Mixotrophic Cultivation Of Microalgae Research Articles

Mixotrophic cultivation of microalgae using agro-industrial waste: Tolerance level, scale up, perspectives and future use of biomass

Alternative culture media make large-scale microalgae biomass production more viable and cost-effective. The aim of this work was to study the addition of whey as an alternative source of organic nutrients in fed-batch mode for the cultivation of microalgae (Spirulina platensis, Chlorella homosphaera and Scenedesmus obliquus). The microalgae demonstrated acceptance of whey at low concentrations, causing the increasing in the maximum specific growth rates (μmax) and biomass concentrations ([X]max) compared with the cultivation without whey in standard medium (μmax of 0.168 d−1 versus 0.075 d−1, and [X]max of 2.274 g L−1 versus 0.970 g L−1, respectively, for S. platensis). Therefore, a cost savings in cultivation media of up to 32 %, 57 %, and 73 % were possible in the cultivation of S. obliquus, S. platensis, and C. homosphaera, respectively, considering the production of 1 kg of biomass. The addition of whey presented different effects in the levels of intracellular protein considering the species of microalgae: an increasing was observed in C. homosphaera (24.18 to 43.96 g/100 g of biomass) and a decreasing in S. platensis (73.75 to 44.06 g/100 g of biomass), comparing with the controls. Scale-up with a natural light source can affect microalgal productivity and biomass composition in a medium supplemented with whey, because of the influence of irradiation. Optimizing the large-scale production of microalgae using agro-industrial residues can lead to significant advancements in biomass production at reduced costs. These biomasses can be integrated into bioenergy production, biofertilizer manufacturing, and animal feed production. By utilizing waste as a nutritional source and optimizing cultivation processes and biomass generation, a closed-loop cycle is established, aligning with the principles of circular bioeconomy and enhancing the sustainability of the process.

A robust multinutrient kinetic model for enhanced lutein and biomass yields in mixotrophic microalgae cultivation: A step towards successful large-scale productions.

Mixotrophic cultivation holds great promise to significantly enhance the productivities of biomass and valuable metabolites from microalgae. In this study, a new kinetic model is developed, explicitly describing the effect of the most influential environmental factors on both biomass growth and the production of the high-value product lutein. This extensive study of multinutrient kinetics for Tetradesmus obliquus in a mixotrophic regime covers various nutritional conditions. Crucial nutrients governing the model include nitrate, phosphate, and glucose. Using seven state variables and 13 unknown parameters, the model's accuracy was ensured through a well-designed two-factor, four-level experimental setup, providing ample data for reliable calibration and validation. Results accurately predict dynamic concentration profiles for all validation experiments, revealing broad applicability. Optimizing nitrogen availability led to significant increases in biomass (up to fourfold) and lutein production (up to 12-fold), with observed maximum biomass concentration of 6.80 g L-1 and lutein reaching 25.58 mg L-1. Noticeably, the model exhibits a maximum specific growth rate of 4.03 day-1, surpassing reported values for photoautotrophic and heterotrophic conditions, suggesting synergistic effects. Valuable guidance is provided for applying the method to various microalgal species and results are large-scale production-ready. Future work will exploit these results to develop real-time photobioreactor operation strategies.

Effect of external acetate added in aquaculture wastewater on mixotrophic cultivation of microalgae, nutrient removal, and membrane contamination in a membrane photobioreactor

The mixotrophic cultivation of microalgae in wastewater has attracted extensive attention due to its many advantages. In this study, acetate, which can be prepared by hydrolysis of aquaculture waste, was used as exogenous organic matter to promote the growth of Chlorella pyrenoidosa cultured in aquaculture wastewater. Microalgae cultivation was carried out in a membrane photobioreactor (MPBR) with continuous inflow and outflow mode. The results showed that exogenous acetate greatly promoted the mixotrophic growth of C. pyrenoidosa. When the dosage of acetate reached 1.0 g L−1, the relative growth rate of microalgae in the logarithmic stage reached 0.31 d−1, which was 4.4 times that of the control. As a result, exogenous acetate also promoted the removal of nutrients from aquaculture wastewater. During the stable operation stage of the MPBR with acetate added in the influent, an average of 87.41%–93.93% nitrogen and 76.34%–88.55% phosphorus was removed from the aquaculture wastewater containing 19.41 mg L−1 total inorganic nitrogen and 1.31 mg L−1 total inorganic phosphorus. However, it was worth noting that adding exogenous acetate also led to an increase in the membrane resistance of the membrane module in the MPBR. Membrane resistance was mainly composed of internal resistance (Ri) and cake resistance (Rc), and with the increase of acetate content in the influent, their proportion in the total resistance gradually increased. Ri contributed the major membrane resistance and was most affected by acetate dosage. Ri reached 32.04 × 1012 m−1 with 1 g L−1 acetate, which accounted for 69.49% of total resistance. Moreover, with the increase of influent acetate concentration of the MPBRs, both the number of insoluble contaminants and dissolved organic contaminants in the membrane modules increased. In addition, the composition of proteins, polysaccharides, and humus in dissolved organic contaminants was close to that in extracellular polymeric substances and soluble microbial products secreted by microalgae. These results suggested that the membrane fouling of membrane modules was closely related to the algal biomass content in the MPBRs. The above results provided a theoretical basis for reducing membrane fouling of MPBR.

