Microalgae as a photosynthetic microorganism have an outstanding ability to use solar energy and convert water and atmospheric CO2 into important biomolecules such as carbohydrates, lipids and other metabolites. Microalgae have high efficiency not only assimilating CO2 and other nutrients, but also sequestering CO2 to mitigate climate change. The cultivation of microalgae is one of the most attractive methods for removing the highly loaded nutrients and simultaneously improving growth rate of biomass in contaminated system. Sivaramakrishnan and Incharoensakdi (2017) utilized freshwater microalgae Scendesmus sp., Chlorella sp., and Chlamydomonas sp. as a potential feedstock for lipid production improvement. However, the application of microalgae in industrial section is challenging. This process may be hindered by mass cultivation and remained costly (Abou-Shanab et al., 2011; Ummalyma et al., 2022).Therefore, it is necessary to develop a low-cost microalgal cultivation approach in order to more effectively supply the demand. Microalgae cultivation in wastewater has gained increasing attention, as one of effective approaches for promoting economic and environmental development, for the production of biofuels (Khan et al., 2019). Microalgae-based wastewater treatment process is a great way to reduce manufacturing costs with low energy needed, which can be a sustainable method of wastewater treatment with reduction of CO2 emission. Owing to the fact that wastewaters contain high content of both organic and inorganic nutrients, i.e., carbon (C) as hydrocarbon, nitrogen (N) as ammonium, and phosphorus (P) as orthophosphates. After the process of anaerobic digestion, the digested sludges can be used as appropriate substrate for microalgae cultivation (Baldisserotto et al., 2023, Ekame et al., 1984). In nutrient assimilation, microalgae consume inorganic substances containing N and P to create biomass. Therefore, microalgae-based system as biological treatment can be efficiently applied in secondary and tertiary treatments for removing nutrient residues from wastewater (Pasqualino et al. 2011). Not only nutrient availability (i.e., N, P and organic carbon sources), but also hydraulic retention time (HRT) in microalgae-based wastewater treatment can play a role in nutrient removal improvement. According to Martínez et al. (2012) and Roleda et al. (2019), high-affinity nutrient uptake in high nutrient loading is upregulated in adaptable algae for rapid growth. N and P removal (i.e., mainly by cell assimilation) was improved by glucose supplementation to the mixotrophic culture of Chlorella vulgaris (Peng et al., 2019). With highly efficient nutrient removal through N and P assimilation by microalgae, this approach makes microalgal cultivation in high nutrient loading well suited for increasing growth with protein-rich biomass (Stedt et al. 2022). However, in nutrient-rich wastewater, too high concentration of ammonia/ammonium (NH3/NH4+) directly inhibits algal growth since oxidative stress and cell metabolism disturbance may occur (Lu et al. 2018). In addition to the effect of nutrient combination on improving bioremediation method, the effect of HRT on wastewater treatment process by algae-based system must also be considered due to its manner regarding limitation of nutrient uptake (Nguyen et al. 2022). Several freshwater green microalgae like Scenedesmus sp. and Chlorella species (i.e., Chlorella sp., Chlorella pyrenoidosa, Chlorella sorokiniana, and Chlorella vulgaris.) were used for wastewater treatment where effective nutrient removal process could be mostly accomplished in mixotrophic culture (Baldisserotto et al., 2023; Chandra et al., 2014; Guldhe et al., 2017; Peng et al., 2019). This method would lead to an increased biomass production.
In the development of sustainable biofuels, microalgae as a probable feedstock for biofuel production are given more attention with their great ability in lipid accumulation and biomass growth. Lipid accumulation in microalgae depends on diverse factors such as microbial species used in the process and nutrients present in the growth medium. It is generally recognized that the microalgae Chlorella species can effectively accumulate starch and lipids. Previously, it has been reported that Chlorella protothecoids cultivated under heterotrophic condition (supplemented with glucose), Chlorella pyrenoidosa grown as mixotrophic culture in piggery wastewater, and Chlorella sp., an autotrophic culture, containing high lipid profile (e.g., palmitic, oleic and linolenic acid) have a potential to produce high lipid yield. (Miao et al., 2004; Wang et al., 2012; Sivaramakrishnan and Incharoensakdi, 2017). One factor triggering lipid accumulation in microalgae is environmental stress such as the depletion of N from growth medium (López García de Lomana et al., 2015; Zhu et al., 2014). However, the period of nutrient depletion and cell harvesting should be controlled for increasing total lipid productivity (Widjaja et al., 2009).
In addition to lipid production in microalgae, H2 production by cyanobacteria which requires photosynthetic products like reducing power and ATP for driving their metabolism is also gaining researchers’ attention. Generally, cyanobacteria as photosynthetic microorganisms are characterized by enormous morphological diversity, such as the unicellular cyanobacteria (spherical, ovoid, or cylindrical cells) and the filamentous cyanobacteria. Most of the filamentous cyanobacteria (heterocyst-forming) like Anabaena siamensis TISTR 8012, Nostoc sp. CU2561, and Fischerella muscicola TISTR 8215 which have both hydrogenase and nitrogenase were characterized as a promising microorganism for high H2 production (Khetkorn et al. 2010; Sukrachan and Incharoensakdi 2020; Wutthithien et al. 2019), in contrast to a lower H2 production in unicellular cyanobacteria such as Aphanothece halophytica (Taikhao et al., 2013).
As for secondary and tertiary wastewater obtained after algae-based treatment process, the spent wastewater with low N/P ration may induce H2 production. An increase in H2 production associated with increased heterocyst frequency was found under N deprived culture (Wutthithien et al. 2019). Organic molecules can be utilized as a source of energy (ATP) whereby the latter is required for promoting H2 production through nitrogenase. Previously, it has been reported that glucose supplemented to Fischerella culture is a suitable substrate for H2 production (Yodsang et al. 2018). Hence, it is possible to use secondary and tertiary wastewater with remaining organic matter for improving H2 yield in cyanobacteria. In this study, characterization of lipid and biomass production by Chlorella sp. was done under different cultivation modes depending on various types of synthetic wastewaters, i.e., petroleum effluent, molasses wastewater, and agro-industrial wastewater. The spent wastewater after algae-based treatment process was used for H2 production by Fischerella muscicola TISTR 8215.