The quest for large-scale production of microalgae biomass as sustainable complements or replacement of some critical raw materials and/or processes in food, energy, agriculture, pharmaceutical, medical and nutraceutical industries has been quite enormous in recent times. In order to produce microalgae biomass and metabolites at competitive prices, several potentially efficient photobioreactors have been developed. However, their potentials can only be realized by selection of appropriate operation modes and culture systems, and optimization of the culture conditions. Operation modes such as batch, fed-batch, semi-continuous, and continuous processes have been investigated for large-scale cultivation of some important freshwater and marine microalgae including several species of Chlorella and Desmodesmus, Spirulina platensis, Isochrysis galbana,Dunaliella salina, and Phaeodactylum tricornutum. The choice of the operation mode depends on the species, the photobioreactor configuration, especially their light supply efficiencies, as well as culture conditions. Although photoautotrophic cultures are extensively used due to their simplicity and reduced risk of contamination, their productivities and standing biomass concentrations are usually very low. However, many species of microalgae can metabolize many organic carbon sources in the presence of light (mixotrophic metabolism) or in dark (heterotrophic metabolism). Thus, mixotrophic and heterotrophic culture systems have been developed for high-density cultures of many species of microalgae. With these culture systems, very simple bioreactors can be used to achieve high cell densities with high productivities. However, the risk of contamination by fast-growing heterotrophic microorganisms is very high while accumulation of light-induced metabolites is very low. These limitations can be overcome by a combination of culture systems. For examples, sequential heterotrophic-photoautotrophic system, sequential heterotrophic-mixotrophic system, and sequential mixotrophic-photoautotrophic system can be used to obtain high cell densities with high concentrations of light-induced metabolites. Furthermore, cyclic photoautotrophic-heterotrophic system and cyclic mixotrophic-heterotrophic system can be used to achieve continuous cell growth under day-night (light-dark) cycles. In all these production modes and culture systems, there is a need to optimize culture conditions such as light intensity and distribution, pH, media components and concentrations, salinity, temperature, mixing and aeration gas composition. The optimal conditions of these variables depend on the species and the target metabolites. In this chapter, the various production modes and culture systems are discussed with an emphasis on their comparative advantages and disadvantages. The effects of various physical (light, temperature, mixing), chemical (nutrient types and concentrations, pH, salinity, aeration gas) and biological (contamination, morphology and size of the cells, as well as light absorption capacity of the cells) conditions on cell growth and productivity in large-scale microalgae cultures are also discussed.