Wed Oct 26, 2016 – Track 3

Dries Vandamme, PhDVisiting Researcher UNSW Sydney AustraliaKU Leuven - BelgiumMolecular and Microbial SystemsLaboratory for Aquatic BiologyDries.vandamme@kuleuven.be

Mixotropic growth of microalgae revisited

opportunities and challenges

Outline

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• Growth modes => cultivation strategy• Species• Hetero- and Mixotrophy: advantages• Limitation and opportunities

o Carbon sourceo Contaminationo Bioreactorso Harvestingo Composition and products

• Conclusions

Growth modes of microalgae cultivation

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Growth mode Energy source

Carbon source

Light requirement

metabolism

Photo-autotrophic Light Inorganic Obligatory no switchHeterotrophic Organic Organic - switchPhotoheterotrophic Light Organic Obligatory switchMixotrophic Light and

organicInorganicand organic

Not Obligatory Simultaneousutilization

Benemann J ABBB 2016

Cultivation strategies: single stage vs sequential

Species dependency

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Growth mode SpeciesPhoto-autotrophic Most species are obligate autotrophsHeterotrophic Amphora, Ankistrodesmus, Chlamydomonas,

Chlorella, Chlorococcum, Crypthecodinium, Cyclotella, Dunaliella, Euglena, Nannochloropsis, Nitzschia, Ochromonas, and Tetraselmis

Mixotrophic Brachiomonas submarina , Chlorella spp., Chlorococcum sp ., Cyclotella cryptica , Euglena gracilis , Haematococcus pluvialis , Nannochloropsisspp ., Navicula saprophila , Nitzschia sp., Ochromonas minima, Phaeodactylum tricornutum , Rhodomonas reticulate and Scenedesmus obliquus Anabaena , Spirulina and Synechococcus

Advantages

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Heterotrophic MixotrophicHigher growth rates and biomass density

Higher growth rates by shortening growth cycles

Higher total lipid content per dry cell weight

Prolonged exponential phase

Higher biomass productivity Reduction of biomass loss from respiration during dark hours

Wastewater remediation: DOC, N, P removal

Reduction of photo-inhibitory effect

Flexibility of cultivation regime

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Babaei et al. 2016

C. vulgaris

Limitations and opportunities

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LimitationsCost for carbon sourceCompetition by fast-growing bacteriaBioreactor implementation/OPEXDownstream processing costs

Opportunities?

Carbon source

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• Glucose• Carboxylic

acids• Alcohols

Perez-Garcia & Bashan 2014

Carbon source

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opportunitiesCheap (waste) organic carbon sourcesStrain bio-prospectionMetabolic engineeringImprove lignocellulosic degradation

Lowrey et al. 2015

Contamination/competition

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opportunitiesMixotrophic cultivation strategiesEstablish cultures in bacteria adverse conditionsBioprospection of fast growing strainsCofactor symbiosis

Deschênes et al. 2016

• Fast growing microorganismso Competition with

microalgaeo Alternate/simultaneous feed

N/glucose

Contamination/competition

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• symbiosis o E. coli's provision of

thiamine derivatives and degradation products

o A. protothecoides growth stimulation, lipid content, glucose uptake

opportunitiesMixotrophic cultivation strategiesEstablish cultures in bacteria adverse conditionsBioprospection of fast growing strainsCofactor symbiosis

Higgins et al. 2016

• Fast growing microorganismso Competition with

microalgae

Bioreactors

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• Oswald et al. (1950s) HRAPopportunitiesCheaper materialsImplementation of renewable energy resourcesNon axenic open pond systemsRA of GMO in large scale facilities

Technical aspect Photo-auto Hetero MixoBioreactor type Photobioreactor

(open/closed)Fermenters Photo-bioreactor

(open/closed)Surface to volume ratio

high low high

Sterility Usually sanitized Sterility required Sterility preferredContamination risk medium high medium

Harvesting using bioflocculation

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opportunitiesExopolysaccharides => bioflocculationImmobilisation strategiesBioflocculation signaling molecules

Alam, M.A., Vandamme, D., Chun, W. et al. Rev Environ Sci Biotechnol (2016). doi:10.1007/s11157-016-9408-8

• Settling Micractinium Benemann et al. 1980

Composition and products

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Compound (%) Photo-autotrophic

Heterotrophic Mixotrophic

Protein 29-60 10-40 32-40Carbohydrates 10-20 15-45 10-30Lipids (total) 3-28 25-60 11-58

Prokop et al. 2015

Composition and products

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Fraction Compound Auto hetero mixoLipids TAG medium high high

isoprenoids medium high mediumOmega-3 FA high medium highhydrocarbons medium high medium

Carbohydrates Starch/glycogen low high lowProteins soluble high low high

insoluble high low mediumPigments chlorophylls high low medium

B-carotene high low mediumLutein medium high highAstaxanthin medium high high

Conclusions

• R&D should focus on:

o Lowering cost of carbon substrateso Bio-prospecting and metabolically engineeringo Determining the potential of consortia in natural co-cultivationo Design of suitable bioreactors in cheap materialso Optimizing downstream processing

