Experimental assessment and mathematical modelling of the growth of Chlorella vulgaris under photoautotrophic, heterotrophic and mixotrophic conditions
Graphical abstract
Introduction
In recent years, the trend towards a circular economy has become more pronounced. In that perspective, conventional wastewater treatment plants are now also considered as resource recovery plants, that recover the energy from available carbon (C) sources (i.e. lipids, cellulose and polyhydroxyalkanoates stored inside the biomass) (Puyol et al., 2017). However, with regard to nitrogen sources, recovery is challenging. A possible solution for this is the use of microalgae for wastewater treatment, since microalgae are capable of storing both nitrogen and phosphorus inside their cells. The harvested cells can then for example be used to extract pigments or lipids for biofuel production and the remaining biomass can be applied as a bio-fertilizer (Arashiro et al., 2020; Cheirsilp and Torpee, 2012). The bottleneck for the use of microalgae, however, is the energy-consuming harvesting step (Parmentier et al., 2020). This is due to the high stability of the microalgae suspension in combination with a low biomass concentration. The latter is inherent to the photoautotrophic growth process, since the light penetration inside the microalgae suspension is inversely proportional to the biomass concentration (Liang et al., 2009).
A possible solution is selecting microalgae species which are capable of growing mixotrophically, i.e. growing autotrophically and heterotrophically at the same time (Pang et al., 2019). This process allows the microalgae to grow on organic carbon (OC) substrates when the incident light becomes limiting, resulting in a higher biomass concentration. With regard to this process, Martinez and Orus (1991) reported that, under non-aerated conditions, the mixotrophic growth rate (μM) is 20% higher than the sum of the autotrophic growth rate (μA) and the heterotrophic growth rate (μH). However, this difference decreased with 10% when the cultures were aerated with air. Furthermore, when aerating with air containing 2% CO2, the difference became negligible (Martinez and Orus, 1991). It can be hypothesized that these differences might be due to the limiting CO2 and O2-concentrations occurring under non-aerated conditions. To test this hypothesis, the inorganic carbon (IC) and dissolved oxygen (DO) concentration was measured in suspensions of the green microalgae species Micractinium inermum under photoautotrophic, heterotrophic and mixotrophic conditions (Smith et al., 2015). The results indicated that the IC and DO-concentration were limiting under photoautotrophic and heterotrophic conditions, respectively. Moreover, during mixotrophic growth, increased levels of IC and DO were measured, together with a 37% increase of μM in comparison with the sum of μA and μH. It was therefore concluded that internal CO2 and O2-exchange led to higher biomass growth during mixotrophic test conditions, and that this growth advantage ceases without limiting IC and DO-concentrations, i.e. when aerating the cultures with air containing 2% CO2. Indeed, endogenous CO2 produced during heterotrophic metabolism could be recycled preferably between the intracellular compartments rather than through the metabolically more energy expensive external incorporation (Grama et al., 2016). However, it was not experimentally assessed to which extent microalgae would recycle these endogenous sources and under which conditions this occurs. It is clear from the experiments of Smith et al. (2015) that a part of the produced CO2 and O2 will be excreted, since increased values of IC and DO were measured during mixotrophic growth. When further increasing the CO2-fraction in the air to 5%, it was observed for Chlorella protothecoides that the metabolization of the organic substrate was blocked under mixotrophic conditions, thus cancelling the (benefits of) mixotrophic growth (Sforza et al., 2012). This indicates that C. protothecoides prefers growing photoautotrophically rather than mixotrophically under elevated IC-concentrations.
On a biochemical level, decreased RuBisCo activity and citrate synthase activity have been observed in Chlorella zofingiensis during mixotrophic growth, indicating a down regulation of the Calvin and tricarboxylic acid (TCA) cycle, respectively (Zhang et al., 2017). Moreover, gene expressions in Chlorella sorokiniana under photoautotrophic and mixotrophic conditions showed an upregulation of the phospoenolpyruvate carboxylase enzyme during mixotrophic growth conditions (Cecchin et al., 2018). This enzyme enables the potential recovery of C-atoms lost by the oxidation of OC-sources. Furthermore, a downregulation of RuBisCo activase was observed but also an upregulation of the RuBisCo accumulation factor (RAF). This indicates that during photoautotrophic growth, RuBisCo activase had to be increased in order to catalyze the carbamylation of RuBisCo due to low CO2-concentrations. In contrast, during mixotrophic growth, the RAF upregulation contributes to the assembly of the RuBisCo complex to improve C-fixation (Cecchin et al., 2018). These findings support the hypothesis of internal CO2-recycling.
Overall, it can be concluded that there is a delicate balance driving the mixotrophic growth of microalgae, enabling a fine regulation of the cell metabolism. However, more research is needed in order to gain insight into the driving factors of this balance. In this research, the mixotrophic microalgae growth was assessed with a combined respirometric-titrimetric unit, using Chlorella vulgaris as an indicator species. The goal was to (1) quantify the amount of CO2 that was internally recycled during mixotrophic growth and (2) to assess the influence of elevated dissolved IC-concentrations on the mixotrophic growth. Finally, a previously developed microalgae model (Manhaeghe et al., 2019) describing the photoautotrophic growth of Chlorella vulgaris was extended with the (photo)heterotrophic growth pathway in order to assess and validate the experimental data.
Section snippets
Combined respirometric-titrimetric unit
The experimental data collection was done using the same respirometric-titrimetric set-up as in Manhaeghe et al. (2019). This set-up consist of a 1.5 L heat-jacketed reactor for temperature control (operating volume of 1.3 L), a stirrer at 250 rpm (IKA Eurostar 20 digital) to obtain a homogenous microalgae suspension and eight fluorescent lamps (Grolux T8-14W, Sylvania) surrounding the reactor providing light (73 μmol m−2.s−1). A DO-probe (Inpro 6860 i/12/120/nA, Mettler Toledo) and a pH-probe
Respirometry-titrimetry for measuring microalgae kinetics under photoautotrophic, heterotrophic and mixotrophic conditions
Fig. 1A and B show the respirometric and titrimetric profile, respectively, measured under dark conditions with the addition of 75 g d-glucose.m−³ (i.e. experiment OC75). It can be seen that the O2-concentration immediately dropped upon glucose addition. Moreover, a pH-decrease of the medium occurred which led to the addition of base to keep the pH within the desired interval 6.90–7.10. Komor and Tanner (1974) reported a pH-increase of the medium upon addition of hexoses which was assigned to
Conclusions
A combined respirometric-titrimetric unit was used to assess the photoautotrophic, heterotrophic and mixotrophic growth of the green microalgae species Chlorella vulgaris. Mass balances revealed that under photoheterotrophic conditions, all the produced CO2 derived from assimilating the organic carbon source was internally recycled for photoautotrophic growth. Furthermore, from the experimental data it was hypothesized that photoautotrophic growth is the preferential growth mechanism of
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
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