Cascading Integration of Electrofermentation and Photosynthesis—Low‐Carbon Biorefinery in Closed Loop Approach

AbstractSingle‐chambered electrofermentation (EF) bioreactors are operated using spent wash to evaluate the effect of applied voltage (EF/AV; −0.6 V) against closed‐circuit (EF/CC; 100 Ω) and control (EF/C; no voltage/resistance) operations using deoiled microalgae biomass‐derived biochar electrodes (anode) on increasing acidogenesis rate/efficiency. Higher total volatile fatty acids (VFA) production (mg L−1) is observed in EF/AV (2866), depicting 31% and 57% increments than EF/CC (1984) and EF/C (1224), respectively. The VFA profiles (C2–C4) in EF/AV are higher with acetic/butyric/propionic acids (1922/704/240 mg L−1) and biogas co‐generation [H2 (22%) and CH4 (3%)] than EF/CC and EF/C. The applied voltage (EF/AV) helps in electron flux regulation for increasing substrates conversion and VFA generation. Residual VFA‐rich effluents from EF process is utilized as substrate to mixotrophic microalgae cultivation (nutrient‐ and stress‐phase) that resulted in biomass production with simultaneous wastewater treatment. EF/AV depicts higher biomass growth (0.72 g L−1; 4th day) and substrate removal (63%) than other operations. Integrations of electrofermentation with photosynthetic processes operated with the residual resources in effluents towards product valorization and wastewater polishing, accounts for low‐carbon economy.

Phytoremediation of Domestic Sewage in Constructed Wetland Integrated with Cultivation of Chlorella sp.: A Novel Technique for Remediation and Resource Recovery

Domestic sewage-based constructed wetland (CW) showed that the effluent from CW-system contain enough plant nutrients and enhanced the growth of microalgae. Hence, a pilot CW system employing Typha latifolia in domestic sewage, integrated with the cultivation wild type Chlorella sp. was investigated. Phytoremediation at 48 hours of detention time caused significant changes in its physicochemical properties and the generated effluent was notably attractive for the cultivation of microalgae. The microalga was grown in 6 treatments: treated-mixotrophic (T1), treated-heterotrophic (T2), treated-autotrophic (T3), control-mixotrophic (T4), control-heterotrophic (T5) and control-autotrophic (T6) conditions for 8 days inside an incubator. The results suggested that phytoremediation effluents integrated with mixotrophic cultivation of microalgae, utilising both light and carbon sources could be the most efficient, environmentally safe, sustainable and novel technique for synergistic resource generation and bioremediation.

Insights into carbon utilization under mixotrophic conditions in Chlamydomonas

Mixotrophic microalgae cultivation with various carbon resources is considered as a strategy that could increase biomass. However, the mechanism of carbon utilization between inorganic carbon (IC) and organic carbon (OC) remains unknown. In this study, IC and OC consumption, chlorophyll fluorescence parameters, intracellular Nicotinamide adenine dinucleotide phosphate content and transcriptional changes in related genes were characterized. The results showed that IC was utilized preferentially, whereas 76% IC was consumed at 8 h. Subsequently, OC was the dominant carbon resource for fermentation. The cell density in the IC group was 100% higher than that in the group without IC at 24 h. Bicarbonate addition enhanced photosynthesis by dissipating less energy and generating more electrons and energy, which benefited OC assimilation. This finding was verified by qRT-PCR analysis. These results elucidate the carbon utilization mechanism under mixotrophic conditions, which provide clues for promoting microalgae growth by regulating carbon utilization.

