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

2. 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

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

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

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

6. 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

7. 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

8. 2.2 Proces optimization
Georgiev et al., 2007

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

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

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

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

13. 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

14. 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

15. 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

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

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

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

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

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

21. 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

22. References
Broekaert, W Cursus: Toepassingsdomeinen in de biotechnologie: Plant.
Council of Agriculture Molecular Farming. [on line]. Accessed on 28/11/2015].
Doran,M.P Biotecnology of hairy root systems. Springer. Berling Heidelberg. 155p.
Eurofins Agro laboratories (2015). 6 tips om Crazy Roots te voorkomen.
[on line]. te-voorkomen Accessed on: [28/11/2015].
Georgiev, M.I., Agostini, E., Ludwig-Müller, J., Xu, J., Genetically transformed roots: from plant disease to biotechnological resource. Trends in Biotechnology; 30,
Georgiev M.I., Pavlov, A.I., Bley, T., Hairy root type plant in vitro systems as sources of bioactive substances. Applied Microbial Biotechnology; 74,
Guillon, S., Trémouillaux-Guiller, J., Pati, P.K., Rideau, M., Gantet, P., Hairy root research: recent scenario and exciting prospects. Current Opinion in Plant Biology; 9,

23. References
Lambert,E., Goossens,A., Panis,B 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., Hairy root biotechnology – indicative timeline to understand missing links and future outlook. Protoplasma; 225,
Ono, N.N., Tian, L., The multiplicity of hairy root cultures: Prolific possibilities. Plant Science; 180,
Paek, K., Hosakatte, N.M., Zhong, J., Production of Biomass and Bioactive Compounds Using Bioreactor Technology. Springer.
Paek, Y. et al Large Scale Culture of Ginseng Adventitious Roots for Production of Ginsenosides. Adv Biochem Engin/Biotechnol 113:
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]. Accessed on: [10/11/2015].