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Cited by 84Algal biology and biofuel productionAquatic Ecosystems and Phytoplankton DynamicsAquaculture Nutrition and Growth

Challenges and Opportunities to Increase Economic Feasibility and Sustainability of Mixotrophic Cultivation of Green Microalgae of the Genus Chlorella

Open access
Dragone, Giuliano·2022·Renewable and Sustainable Energy Reviews
DOI 10.1016/j.rser.2022.112284
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Abstract

Green microalgae belonging to the genus Chlorella have long been used as health foods and additives for human consumption, as well as animal feed. These photosynthetic microorganisms have also found application in the bioremediation of wastewater and bio-mitigation of CO2. In the last decades, Chlorella microalgae have attracted considerable attention due to their great potential as feedstock for production of biofuels and high-value products. This review explores key aspects related to the cultivation of different Chlorella species under mixotrophic conditions. Common organic compounds and cheap substrates used for mixotrophic microalgal growth are presented. Bottlenecks and strategies to increase economic feasibility and sustainability of mixotrophic cultivation of Chlorella for future commercial production are also discussed. In this sense, the use of wastewater as a nutrient source for microalgae cultivation seems to be a promising alternative to improve the sustainability and economic feasibility of microalgae production systems. However, since wastewater presents a complex composition, methods to minimize the risk of contamination should be developed. Additionally, the screening of novel mixotrophic species of Chlorella capable of both assimilating a wide range of organic compounds from wastewater and accumulating high content of molecules of interest (e.g., lipids) would be highly recommended.

AI snapshot

Written from the paper’s full text.

TL;DR

This review examines mixotrophic cultivation of Chlorella as a route to improve biomass and metabolite productivity relative to photoautotrophic and heterotrophic modes, while reducing costs through cheap organic substrates and wastewater. It concludes that commercial-scale feasibility remains limited by contamination risk, organic substrate cost, and lack of integrated techno-economic and life-cycle assessments.

Key findings

  • Mixotrophic growth often raises biomass and lipid productivity: C. sorokiniana reached 1.325 g L⁻¹ with 10 g L⁻¹ glucose, 4.2× photoautotrophic growth, while C. minutissima reached 0.216 g L⁻¹ with 1% w/v glucose, 2× heterotrophic growth.
  • Organic carbon source performance is species- and strain-specific; glucose is often best, but high concentrations can inhibit growth (e.g., glucose above 100 g L⁻¹ or glycerol above 2% v/v significantly inhibited C. vulgaris biomass productivity; 5% v/v methanol was toxic to C. minutissima).
  • Wastewater and agro-industrial by-products are promising cheap substrates: hydrolysed cheese whey gave C. vulgaris specific growth rate 0.43 d⁻¹, final biomass 3.58 g L⁻¹, and biomass productivity 0.75 g L⁻¹ d⁻¹, which were 3.5, 2.9, and 7.5 times higher than photoautotrophic conditions; immobilized Chlorella on textile wastewater achieved NH₄⁺-N, COD, and colour removal efficiencies as high as 90%, 75%, and 60%.
  • Sustainability assessments suggest benefits but remain limited: mixotrophic C. vulgaris with glucose in a hybrid system led to net GWP and FER savings approaching 76% and 75% relative to fossil diesel, while glucose cost has been estimated at about 80% of total medium cost; comprehensive industrial-scale economic evaluations of mixotrophic versus autotrophic or heterotrophic cultivation have not been reported.

Methods

  • Design: narrative review of Chlorella mixotrophic cultivation, covering nutritional modes, organic carbon sources, low-cost substrates, contamination strategies, metabolites of interest, and sustainability indicators.
  • Data or sample: no primary experimental sample; the review synthesizes published studies and includes tables of organic compounds used for mixotrophic growth, wastewater compositions (COD, TN, TP), and biomass/lipid productivity under mixotrophic conditions.
  • Main analysis: qualitative synthesis and comparison of reported growth, metabolite, economic, and environmental metrics; no systematic review protocol or meta-analysis is described.