Enhancement of microalgal biomass productivity through mixotrophic culture process utilizing waste soy sauce and industrial flue gas

Wastewater treatment plants are indispensable facilities, which emit a massive amount of greenhouse gases. To boost CO2 mitigation and wastewater treatment performance, mixotrophic microalgae cultivation using wastewater has recently been proposed. In this study, food industry wastewater (waste soy sauce) was applied to Chlorella sorokiniana UTEX 2714 cultivation. By using a medium with 20% (v/v) of 10-fold diluted soy sauce, the biomass and fatty acid methyl ester (FAME) productivity enhanced by 1.93 and 1.76 times, respectively. Biomass productivity increased up to 5.2 times when using medium with high soy sauce content under high-intensity light that inhibits cell growth in photoautotrophic environments. Furthermore, industrial flue gas treatment with wastewater was demonstrated by outdoor semi-continuous cultivation with 42% improved biomass production. Consequently, these results suggest that mixotrophic microalgal cultivation has great potential to address both climate change and water pollution while producing valuable products and can contribute to building a sustainable society.

Use of crude glycerol for mixotrophic culture of Phaeodactylum tricornutum

Crude glycerol is a waste stream from biodiesel production, which has previously been shown to be useable for mixotrophic cultivation of microalgae; however, at high concentrations the presence of contaminants may limit culture growth. In this study two previously identified methods of contaminant removal from crude glycerol were trialled for use in mixotrophic culture of Phaeodactylum tricornutum. Neither crude glycerol without contaminant removal, nor crude glycerol which has had contaminants removed through calcium precipitation, lead to culture growth of P. tricornutum. However, pH adjusted crude glycerol gave comparable cell densities (6.03 ± 0.24 × 106 cells ml−1 compared to 5.66 ± 0.15 × 106 cells ml−1), growth rates (3.25 ± 0.26 days compared to 2.85 ± 0.21 days) and fatty acid profiles compared to reagent grade glycerol. There were alterations in the carbon partitioning of the microalgae, in addition to changes in cell widths. Cell widths increasing when harvested at stationary phase, compared to reagent grade glycerol (4.88 compared to 4.28 μm), while chlorophyll (11.38 compared to 6.25 %DW) and carbohydrate contents decreased (17.29 compared to 14.15 %DW respectively). As a result, it can be concluded that this method of contaminant removal meant that crude glycerol may be successfully used for culture of P. tricornutum, which may reduce the costs of microalgal culture, depending upon the end use.

A review on the sustainable procurement of microalgal biomass from wastewaters for the production of biofuels

The feasibility of microalgal biomass as one of the most promising and renewable sources for the production of biofuels is being studied extensively. Microalgal biomass can be cultivated under photoautotrophic, heterotrophic, photoheterotrophic, and mixotrophic cultivation conditions. Photoautotrophic cultivation is the most common way of microalgal biomass production. Under mixotrophic cultivation, microalgae can utilize both organic carbon and CO2 simultaneously. Mixotrophic cultivation depicts higher biomass productivity as compared to photoautotrophic cultivation. It is evident from the literature that mixotrophic cultivation yields higher quantities of polyunsaturated fatty acids as compared to that photoautotrophic cultivation. In this context, for economical biomass production, the organic carbon of industrial wastewaters can be valorized for the mixotrophic cultivation of microalgae. Following the way, contaminants’ load of wastewaters can be reduced while concomitantly producing highly productive microalgal biomass. This review focuses on different aspects covering the sustainable cultivation of different microalgal species in different types of wastewaters.

Cultivation of Chlorella pyrenoidosa with different phosphorus forms under photoautotrophic and mixotrophic modes: Biochemical component synthesis and phosphorus bioavailability appraisement

Microalgae cultivation with wastewater could reduce the water pollution and relieve global energy crisis. Nowadays, there is few study on the bioavailability of phosphorus forms in wastewater for microalgae growth and biochemical component synthesis. In this study, Chlorella pyrenoidosa was cultivated under photoautotrophic and mixotrophic modes with five phosphorus forms, including K 2 HPO 4 (Pi), sodium tripolyphosphate (STPP), glucose-6-phosphate (G-6-P), β-sodium glycerophosphate (β-glycerol-P), and adenosine triphosphate (ATP). Results showed that Chlorella pyrenoidosa could utilize all phosphorus forms to sustain its growth. Pi was the preferred phosphorus form that could be absorbed directly, while monophosphates (G-6-P and β-glycerol-P) could promote the accumulation of carbohydrate, protein and chlorophyll- a . Regarding phosphorus absorption and utilization, most of phosphorus in mixotrophic bulk were transformed and stored in intracellular phosphorus pool, whereas photoautotrophic microalgae absorbed excess phosphorus in a short time and released subsequently. The G-6-P enhanced the AKP activity and improved organic phosphorus hydrolysis. Results will provide valuable information for mixotrophic microalgae cultivation with different phosphorus forms, and further for phosphorus recovery and high density microalgae cultivation in wastewater. • Effect of phosphorus forms on microalgae growth and intracellular component. • Different bioavailability and utilization strategy of phosphorus forms. • Mixotrophy improved the performance of phosphorus absorption and utilization. • G-6-P was more effective for protein and carbohydrate accumulation.

