Use of Hairy Root Cultures for bioreactor processes Microbial Process Technology Seminar 2015-2016 Kasper Deflem, Kevin Gui, Milan Reyn, Steven van den Bussche

Summary 1. Introduction: What are “Hairy Root Cultures (HRC)” 2. General procedure: 2.1 Transformation 2.2 Process optimization 2.3 Bioreactor design 3. Industrial applications

1. Introduction Origin: natural disease (Higher plants) Cause: Agrobacterium Rhizogenes infection Adventitious roots + a lot of root hairs Eurofins Agro Laboratories, 2014

1. Introduction Target higher plant  HRC as “mini biofactory” - Secondary metabolites - Recombinant proteins - Phytoremediation Guillon et al., 2006 Council of Agriculture, 2011

1. Introduction Why (or why not) use HRCs for bioreactor processes?

2. General procedure Research and development: Inocculation: wounded plant + A. rhizogenes Transformation (transfer T-DNA) Selection, screening, optimization Production: Upscaling to bioreactor stage Downstream processing Rootec, 2015

2.1 Transformation Root inducing plasmid Transfer of T-DNA segment Integration in plant genome (RANDOM!) Possibility to add genes in T-DNA segment Recombinant proteins Broekaert, 2015

2.2 Proces optimization Georgiev et al., 2007

Our Focus Genetic engineering Optimization of the nutrient medium Permeabilizitation and cultivation in two phase systems

Genetic engineering Adding extra genes to T-DNA Enhancing metabolic pathway Inhibiting competing pathways Complicated

Optimization of the nutrient medium Medium composition Usage of two stages Elicitation Chemical Biological Georgiev et al., 2007

Permeabilizitation and cultivation in two phase systems Metabolites storage Extraction without damaging When to use which method?

2.3 Bioreactor design Important design considerations Shear stress Mass and O2 transfer problems Growth support HR are shear sensitive Sometimes HR make roteclumps  mass and oxygen transfer problems Growth support: vertical or horizontal meshes

Types of reactors Liquid phase Stirred tank Air lift Bubble column HR roots are suspended in liquid phase Stirred tank: good mixing air and nutriens, high shear stress!! Air lift (pictures): low shear stress, easy design, HR not uniform Problems with upscaling (10 g/L) Paek et al., 2014

Types of reactors Gas phase Mist Nutrient sprinkle Trickle bed Hybrid Smaller droplets  less problems with mass and oxygen transfer (mist is the smallest, trickle bed the biggest droplets) Work intensive, placing HR Mor complex construction Hybrid: best of both worlds: start as liquid, later gas Paek et al., 2014

3. Industrial applications Swiss company: ROOTec Industrial production phytochemicals with HRC’s E.g. linarin, camptothecin Rootec,2015

3. Industrial applications Ginseng case Successfully upscaling Pilot study using 10 ton bioreactors ginsenosides Paek et al., 2009

Take home messages Hairy root culture caused by A. rhizogenes infection Production of: - Secondary metabolites - Recombinant proteins - Phytoremediation

Take home messages Research and development: Inocculation: wounded plant + A. rhizogenes Transformation (transfer T-DNA) Production: Upscaling to bioreactor stage Rootec, 2015

Take home messages Hairy roots: Need oxygen Are shear sensitive Have problems with mass transfer and oxygen limitations

Take home messages Liquid CSTR Phase Reactor Advantages Disadvantages Liquid CSTR Mixing and breaking up air bubbles prevent root clump forming and enhance oxygenation High shear force Air lift/bubble column Low shear stress Simple design Low maintenance HR not uniformly distributed; makes upscalling difficult Gas Mist Abundant oxygen supply almost no mass transfer limitations High space utilization Complex and labor intensive set-up with high energy consumption Trickle-bed Lower energy consumption May produce a viscous liquid film on the roots; mass transfer limitations Labor intensive set-up

References Broekaert, W. 2015. Cursus: Toepassingsdomeinen in de biotechnologie: Plant. Council of Agriculture. 2011. Molecular Farming. [on line]. http://flora.coa.gov.tw/view_eng.php?id=75&view=Y Accessed on 28/11/2015]. Doran,M.P. 2013. Biotecnology of hairy root systems. Springer. Berling Heidelberg. 155p. Eurofins Agro laboratories (2015). 6 tips om Crazy Roots te voorkomen. [on line]. http://blgg.agroxpertus.nl/expertise/gewasgezondheid/artikelen/6-tips-om-crazy-roots- te-voorkomen Accessed on: [28/11/2015]. Georgiev, M.I., Agostini, E., Ludwig-Müller, J., Xu, J., 2012. Genetically transformed roots: from plant disease to biotechnological resource. Trends in Biotechnology; 30, 528-537. Georgiev M.I., Pavlov, A.I., Bley, T., 2007. Hairy root type plant in vitro systems as sources of bioactive substances. Applied Microbial Biotechnology; 74, 1175-1185. Guillon, S., Trémouillaux-Guiller, J., Pati, P.K., Rideau, M., Gantet, P., 2006. Hairy root research: recent scenario and exciting prospects. Current Opinion in Plant Biology; 9, 341-346.

References Lambert,E., Goossens,A., Panis,B. 2009. Cryopreservation of hairy root cultures of Maesa lanceolata and Medicago truncatula. Plant Cell Tiss Organ Cult: 96:289–296. Mehrotra, S., Srivastava, V., Ur Rahman, L., 2015. Hairy root biotechnology – indicative timeline to understand missing links and future outlook. Protoplasma; 225, 1189-1201. Ono, N.N., Tian, L., 2011. The multiplicity of hairy root cultures: Prolific possibilities. Plant Science; 180, 439-446. Paek, K., Hosakatte, N.M., Zhong, J., 2014. Production of Biomass and Bioactive Compounds Using Bioreactor Technology. Springer. Paek, Y. et al. 2009. Large Scale Culture of Ginseng Adventitious Roots for Production of Ginsenosides. Adv Biochem Engin/Biotechnol 113: 151-176. Vashishtha, M. and Saurabh,K. Modelling of mist reactor: effect of packing fraction and film thickness on the growth of hairy roots. Malaviya National Institute of Technology. ROOTec bioactives Ltd. [on line]. http://www.rootec.com/en/home. Accessed on: [10/11/2015].