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Key referencesAndrade, M.R., Costa, J.A. V, 2007. Mixotrophic cultivation of microalga Spirulina platensis using molasses as organic substrate. Aquaculture 264, 130–134. doi:10.1016/j.aquaculture.2006.11.021

Babaei, A., Mehrnia, M.R., Shayegan, J., Sarrafzadeh, M.H., 2016. Comparison of different trophic cultivations in microalgal membrane bioreactor containing N-riched wastewater forsimultaneous nutrient removal and biomass production. Process Biochemistry 51, 1568–1575. doi:10.1016/j.procbio.2016.06.011

Ceron Garcia, M.C., Garcia Camacho, F., Sanchez Miron, A., Fernandez Sevilla, J.M., Chisti, Y., Molina Grima, E., 2006. Mixotrophic production of marine microalga Phaeodactylumtricornutum on various carbon sources. Journal of Microbiology and Biotechnology 16, 689–694.

Chaiprapat, S., Sasibunyarat, T., Charnnok, B., Cheirsilp, B., 2016. Intensifying Clean Energy Production Through Cultivating Mixotrophic Microalgae from Digestates of Biogas Systems: Effects of Light Intensity, Medium Dilution, and Cultivating Time. BioEnergy Research. doi:10.1007/s12155-016-9780-9

Chojnacka, K., Noworyta, A., 2004. Evaluation of Spirulina sp. growth in photoautotrophic, heterotrophic and mixotrophic cultures. Enzyme and Microbial Technology 34, 461–465. doi:10.1016/j.enzmictec.2003.12.002

Deschênes, J.S., Boudreau, A., Tremblay, R., 2015. Mixotrophic production of microalgae in pilot-scale photobioreactors: Practicability and process considerations. Algal Research 10, 80–86. doi:10.1016/j.algal.2015.04.015

Deschênes, J.-S., 2016. A Bacteriostatic Control Approach for Mixotrophic Cultures of Microalgae. IFAC-PapersOnLine 49, 1074–1078. doi:10.1016/j.ifacol.2016.07.345

Higgins, B.T., Gennity, I., Samra, S., Kind, T., Fiehn, O., VanderGheynst, J.S., 2016. Cofactor symbiosis for enhanced algal growth, biofuel production, and wastewater treatment. AlgalResearch 17, 308–315. doi:10.1016/j.algal.2016.05.024

Kandimalla, P., Desi, S., Vurimindi, H., 2016. Mixotrophic cultivation of microalgae using industrial flue gases for biodiesel production. Environmental Science and Pollution Research 23, 9345–9354. doi:10.1007/s11356-015-5264-2

Kim, B., Praveenkumar, R., Lee, J., Nam, B., Kim, D.M., Lee, K., Lee, Y.C., Oh, Y.K., 2016. Magnesium aminoclay enhances lipid production of mixotrophic Chlorella sp. KR-1 whilereducing bacterial populations. Bioresource Technology 219, 608–613. doi:10.1016/j.biortech.2016.08.034

Lee, Y.K., 2001. Microalgal mass culture systems and methods: Their limitation and potential. Journal of applied phycology 13, 307–315.

Lowrey, J., Brooks, M.S., McGinn, P.J., 2015. Heterotrophic and mixotrophic cultivation of microalgae for biodiesel production in agricultural wastewaters and associatedchallenges???a critical review. Journal of Applied Phycology 27, 1485–1498. doi:10.1007/s10811-014-0459-3

Mahapatra, D.M., Chanakya, H.N., Ramachandra, T. V., 2014. Bioremediation and lipid synthesis through mixotrophic algal consortia in municipal wastewater. Bioresource Technology 168, 142–150. doi:10.1016/j.biortech.2014.03.130

Perez-garcia, O., Bashan, Y., 2014. Algal Biorefineries. Springer Netherlands, Dordrecht. doi:10.1007/978-94-007-7494-0

Perez-Garcia, O., Escalante, F.M.E., De-Bashan, L.E., Bashan, Y., 2011. Heterotrophic cultures of microalgae: metabolism and potential products. Water research 45, 11–36. doi:10.1016/j.watres.2010.08.037

Prokop, A., Bajpai, R.K., Zappi, M.E., 2015. Algal biorefineries: Volume 2: Products and refinery design, Algal Biorefineries: Volume 2: Products and Refinery Design. doi:10.1007/978-3-319-20200-6

Subramanian, G., Yadav, G., Sen, R., 2016. RSC Advances Rationally leveraging mixotrophic growth of microalgae in di ff erent photobioreactor con fi gurations for reducing the carbon footprint of an algal biore fi nery : a techno-economic perspective. RSC Advances 72897–72904. doi:10.1039/C6RA14611B

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Wed Oct 26, 2016 – Track 3

Dries Vandamme, PhDDries.vandamme@kuleuven.be

Follow me on twitter: @DriesVandamme

Mixotropic growth of microalgae revisited

opportunities and challenges