Limitations

  • Stated: examples of successful commercial-scale facilities using mixotrophic Chlorella cultivation are scarce; comprehensive economic evaluations of mixotrophic growth compared with autotrophic or heterotrophic modes at industrial scale have not been reported; TEA and LCA are needed using data from experimental mixotrophic trials with cheap organic carbon sources and large-scale systems.
  • Stated: wastewater use is constrained by complex and variable composition, contamination risk, and the fact that biomass produced on wastewater should mainly be used for biofuels rather than food or feed applications.
  • Evident from the text: as a narrative review, it does not report a systematic search strategy, inclusion criteria, or quantitative meta-analysis, so conclusions depend on the selected cited studies.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1016/j.rser.2022.112284

Cites

132 of 132 references verified0 citation errors · 0 not checkable

Statistics

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Claims the paper makes

0 of 108 cited sources checked · 0 supported · 0 flagged

  • Interpretationreasoning

    Mixotrophic processes are a feasible alternative to overcome the limitations of pure photoautotrophic cultures.

    “Consequently, significant attention has been given to mixotrophic processes as a feasible alternative to overcome the limitations of pure photoautotrophic cultures.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Commercial production of biofuels and high-value products from microalgae is still limited.
  • Premisereasoning

    Photoautotrophic cultivation has difficulty achieving high biomass concentration because light penetration is inversely proportional to the turbidity of the culture medium.

    “However, photoautotrophic cultivation has difficulty achieving high biomass concentration since light penetration is inversely proportional to the turbidity of the culture medium.”
    Quote in the paperSubstantial claim
  • Premisecitation

    Several studies have demonstrated that cultivation of mixotrophic microalgae results in greater biomass concentration when compared to photoautotrophic growth.

    “Several studies have demonstrated that cultivation of mixotrophic microalgae results in greater biomass concentration when compared to photoautotrophic growth [27][28][29].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [27][28][29]

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  • Premisecitation

    The economic viability of producing microalgal biomass remains a major hurdle to its commercialization.

    “The economic viability of producing microalgal biomass remains a major hurdle to its commercialization [13][14][15].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [13][14][15]

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  • Premisereasoning

    Net photosynthesis cannot take place in deep regions of microalgal cultivation systems at high cell densities due to strong attenuation of light.

    “Therefore, net photosynthesis cannot take place in deep regions of microalgal cultivation systems at high cell densities due to the strong attenuation of light.”
    Quote in the paperSubstantial claim
  • Classified as probably not a substantial claim. 17
  • May combine two claims. 31

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Chlorella can remove soluble organic compounds and nutrients such as N and P from industrial and municipal effluents.

    “Moreover, Chlorella can be used to remove soluble organic compounds and other nutrients (e.g., N and P) from industrial and municipal effluents [7,8].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [7] Mohsenpour, 2021, Integrating micro-algae into wastewater treatment: a review, Sci Total Environ source

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    • cites [8] Koutra, 2021, Assessing the potential of Chlorella vulgaris for valorization of liquid digestates from agro-industrial and municipal organic wastes in a biorefinery approach, J Clean Prod source

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  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Under mixotrophic conditions, both organic compounds and CO2 are necessary for growth.

    “Under mixotrophic conditions, both organic compounds and CO 2 are necessary for growth [22].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [22] Dragone, 2010, Third generation biofuels from microalgae

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  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Chlorella biomass is used in aquaculture as a feed supplement to enhance skin pigmentation of ornamental fish and improve immunity and growth of fish and crustaceans.

    “Chlorella biomass also finds application in aquaculture as feed supplement to enhance the skin pigmentation of ornamental fish or to improve the immunity and growth of fish and crustaceans, among others [2,3].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [2] Ahmad, 2020, Applications of microalga Chlorella vulgaris in aquaculture, Rev Aquacult source

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    • cites [3] Sergejevová, 2012, Chlorella biomass as feed supplement for freshwater fish: sterlet, Acipenser ruthenus, Aquacult Res source

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  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Chlorella has been cultivated at commercial scale in Taiwan and Japan since the early 1960s to produce nutritional supplements and healthy foods, owing to its high content of proteins, vitamins and minerals.

    “These microalgae have been cultivated at commercial scale in Taiwan and Japan since the early 1960s to produce nutritional supplements and healthy foods, owing to their high content of proteins, vitamins and minerals [1].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [1] Spolaore, 2006, Commercial applications of microalgae, J Biosci Bioeng source

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    Premisecitation

    Some Chlorella species accumulate large amounts of starch under stressful conditions, while others synthesize neutral lipids as triacylglycerol; their biomass is a promising feedstock for bioethanol or biodiesel respectively.

