Thesis title: Development of strategies for the contamination control in heterotrophic microalgal cultures
Microalgae are photosynthetic microorganisms well recognized for their capacity to produce a wide range of biomolecules of industrial interest, such as carotenoids, vitamins, starch, phenols and fatty acids. At industrial scale, microalgae are mainly cultivated in fully photoautotrophic or heterotrophic conditions. In comparison with photoautotrophic processes, heterotrophic cultivation can ensure higher biomass productivities (more than 20 gl-1d-1) and lower production costs. Particularly, heterotrophy makes it possible to employ wastewaters as a source of nutrients, which can further enhance process sustainability. However, in heterotrophy, the use of organic substrates increases the risk of contamination by competing microorganisms, including bacteria and fungi, which can outgrow microalgae or compromise the quality of produced biomass. This problem could be solved working in sterile conditions, however in the wastewater treatment it would be very difficult and expensive.
This PhD project is devoted to the heterotrophic production of microalgae biomass from agro- industrial wastes. The main objective is to develop strategies that can generate a selective pressure for microalgal biomass production, allowing the control and the mitigation of the contamination by bacteria and fungi, without the recourse to sterilization.
Firstly, the uncoupled feeding strategy has been applied in the integration between microalgal heterotrophic cultivation and wastewaters treatment. That has been done at pilot scale, in the MEWLIFE project, which stands for MicroalgaE biomass from phototrophic-heterotrophic cultivation using olive oil Wastewaters (OMW), however in the project the cheese whey was also tested as wastewater. The strategy was applied in a first heterotrophic section to increase the microalgae to bacteria ratio, so that compounds in the wastewaters could be almost entirely consumed by microalgae in a second heterotrophic section. The application of the strategy couldn’t always allow to maintain a high microalgae to bacteria ratio in the first step. However, concerning the wastewater treatments, multiple significative correlations have been identified among data collected in this section. In particular, a positive correlation was found between phenols’ removal and batch duration, OMW volume added and final phenols’ concentration, produced biomass and initial biomass concentration; while negative correlations were identified between OMW volume added and chlorophyll a content in microalgal biomass and chlorophyll a content in the microalgal biomass and final phenols’ concentration. Also, it was found out that the type of wastewater can affect the COD removal and the starch and pigments content.
The uncoupled feeding of organic carbon and nitrogen can be hardly applied by employing wastewaters, since carbon and nitrogen are typically simultaneously present in them. Therefore, a second strategy has been developed, to control the contamination, in microalgae cultivation, directly in wastewaters. The strategy is based on the high ability of microalgae to accumulate carbon, in nitrogen depletion conditions, differently from what many bacteria can do. The process where the strategy can be applied is described by an exponential phase with carbon and nitrogen, that ends when the nitrogen is depleted, giving a N-starvation phase. Here the microalgae store energy reserve materials, until the depletion of carbon, resulting then in a carbon and nitrogen depletion phase. After that, a nitrogen and energy starvation starts, where microalgae are advantaged, since they can consume the stored material. It is imposed that the last phase ends when the contamination ratio is at least the same as the beginning. Two alternative scenarios can occur: when the carbon concentration is not high enough and the process is composed of exponential and famine phases only. On the contrary, when the carbon concentration is very high, the process is composed of exponential and N-starvation phases only. This process has been described by a mathematical model that was used to simulate the process for different C/N ratios, that represent different wastewaters, and in different conditions: initial biomass concentration, maximum fattening factor and contamination ratio. In this way, identifying the best conditions for the treatment of different wastewaters was possible. In particular, for low values of C/N ratio, low productivity values can be obtained. Furthermore, the variation of the contamination ratio affects in a very slight way the process, differently from what happens when the maximum fattening factor and initial biomass concentration are variated. Higher values of maximum fattening factor and initial biomass concentration, allow to obtain higher total biomass productivities and higher C/N values. Therefore, working with higher values of maximum fattening factor and initial biomass concentration would be more convenient. In the end, a validation experiment has been conducted to verify the feasibility of the model, with a C/N equal to 60. The experimental values of total biomass productivity, total produced biomass, batch duration and final biomass concentration were higher than the expected values. Furthermore, the final contamination ratio was higher than the initial one and, thus, higher than the expected. Therefore, the model should be modified, considering a famine phase also for high C/N values, or considering the presence of two or more microalgal populations with different accumulation capacities.
The contamination problem in the wastewater treatment integrated with microalgae cultivation can be overcome by extreme cultivation conditions that can hamper the contaminants’ growth. For this reason, at the end of this PhD work, the heterotrophic growth of the extremophile Galdieria sulphuraria ACUF_064 (T=37 °C and pH=1.8) has been investigated, in presence of glucose, fructose, these two together and sucrose. The study was conducted in a stirred-tank bioreactor at laboratory scale, as a first step to evaluate the possible applicability of this microalga in the wastewater treatment. The most relevant results are that: G. sulphuraria grows faster on glucose than the other C-sources. Also, when glucose and fructose are simultaneously present in the medium, this microalga prefers glucose over fructose, and the presence of fructose doesn’t affect the growth rate of G. sulphuraria, when combined with glucose. However, the fructose uptake seems to be repressed by glucose. To conclude, the growth rate on sucrose doesn’t look to be affected by its hydrolysis.