Mixotrophic cultivation of microalgae for carotenoid production

AbstractThe intrinsic ability of microalgae to accumulate high amounts of carotenoids has made them the preferred aquatic organisms of biotechnological exploration for carotenoid production. To continuously innovate and modify microalgal bioprocesses, aquaculture scientists have been working hard for the past decades in a forward‐looking way, and mixotrophic cultivation of microalgae is deemed as a promising strategy to decrease production cost. This review is intended to summarise the recent research advancement of carotenoids production from mixotrophically cultivated microalgae, starting from the structure, biosynthesis, physiological roles and applications of carotenoids and followed by the production processes both currently established and under development. Most importantly, the microalgal physiology of mixotrophic cultivation is reviewed in depth both in general and specifically for the most studied species, and the prospects of commercially viable mixotrophic microalgal processes for carotenoid production along with the insight of future research are of course discussed. Finally, we conclude that mixotrophy might be a promising strategy for large‐scale cultivation of microalgae to produce carotenoids although some technical obstacles need to be overcome.

Mixotrophic Cultivation of Microalgae Using Biogas as the Substrate.

Biogas utilization through biotechnology represents a potential and novel technology. We propose the microalgal mixotrophic cultivation to convert biogas to microalgae-based biodiesel, in which methanotroph was co-cultured to convert CH4 to organic intermediate (and CO2) for microalgal mixotrophic growth. This study constructed a co-culture of Methylocystis bryophila (methanotroph) and Scenedesmus obliquus (microalgae) with biogas feeding. Compared with the single culture of S. obliquus, higher microalgal biomass but a lower chlorophyll concentration was observed. The organic metabolism-related genes were upregulated, verifying microalgal mixotrophic growth. The stoichiometric calculation of M. bryophila culture shows that M. bryophila tends to release organic matter rather than grow under a low O2 content. M. bryophila rarely grew under five different light intensities, indicating that M. bryophila acts as a biocatalyst in the co-culture. The organic intermediate released by methanotroph increased the maximum biomass of microalgal culture, accelerated nitrogen absorption, accumulated more monounsaturated fatty acids, and improved the adaptation to light. The co-culture of microalgae and methanotroph may provide new opportunities for microalgae-based biodiesel production using biogas as a substrate.

Heterotrophic and mixotrophic cultivation of microalgae to simultaneously achieve furfural wastewater treatment and lipid production

Furfural wastewater (FWW) treatment is important in modern chemical production. However, traditional wastewater treatment methods are inappropriate for the treatment of FWW. In this work, Chlorella pyrenoidosa was employed to study the growth and pollutants removal with both heterotrophic and mixotrophic cultures. The results show that the biomass and removal efficiency for COD and TN were the highest under 10-fold dilution. However, TP removal were inconsistent when the algae were cultivated in both mixotrophic and heterotrophic modes. Compared to high nitrogen (0.75 g/L NaNO3), the algal cells grew faster when adding 0.25 g/L NaNO3 to the FWW, whether in mixotrophic or heterotrophic conditions. The total lipid content in heterotrophic conditions was 18.53%, which was higher than the values in mixotrophy when the concentration of NaNO3 was 0.75 g/L. Different carbon assimilation mechanisms of the algal cells result in a discrepancy in cell growth and pollutant removal, under different culture modes.