    “Some species of Chlorella have the ability to accumulate large amounts of starch under stressful culture conditions (e.g., nitrogen starvation), while others synthesize neutral lipids in the form of triacylglycerol (TAG) instead; thus, the biomass of these microalgal species has been considered as a promising feedstock for producing bioethanol or biodiesel, respectively [4][5][6].”
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    • cites [4][5][6]

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    Premisecitation

    Several studies have suggested that wastewater can be used as a nutrient source for microalgae cultivation and this could significantly improve the sustainability and economic feasibility of microalgae production systems at large scale.

    “Several studies have suggested that wastewater can be used as a nutrient source for microalgae cultivation and this could significantly improve the sustainability and economic feasibility of microalgae production systems at large scale [78][79][80].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [78][79][80]

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    • Rests on an assumption: Contamination risks in wastewater-based mixotrophic cultivation can be managed without prohibitive cost.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Nutrient uptake also depends on environmental factors that affect microalgae growth such as light intensity, temperature, pH, turbidity and colour.

    “Moreover, nutrient uptake also depends on environmental factors that affect microalgae growth such as light intensity, temperature, pH, turbidity and colour, among others [89].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [89] Molinuevo-Salces, 2019, Microalgae and wastewater treatment: advantages and disadvantages

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  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Interpretationnone given

    Immobilization of Chlorella cells within natural biopolymers such as alginate could potentially overcome microalgae harvesting issues at commercial scale by preventing biomass from sticking on external surfaces.

    “Immobilization of Chlorella cells within natural biopolymers, such as alginate, could potentially overcome microalgae harvesting issues at commercial scale by preventing biomass from sticking on external surfaces.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    An N:P ratio ranging between 8:1 and 45:1 is an important macronutrient requirement for freshwater microalgae cultivation.

    “Other study [92] suggested N:P ratio, ranging between 8:1 and 45:1, is an important macronutrient requirement for freshwater microalgae cultivation.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [92] Whitton, 2016, Influence of microalgal N and P composition on wastewater nutrient remediation, Water Res source

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  • May combine two claims.

    Interpretationreasoning

    Under high light intensities exceeding saturation, mixotrophic and photoautotrophic growth rates of C. pyrenoidosa tend to equalize, implying that no benefit might be derived by using an organic carbon source.

    “Under high light intensities (e.g., those exceeding the intensity of saturation), the mixotrophic and photoautotrophic growth rates of that microalga tend to equalize, implying that no benefit might be derived by using an organic carbon source.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Mixotrophic microalgae belonging to Chlorella, Chlamydomonas and Scenedesmus were capable of growing on wastewater and poultry litter extract and produced higher biomass concentration than that achieved on freshwater-based enriched growth media (BG11).

    “Mixotrophic microalgae belonging to the genera Chlorella, Chlamydomonas and Scenedesmus were also capable of growing on wastewater and poultry litter extract (PLE) and produced higher biomass concentration than that achieved on freshwater-based enriched growth media (BG11) [29].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [29] Bhatnagar, 2011, Renewable biomass production by mixotrophic algae in the presence of various carbon sources and wastewaters, Appl Energy source

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    Premisereasoning

    The overall wastewater composition affects nutrient uptake, with an optimal C:N:P ratio that differs between microalgae species.

    “The overall wastewater composition affects nutrient uptake, existing an optimal C:N:P ratio, which differs between microalgae species.”
    Quote in the paperNot a substantial claim

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    Premisecitation

    Although centrate had higher concentrations of nitrogen, phosphorus and COD than primary and secondary effluents, the growth rate of C. vulgaris was significantly higher in primary effluent (0.61 d-1) with a C:N:P ratio of 24:5:1 than in centrate (0.52 d-1) and secondary effluent (0.25 d-1).

