# Propagation of Inoculum for _Haematococcus pluvialis_
## 1 Introduction
Scaling-up the process to the commercial level is a complex task since it is difficult to assess which factors will have a deciding impact on the scaling-up process. As a result, most large-scale cultivations systems provide a lower yield than is expected, considering the results obtained on a laboratory scale [8]. One of the fundamental problems that needs to be solved is the problem of optimizing the process of producing a sufficient amount of inoculum for large volume open ponds or photobioreactors. The two most relevant criteria, rarely analyzed in a laboratory scale, are minimizing the required time and costs. Coming up with suitable methods and procedures is necessary for the processes conducted on a large-scale. It is closely related to cultivated microorganisms, used cultivation systems, and the location of the production plant. The efficiency of such systems and their reliability gets better with time as the system operators, who manage the cultures of cultivated microorganisms, gather knowledge, and gain experience. Commercial producers are not eager to disclose the details of industrial solutions [13]. Improvement in efficiency while scaling-up bioprocesses can be achieved by manipulating the key process parameters [8]. In the case of microalgae cultivation, the parameters that influence the biomass growth and secondary metabolites accumulation are time profile of light intensity, coupled with culture agitation time-profile, aeration rate, availability of nitrogen, and carbon dioxide source, and temperature. It is also essential to progressively adapt the abovementioned process parameters, along with the culture growth rate and its physiological state [14]. At the stage of research and development, the experiments should be conducted long enough to reveal any problems related to the aging of the system. Such issues, for example, contamination, biofouling, and degradation of material are noticeable only after a minimum of a few months of continuous work [8]. It is recommended to gradually propagate the culture of microorganisms when cultivating microalgae on a larger scale. Generally, in microalgae cultivations at the point of moving to the following stages of scaling-up, it is advised to use inoculation levels of 1% to 10% of the volume of tanks used for the next stage of cultivation. For this reason, it is necessary to scale-up cultivation in stages from a lower volume of the initial production to the final stage that is commercial-scaleproduction [15]. At every stage of scaling-up, it is necessary to check the immunity and viability of cells of a particular microalgae strain. The density of cells is another important parameter that should be higher than 10-million cells per mL to avoid the long lag phase after inoculation [8]. Many stages and the extended time required to carry out such process increase costs, but also the risk of contamination [13]. When conducting experiments, or producing microalgae, a pure culture of microorganisms should be cultivated in consistent and short time intervals (every week or every fortnight) in liquid culture to be able to quickly provide \"healthy\" inoculum ready for immediate use. In the case of a medium storage period (6 months), it is recommended to cultivate cultures on solid-state medium, preferably under relatively low light (PAR \(<\) 25 umol m\({}^{2}\)s\({}^{-1}\)). Long-term storage of strains should be conducted by freezing the strains using cryoprotectants [15]. Conducting scientific experiments is very often closely related to the possibility of using the results in the economic environment, in large scale industrial production. Sooner or later, scientists come across issues related to moving experiments from laboratory-scale to semi-technical and industrial scale. When conducting research on a laboratory-scale, one can buy suitable devices from companies that produce them commercially.