Mixotrophic cultivation of microalgae to enhance the biomass and lipid production with synergistic effect of red light and phytohormone IAA

Microalgal cultivation still presents a big challenge in commercial application due to the small size and low density of microalgae. The effect of phytohormones or light spectra on enhancing microalgal growth has been individually demonstrated at the bench scale. However, the synergistic action of phytohormones and light spectra on algal growth and lipid production remains unclear. In this study, the optimal carbon and nitrogen concentrations, as well as the optimum dosage of the phytohormone indole-3-acetic acid (IAA), for the growth of Chlorella vulgaris were first evaluated under mixotrophic cultivation. A light spectrum experiment was then conducted in an incubator equipped with white and red light. Results show that optimal mixotrophic cultivation conditions were 10 g L−1 glucose, 2250 mg L−1 sodium nitrate and 10 mg L−1 IAA. Under the optimum IAA treatment and irradiation with red light, the highest biomass, lipid content and lipid productivity were 28.34%, 3.53 g L−1 and 140.04 mg L−1 d−1, respectively. As compared to white light, the synergistic action of red light and IAA stimulated biomass production and lipid accumulation by algal cell metabolism and carbon source bioconversion. Furthermore, the post-optimized C. vulgaris had the potential for biodiesel production. These results provide new insights for the highly efficient cultivation of microalgae.

Mixotrophic Cultivation of Microalgae in Cassava Processing Wastewater for Simultaneous Treatment and Production of Lipid-Rich Biomass

Cassava processing wastewater (CPW) is a highly polluting, liquid residue of cassava processing, usually discarded or treated anaerobically. However, it can serve as a low-cost culture medium for microalgae. After a preliminary evaluation of the growth of 10 microalgal strains in diluted CPW, the microalgae Haematococcus pluvialis SAG 34−1b and Neochloris (Ettlia) oleoabundans UTEX 1185 were selected for cultivation in CPW without a supply of additional nutrients and evaluated for their growth, lipid production, and nutrients removal. Maximal biomass concentrations of 1.79 g·L−1 for H. pluvialis and 3.18 g·L−1 for N. oleoabundans were achieved with 25% CPW medium on the 13th day of growth. The algae H. pluvialis and N. oleoabundans removed 60.80 and 69.16% of the chemical oxygen demand, 51.06 and 58.19% of total nitrate, and 54.68 and 69.84% of phosphate, respectively. On average, lipid productivities reached 0.018 and 0.041 g·L−1 day−1 for H. pluvialis and N. oleoabundans, respectively. Therefore, cultivating these microalgae in diluted CPW is a promising treatment for cassava wastewater with simultaneous valuable biomass production.

Regulation of carbon source metabolism in mixotrophic microalgae cultivation in response to light intensity variation

Microalgae are one of the promising sources for renewable energy production, and the light intensity variation can affect the biofuel generation and carbon assimilation of mixotrophic microalgae. To reveal the response of carbon assimilation to light intensity, the effect of light intensity on the carbon source metabolism of Chlorella vulgaris under mixotrophic cultivation was investigated in this study. Moreover, the optimal carbon source composition for mixotrophic microalgae cultivation was evaluated using bicarbonate (HCO3−) and carbonate (CO32−) as inorganic carbon sources, and glucose and acetate as organic carbon sources. The optimal light intensity for Chlorella vulgaris growth was at the range of 8000–12000 lux. For the accumulation of biochemical components, low light intensity was beneficial to protein accumulation, and high light intensity was advantageous for carbohydrate and lipid accumulation. With HCO3− and glucose, the maximum lipid content reached 37.0% at a light intensity of 12000 lux. The citrate synthase activity was negatively correlated with light intensity, showing an opposite trend to biomass production. High light intensity had a positive impact on Rubisco expression, which promoted the microalgae growth and carbon fixing. The energy produced by heterotrophic metabolic activities increased at low light intensity, and the enhancement of biomass production with high light intensity was mainly caused by the improved photoreaction efficiency during the mixotrophic cultivation.

Sustainable biodiesel production from microalgae Graesiella emersonii through valorization of garden wastes-based vermicompost

Biodiesel production from microalgae has gained significant interest recently due to the growing energy demand and non-renewable nature of petroleum. However, high cost of production and environmental health related issues like excess use of inorganic fertilizers, eutrophication are the major constraints in commercial-scale biodiesel production. Besides this, solid wastes (garden-based) management is also a global concern. In the present study, to overcome these limitations vermicompost extract was tested as nutrient source to enhance growth performance and lipid production from a freshwater microalga (Graesiella emersonii MN877773). Garden wastes were first converted into vermicompost manure and its extract (aerobic and anaerobically digested) was prepared. The efficacy of the extract was then tested in combination with BG11 medium. The mixotrophic cultivation of microalgae in anaerobically digested vermicompost extract at 50:50 combination with BG11 medium enhanced the cell biomass (0.64 g d. wt. L−1) and lipid productivity (3.18 mg L−1 day−1) of microalgae by two times. Moreover, the combination also improved the saturated (methyl palmitate) and monounsaturated fatty acids (oleic acid) content in the test algae. The quality of biodiesel also complies with all the properties of biodiesel standard provided by India, the USA, and Europe except the cold filter plugging property. The combination was also found to improve the cell biomass (0.041 g L−1) as compared to BG11 medium in mass-scale cultivation. Hence, the study proved that G. emersonii grown in media supplemented with garden waste-based vermicompost extract had significant potential for mass-scale biodiesel and bioproduct production.