    “On the other hand, AlMomani and Örmeci [82] found that although centrate had higher concentrations of nitrogen, phosphorus and COD than the primary and secondary effluents (Table 2), the growth rate of C. vulgaris was significantly higher in primary effluent (0.61 d -1 ) with a C:N:P ratio of 24:5:1 than those obtained in centrate (0.52 d -1 ) and secondary effluent (0.25 d -1 ).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [82] AlMomani, 2016, Performance of Chlorella vulgaris, Neochloris oleoabundans, and mixed indigenous microalgae for treatment of primary effluent, secondary effluent and centrate, Ecol Eng source

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  • May combine two claims.

    Interpretationreasoning

    In mixotrophic cultures, as organic substrates are assimilated, cell growth is not strictly dependent on photosynthesis, so light is no longer a limiting factor and photo-limitation can be reduced at low and moderate light intensities.

    “As organic substrates are assimilated in mixotrophic cultures, cell growth is not strictly dependent on photosynthesis and hence, light is no longer a limiting factor for microalgal growth; thus, photo-limitation can be reduced in mixotrophic cultivation systems at low and moderate light intensities.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Mixotrophic cultivation may increase biomass production and improve lipid content, which is important for production of microalgal biodiesel.

    “Mixotrophic cultivation may not only increase biomass production, but also improve lipid content which is of great importance for the production of microalgal biodiesel [28].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [28] Wan, 2011, The effect of mixotrophy on microalgal growth, lipid content, and expression levels of three pathway genes in Chlorella sorokiniana, Appl Microbiol Biotechnol source

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    Premisecitation

    Monosaccharides had more significant growth-promoting effect on C. pyrenoidosa than disaccharides at different light intensities (1080 and 1800 lux); among hexoses, galactose gave the highest mixotrophic cell growth, followed by glucose and fructose.

    “A different study found that monosaccharides had more significant growth-promoting effect on C. pyrenoidosa than disaccharides (cellobiose, maltose and sucrose) at different light intensities (1080 and 1800 lux); and, among the hexoses, galactose gave the highest mixotrophic cell growth, followed by glucose and fructose [70].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [70] Dvořáková-Hladká, 1966, Utilization of organic substrates during mixotrophic and heterotrophic cultivation of algae, Biol Plant (Prague) source

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    Premisecitation

    Supplementing photosynthetic cultures with an organic carbon source provides an extra source of energy and reduced carbon, stimulating biomass production while reducing biomass losses associated with respiration.

    “Supplementing photosynthetic cultures with an organic carbon source provides an extra source of both energy and reduced carbon, which stimulate the production of biomass while simultaneously reducing biomass losses associated with respiration [30].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [30] Park, 2012, Mixotrophic and photoautotrophic cultivation of 14 microalgae isolates from Saskatchewan, Canada: potential applications for wastewater remediation for biofuel production, J Appl Phycol source

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    Premisenone given

    Carbohydrates (e.g., glucose), acetate and glycerol have been the most common organic compounds used for the growth of mixotrophic species of Chlorella.

    “Carbohydrates (e.g., glucose), acetate and glycerol have been the most common organic compounds used for the growth of mixotrophic species of Chlorella (Table 1).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Specific growth rate (0.43 d-1), final biomass concentration (3.58 g/L) and biomass productivity (0.75 g/L/d) under mixotrophic conditions with hydrolysed cheese whey were 3.5, 2.9 and 7.5 times higher respectively than under photoautotrophic conditions.

    “Specific growth rate (0.43 d -1 ), final biomass concentration (3.58 g L -1 ) and biomass productivity (0.75 g L -1 d -1 ) under mixotrophic conditions were found to be 3.5, 2.9 and 7.5 times higher respectively than those obtained under photoautotrophic conditions [16].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [16] Abreu, 2012, Mixotrophic cultivation of Chlorella vulgaris using industrial dairy waste as organic carbon source, Bioresour Technol source

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  • May combine two claims.

    Premisecitation

    Acetate is also a non-fermentative substrate and does not support anaerobic growth of bacteria.

    “Acetate is also a non-fermentative substrate and does not support anaerobic growth of bacteria [103].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [103] Ganuza, 2016, Heliae development, LLC: an industrial approach to mixotrophy in microalgae

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    Premisecitation

    For some Chlorella strains and culture conditions, mixotrophic culture has no positive effect on cell lipid content, so the increase in lipid productivity is mainly due to increased biomass productivity.

