and other pigments are not being used for photosynthesis and instead must be dissipated as heat and fluorescence. Consequently, the efficiency of light utilization usually drops from a theoretical value of
20% (based on photosynthetically active irradiance) to lower than 4%, roughly corresponding to an annual biomass yield of about 40tha~ .
After light, temperature is the most important parameter to measure and control the microalgal culture. Some microalgal strains tolerate a broad temperature range between 15 and 35 °C (e.g., Chlorella and Spirulina), while Haematococcus usually requires a more rigorous regulation between 25 and 27 °C. However, for the majority of freshwater microalgae, the optimum temperature ranges within 25 and 30 °C.
Successful cultivation requires continuous monitoring of physicochemical parameters, that is, pH, temperature, oxygen concentration, and nutrient status. The basic biological method used is microscopic examination to detect morphological changes and contamination by other microalgae and protozoa. Nutrient status can be followed by monitoring the concentration of nitrogen, using it as a measure for adding the proportional amounts of other nutrients. In mass cultivation of microalgae, monocultures are usually required for biomass exploitation. The appearance of 'contaminants' (other microalgae as well as protozoa, bacteria, or fungi) might indicate that the cultivated culture has come under stress. Contaminants often represent one of the major limitations to large-scale production in microalgal cultures, particularly with strains that cannot be grown in a selective medium outdoors. For the cultivation of some microalgae (e.g., Haematococcus), the use of a closed system becomes mandatory.
A sufficient mixing of the microalgal suspension is necessary to ensure nutrient diffusion and a homogeneous light supply to the cells, as well as to prevent the accumulation of oxygen in the culture, particularly when they are grown in a closed system. Indeed, excessive oxygen accumulation in a culture can promote photoinhibition of photosynthesis and a decline in growth. On the other hand, excessive mixing can cause hydrodynamic or sheer stress to the cells, and consequently a similar reduction of productivity.
Biophysical and biochemical monitoring methods generally reflect the status of the cells' photosynthetic apparatus and are used to adjust the appropriate cultivation conditions for the production of biomass or certain compounds. The concentration of dissolved oxygen measured by an oxygen electrode is considered as a reliable and sensitive indicator of photosynthetic activity in microalgal cultures.
Recently, chlorophyll fluorescence has become one of the most common and useful approaches used for monitoring the physiological status of cultures. Its non-invasiveness, sensitivity, ease of use, as well as its promptness make it a convenient and suitable technique in microalgal biotechnology. The ratio Fv/Fm (variable to maximum fluorescence yield) is considered to be a convenient measure of the performance of photochemical processes in photosystem II (the PSII photochemical yield): it relates the utilization of absorbed light energy to primary production. A decline in the Fv/Fm ratio can be considered as a reliable warning signal of culture stress.
Culture growth might be estimated as changes of optical density (OD) at 750 nm, biomass dry weight, or the number of cells. The specific growth rate is usually estimated as p (h_1) = (ln X2 — ln X1)/(f2 — t1), where X is cell number or dry weight at various times. Biomass productivity can be expressed as the areal or volumetric yield per unit time, that is, in gm—2 day—1 or in gl—1day—1, respectively.
Basically, two cultivation regimes are used for the growth of microalgal cultures. In the batch regime, the culture is inoculated and at a certain point of growth it is harvested. In the continuous regime, the culture is harvested continuously according to its growth rate and fresh medium is added to replace nutrients. In practice, semi-continuous or semibatch regimes are usually adopted, that is, where a part of the culture is harvested at regular intervals.
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