Enhancement of microalgal biomass, lipid production and biodiesel characteristics by mixotrophic cultivation using enzymatically hydrolyzed chitin waste

There is a growing interest for the mixotrophic cultivation of microalgae using sustainable natural carbon sources. Chitin from shrimp waste was hydrolyzed using chitinase from Trichoderma asperellum and was exploited for the mixotrophic cultivation of microalgae. Chitin saccharification was optimized using Box-Behnken design to produce 50.41% (w/w) of reducing sugars using chitin, 0.83% (w/v), chitinase, 2.92 (v/v), temperature, 40 °C and time, 4.48 h. The biomass productivity of Tetradesmus obliquus and Aphanocapsa sp. was promoted to 39.1 and 40.57 mg L−1 day−1 in the presence of 0.5 g L−1 of N-acetylglucosamine oligomers (GlcNAc), which was estimated to be ∼1.9 and ∼2-folds higher than the autotrophic conditions, respectively. Modified Logistic kinetic model confirmed higher growth rates and shortened lag-time of the mixotrophic cultures. The lipid productivity of both microalgae species was >1.5-times higher than the autotrophic conditions. Nitrate removal from the culture medium was found to be an effective strategy to increase both lipid content and lipid productivity for T. obliquus, but this strategy was not effective for Aphanocapsa sp. Fatty acid analysis revealed an increase of saturated fatty acids under mixotrophic cultivation for both microalgae species. Additionally, several biodiesel evaluation parameters indicated superior characteristics for the mixotrophic culture compared to the autotrophic conditions. Accordingly, mixotrophic growth of microalgae using GlcNAc as a new, sustainable carbon and nitrogen source opens a new avenue for biofuel production with an effective waste conversion into valuable commodities.

Mixotrophic biorefinery: A promising algal platform for sustainable biofuels and high value coproducts

Mixotrophic microalgae cultivation is becoming the most promising and sustainable process for sustainable biosynthesis of biochemicals and biofuels. It offers significantly higher productivity to cope up the key challenges to develop industrial algal processes. Mixotrophic cultivation strategy (MCS) leads to better productivity due to the unique metabolic capacity of microalgae by combining both photosynthesis and oxidative metabolic pathways for biomass generation. The capacity of the mixotrophic phenomenon is being investigated for sustainable bioprocess development ensuring higher energy recovery, economy, environmental and social benefits. However, developments are in pipeline and still to attain a commercial phase. In this article, recent technological developments in MC bioprocess for biofuels production are discussed; synergistic carbon and energy regulation, critical discussion up on commercially important organic carbon sources and their effects on MC bioprocessing have been demonstrated; Moreover, the prospective and challenges of higher-scale MC-linked bioprocessing and future direction on technology development have been addressed.

Optimum operational conditions for mixotrophic microalgae growth and nutrient recovery

Microalgae have been presented as microorganisms with great potential to recover nutrient from wastewater. Mixotrophic cultivation of microalgae in nutrient rich wastewater can help eliminating the deficiencies of both phototrophic and heterotrophic growth by allowing the independent optimisation of respiration and photosynthesis processes. Nutrient control and uptake by mixotrophic microalgae can be achieved either in a single or two-stage process using sequential reactors in a continuous flow system. Therefore, this work aims at studying mixotrophic microalgae growth in a two-stage biological process under continuous flow conditions with biomass recycle to recover nutrients from wastewater, considering the effects of different operational conditions (hydraulic retention time (HRT), cell retention time (CRT) and different nitrogen sources). The optimum operational conditions for algal nutrient uptake were identified to be 48 h HRT and 14 d CRT, using a mix of nitrogen sources (Ammonium-N to Nitrate-N ratio of 1:1) with 40.0% and 93.2% of phosphorus of nitrogen recovery in algal biomass, respectively.