    “For some Chlorella strains and under certain culture conditions (e.g., C. sorokiniana cultivated in secondary municipal wastewater supplemented with glycerol [30]), there is no positive effect of mixotrophic culture on cell lipid content; thus, the increase in lipid productivity is mainly due to increases in biomass productivity [105].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [30] Park, 2012, Mixotrophic and photoautotrophic cultivation of 14 microalgae isolates from Saskatchewan, Canada: potential applications for wastewater remediation for biofuel production, J Appl Phycol source

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    • cites [105] Ogbonna, 2015

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    Interpretationcitation

    Acetate may be a good choice as organic carbon source; continuous supplementation of acetate in small quantities may reduce the chance of contamination in Chlorella cultures.

    “In this context, acetate may be a good choice as organic carbon source; continuous supplementation of acetate in small quantities may reduce the chance of contamination in Chlorella cultures [12].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [12] Liu, 2014

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    Premisecitation

    At the end of the experiment, no differences in cell densities among the three cultures were observed.

    “At the end of the experiment, no differences in cell densities among the three cultures were observed.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [106] Marchello, 2018, Physiological and ecological aspects of Chlorella sorokiniana (Trebouxiophyceae) under photoautotrophic and mixotrophic conditions, Microb Ecol source

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  • May combine two claims.

    Premisecitation

    Methods for avoiding undesired contamination include sterilization and operation under sterile conditions, use of excessive inoculation concentration, and multi-stage culture systems; however, all these methods increase production costs at large commercial scale.

    “Methods for avoiding this type of undesired contamination include sterilization and operation under sterile conditions, use of excessive inoculation concentration as well as multi-stage culture systems; however, all these methods will increase production costs at large commercial scale [27].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [27] Lowrey, 2015, Heterotrophic and mixotrophic cultivation of microalgae for biodiesel production in agricultural wastewaters and associated challenges—a critical review, J Appl Phycol source

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    Premisecitation

    The cost of glucose is estimated to be about 80% of the total medium cost, making mixotrophic cultivation systems economically unfeasible.

    “For instance, the cost of glucose has been estimated to be about 80% of the total medium cost [108], making mixotrophic cultivation systems economically unfeasible.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [108] Li, 2007, Large-scale biodiesel production from microalga Chlorella protothecoides through heterotrophic cultivation in bioreactors, Biotechnol Bioeng source

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  • May combine two claims.

    Premisecitation

    For mixotrophic cultivation of C. pyrenoidosa, increasing acetate concentration up to 10 g/L improved neutral lipid content and increased concentrations of saturated (C16:0, C18:0) and monosaturated (C16:1, C18:1) fatty acids.

    “An improvement of the neutral lipids content and higher concentrations of saturated (e.g., C16:0 and C18:0) and monosaturated fatty acids (e.g., C16:1 and C18:1), was also observed for mixotrophic cultivation of C. pyrenoidosa when acetate concentration was increased up to 10 g L -1 [45].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [45] Liu, 2018, Lipid accumulation of Chlorella pyrenoidosa under mixotrophic cultivation using acetate and ammonium, Bioresour Technol source

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    Premisecitation

    Enclosed photobioreactor systems are generally costly to build and operate, and are technically very difficult to scale-up, mainly due to challenges in maintaining optimum cultivation conditions at large scale.

    “However, these culture systems are generally costly to build and operate, and are technically very difficult to scale-up, mainly due to the challenges in maintaining optimum cultivation conditions at large scale [99].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [99] Wang, 2014, Mixotrophic cultivation of microalgae for biodiesel production: status and prospects, Appl Biochem Biotechnol source

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    Premisecitation

    Microalgae synthesize and accumulate carbohydrates that act as energy and organic carbon storage products inside cells and as structural components in cell walls.

    “Microalgae synthesize and accumulate a wide range of carbohydrates that serve two main purposes: they act as energy and organic carbon storage products inside microalgal cells and as structural components in cell walls [119].”
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    • cites [119] Raven, 2003, Carbohydrate metabolism and respiration in algae source

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    Premisecitation

    The addition of organic substrates can be carried out in a controlled way in enclosed photobioreactors, allowing much higher areal productivities.

    “The addition of organic substrates can be carried out in a controlled way, allowing much higher areal productivities [100].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [100] Bassi, 2014, Mixotrophic algae cultivation for energy production and other applications

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    Premisereasoning

    Enclosed photobioreactor systems offer the advantages of microalgae cultivation under controlled conditions, preventing the invasion of undesirable microorganisms.

    “Enclosed photobioreactor systems offer the advantages of microalgae cultivation under controlled conditions, preventing the invasion of undesirable microorganisms.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Triglycerides can be extracted from microalgal cultures and transesterified to produce fatty acid methyl esters (FAMEs), which are one of the primary molecular components of biodiesel.

    “Triglycerides can be extracted from microalgal cultures and transesterified to produce fatty acid methyl esters (FAMEs), which are one of the primary molecular components of biodiesel [111].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [111] Cameron Coates, 2013, Bioactive and novel chemicals from microalgae

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  • May combine two claims.

    Interpretationreasoning

    Higher lipid productivity under mixotrophic growth compared with photoautotrophic growth can result from combined increases in biomass productivity and lipid content, or from increased biomass productivity at relatively constant lipid content.

    “It is worth noting that the higher lipid productivity under mixotrophic growth conditions as compared to photoautotrophic conditions can result from the combined increases in biomass productivity and lipid content, or from increased biomass productivity at relatively constant lipid content.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Chlorella was successfully cultivated under mixotrophic mode in a 10-L outdoor enclosed tubular photobioreactor sterilized with 2-6% v/v hydrogen peroxide solution, but axenic microalgal cultivation could not be maintained when the culture volume was scaled-up to 300 L.

    “Chlorella was successfully cultivated under mixotrophic mode in a 10-L outdoor enclosed tubular photobioreactor, sterilized previously with 2-6% v/v hydrogen peroxide solution [98]; but, axenic microalgal cultivation could not be maintained when the culture volume was scaled-up to 300 L [101].”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [98] Lee, 1996, Mixotrophic growth of Chlorella sorokiniana in outdoor enclosed photobioreactor, J Appl Phycol source

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    • cites [101] Lee, 2001, Microalgal mass culture systems and methods: their limitation and potential, J Appl Phycol source

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  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Methanol has been proposed as a relatively cheap organic substrate that could maintain both the sterility of the mixotrophic culture and high biomass concentration of Chlorella sp. in long-term open pond systems.

    “Methanol has also been proposed as a relatively cheap organic substrate that could maintain both the sterility of the mixotrophic culture and high biomass concentration of Chlorella sp. in long-term open pond systems [104].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [104] Choi, 2011, Effects of methanol on cell growth and lipid production from mixotrophic cultivation of Chlorella sp, Biotechnol Bioproc Eng source

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  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    The major part of the nonpolar lipids (neutral lipids) of microalgae are triglycerides and free fatty acids, whereas the polar lipids are essentially glycerides in which one or more fatty acids has been replaced with a polar group.

    “The major part of the nonpolar lipids (neutral lipids) of microalgae are triglycerides and free fatty acids, whereas the polar lipids are essentially glycerides in which one or more of the fatty acids has been replaced with a polar group, for instance phospholipids and glycolipids [110].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [110] Becker, 2003, Microalgae in human and animal nutrition

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  • May combine two claims.

    Premisecitation

    When glucose was supplemented for mixotrophic cultivation of C. protothecoides, cells accumulated storage neutral lipids rather than membrane polar lipids, and fatty acid composition changed from polyunsaturated to saturated and monosaturated, favouring biodiesel production.

    “When glucose was supplemented as an organic carbon source for mixotrophic cultivation of C. protothecoides, microalgal cells accumulated storage neutral lipids rather than membrane polar lipids, while the composition of fatty acids changed from polyunsaturated to saturated and monosaturated, which favours the production of biodiesel [42].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [42] Ren, 2016, Glucose feeding recalibrates carbon flux distribution and favours lipid accumulation in Chlorella protothecoides through cell energetic management, Algal Res source

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    Premisecitation

    Almost 90% of BOD from unsterilized secondary wastewater was reduced by a co-existing C. vulgaris-bacteria system under mixotrophic conditions, whereas no substantial reduction in BOD concentration was recorded for sterile secondary wastewater treated by C. vulgaris alone.

    “It was reported that almost 90% of BOD from unsterilized secondary wastewater was reduced by a co-existing C. vulgarisbacteria system under mixotrophic conditions, whereas no substantial reduction in BOD concentration was recorded for sterile secondary wastewater treated by C. vulgaris alone [97].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [97] He, 2013, The combined effect of bacteria and Chlorella vulgaris on the treatment of municipal wastewaters, Bioresour Technol source

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  • May combine two claims.

    Premisecitation

    Addition of 5 up to 15 g/L glucose led to a substantial increase in lipid accumulation in C. sorokiniana, but lipid content declined at glucose concentrations higher than 20 g/L.

    “The addition of 5 up to 15 g L -1 glucose led to a substantial increase in the accumulation of lipids, but the lipid content declined at glucose concentrations higher than 20 g L -1 [28].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [28] Wan, 2011, The effect of mixotrophy on microalgal growth, lipid content, and expression levels of three pathway genes in Chlorella sorokiniana, Appl Microbiol Biotechnol source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Both the biomass productivity (2.5 g/L/d) and lipid content (43%) of microalgal cells grown mixotrophically on dry-grind ethanol thin stillage were higher than those obtained on modified basal medium containing glucose.

    “Both the biomass productivity (2.5 g L -1 d -1 ) and lipid content (43%) of microalgal cells grown mixotrophically on TS were higher than those obtained on modified basal medium containing glucose [95].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [95] Mitra, 2012, Heterotrophic/mixotrophic cultivation of oleaginous Chlorella vulgaris on industrial co-products, Algal Res source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S4 (based).

    Interpretationnone given

    To date, examples of successful facilities using mixotrophic cultivation of Chlorella at commercial scale are scarce.

    “Cultivation of Chlorella species under mixotrophic conditions offers the possibility of greatly increasing microalgal biomass and accumulation of valuable intracellular metabolites but, to date, examples of successful facilities using this culture regime at commercial-scale are scarce.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S2 (checkable). May combine two claims.

    Interpretationreasoning

    Although mixotrophic growth of Chlorella strains has potential to improve environmental and economic aspects of microalgal biofuel production, more research is needed to demonstrate the sustainability of this cultivation system.

    “These examples clearly show that although mixotrophic growth of Chlorella strains has potential to improve environmental and economic aspects of microalgal biofuel production, more research is needed to demonstrate the sustainability of this cultivation system.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationnone given

    At high cell densities, light becomes limiting and photoautotrophic metabolic activity is reduced compared with heterotrophic mode.

    “At high cell densities, light becomes limiting and the photoautotrophic metabolic activity is reduced in comparison to that of heterotrophic mode.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Photosynthetic pigments in microalgae exhibit beneficial biological activities such as anti-carcinogenic, anti-inflammatory, antioxidant, anti-obesity, anti-angiogenic and neuroprotective.

    “These pigments have received great attention since they exhibit several beneficial biological activities such as anti-carcinogenic, anti-inflammatory, antioxidant, anti-obesity, anti-angiogenic and neuroprotective [122].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [122] Cuellar‐Bermudez, 2015, Extraction and purification of high‐value metabolites from microalgae: essential lipids, astaxanthin and phycobiliproteins, Microb Biotechnol source

      Not checked in the free run.

  • May combine two claims.

    Interpretationcitation

    Higher capital costs of the cultivation system require a greater amount of biomass to be diverted to achieve economic parity, which can prevent the system from functioning primarily as a biofuel production platform.

    “On the other hand, the higher the capital costs of the cultivation system (e.g., increased investment in costly photobioreactors), the greater the amount of biomass that should be diverted to achieve economic parity, thus no longer making the microalgae cultivation system to function primarily as a platform for biofuel production [132].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [132] Somers, 2021, Techno-economic and life-cycle assessment of fuel production from mixotrophic Galdieria sulphuraria microalgae on hydrolysate, Algal Res source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S4 (based).

    Interpretationnone given

    Integration of microalgal biomass production with wastewater treatment will improve the economic feasibility of commercial-scale cultivation of Chlorella species.

    “Integration of microalgal biomass production with wastewater treatment will improve the economic feasibility of commercial scale cultivation of Chlorella species.”
    Quote in the paperNot a substantial claim
45 of 45

  • The paper relies on this without support.

    Commercial production of biofuels and high-value products from microalgae is still limited.

    “However, although several companies, research institutes and governments have invested in microalgae cultivation for many years, at present the commercial production of biofuels and high-value products from these photosynthetic microorganisms is still limited.”

  • The paper relies on this without support.

    Contamination risks in wastewater-based mixotrophic cultivation can be managed without prohibitive cost.

    “However, since wastewater presents a complex composition, methods to minimize the risk of contamination should be developed.”

  • The paper relies on this without support.

    The composition of wastewater is complex and diverse.

    “as the composition of wastewater is complex and diverse”
3 of 3

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • In PBRs with high concentrations of organic carbon, the proliferation of competing microorganisms such as bacteria can lead to medium turbidity and reduced light penetration, which directly suppresses the growth of the microalgal biomass.

    “In PBRs with high concentrations of organic carbon, the proliferation of competing microorganisms such as bacteria can lead to medium turbidity and reduced light penetration, which directly suppresses the growth of the microalgal biomass [123].”

    From Multi-Sensing Monitoring of the Microalgae Biomass Cultivation Systems for Biofuels and Added Value Products Synthesis—Challenges and Opportunities (2025) · cites this paper as [123]

    Section 6 says fast-growing competing microorganisms, including bacteria, can reduce microalgal biomass yields. Section 3 says turbidity reduces light penetration and describes this problem in raw wastewater, but it does not say that bacterial proliferation causes the turbidity. The claimed chain from bacteria to turbidity to suppressed growth is therefore not fully supported. (LLM, conf=0.86)

    Experimental — accuracy not yet measured

  • Microalgae require significantly fewer chemical inputs such as pesticides and synthetic fertilisers, minimising risks of soil degradation and water pollution.

    “microalgae require significantly fewer chemical inputs, such as pesticides and synthetic fertilisers, thus minimising the risks of soil degradation and water pollution [116].”

    From Microalgae Bioactives for Functional Food Innovation and Health Promotion (2025) · cites this paper as [116]

    Section 5 (page unavailable) says wastewater can supply nutrients for microalgae cultivation as an alternative to defined synthetic culture media. It does not establish that microalgae require significantly fewer pesticides or synthetic fertilisers, or that their cultivation minimises soil degradation and water pollution. (LLM, conf=0.78)

    Experimental — accuracy not yet measured

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Glucose-grown C. protothecoides lipid content differs between text and table

    Major

    The text reports about 25% lipid content for C. protothecoides grown with 15 g L -1 glucose, but Table 3 lists 14.1% for that condition. This discrepancy affects the discussion of lipid production under mixotrophic cultivation.

1 of 1

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Authors
Dragone, G.
Affiliations
Technical University of Denmark (DK)
Corresponding author
Dragone, Giuliano
ORCID
Dragone, Giuliano
Venue
Renewable and Sustainable Energy Reviews
Publisher
Elsevier BV
Published
2022-05
Volume
160
Pages
112284
ISSN
1364-0321 (ISSN-L)
DOI
10.1016/j.rser.2022.112284
OpenAlex
W4214835808
Semantic Scholar
1e17792dc7ffba2da5ea918855ad92bb30b798fb
Cited by
84
References
131
Citations by year
2026: 12 · 2025: 19 · 2024: 22 · 2023: 22 · 2022: 9
FWCI
3.25
Type
journal article
Language
en
Open access
Licence
Locations
  • Renewable and Sustainable Energy Reviews (publishedVersion, cc-by)
  • RePEc: Research Papers in Economics (submittedVersion)
  • Technical University of Denmark, DTU Orbit (Technical University of Denmark, DTU) (submittedVersion, cc-by)
Funders
Novo Nordisk Fonden (NNF20SA0066233)
Topics
  • Physical Sciences › Energy › Renewable Energy, Sustainability and the Environment › Algal biology and biofuel production
  • Physical Sciences › Environmental Science › Environmental Chemistry › Aquatic Ecosystems and Phytoplankton Dynamics
  • Life Sciences › Agricultural and Biological Sciences › Aquatic Science › Aquaculture Nutrition and Growth
Keywords
Chlorella, mixotrophic cultivation, green microalgae, economic feasibility, sustainability, water reuse, organic substrates, biofuels, microalgal lipids, contamination risk, CO2 emission reduction, bioremediation

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Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-07.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-07.

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