Scalable and continuous in-stream sclareol extraction from

genetically modified chlamydomonas reinhardtii

Student Antonio Fernando Moncayo

Guzman
Study programme MSc Biotechnology
Reg. number 1043540
Start date 01-03-2021
End date 31-08-2021
Duration 26 weeks (36 ECTS)
Supervisor(s) Rafael García Cubero
Examiner(s) Iago Dominguez Teles

Introduction

Sclareol is a diterpenoid, which fulfills metabolic functions in the photosynthetic system

of the plant as a light receptor, photo protector, and plant growth regulator. Nowadays, it
has a commercial relevance in the pharmaceutical industry due to its anticancer
properties and antimicrobial activity, as well as in cosmetic and food additives industries.
However, the current production and extraction process of sclareol faces several
challenges. Some of them are: slow growth of plants, use of arable land, low extraction
yield, and an energy-intense purification process (Caniard et al, 2012; Wang et al, 2018).
These challenges could be solved with new ideas to reach an economically feasible and
eco-friendly production and extraction process of sclareol.

In recent years, studies have been conducted on genetically modified bacteria and yeast
to produce terpenoids. These alternatives have shown relatively high production yields,
which has made it possible to obtain continuous production process (Wang et al, 2018).
Nevertheless, it is important to mention that both microorganisms need carbohydrates
sources to grow, which means there will be additional raw material costs.

In contrast, microalgae show great potential as light-driven microbial factories to the

sustainable production of value-added products. Their quick-growing capacities,
phototrophic nature, carbon dioxide consumption, but especially their native terpenoid
biosynthesis pathways make them the most suitable platform for sclareol production
(Davies et al, 2015; Khan et al, 2018; Lauersen et al, 2018, Papaefthimiou et al, 2019).
Chlamydomonas reinhardtii has received special interest to be genetically modified to
produce sclareol, due to its ease of culturing and because it is one of the most well-
studied microalgae.

The Merit Project (https://merit-project.net/), looking for renewable innovative solutions,

has carried out several approaches for the production and extraction of terpenoids by
microalgae (Kovács, 2020). So far, some key points for further research have been
defined. First, sclareol is not excreted outside the cell but is accumulated inside.
Furthermore, sclareol shows product inhibition, which could be an obstacle to developing
a continuous extraction process. However, it is possible to use organic solvents to
continuously remove sclareol from microalgae, maintaining cell viability and integrity.
This would reduce the cost associated with cell cultivation and cell disruption in the
downstream process. Dodecane has shown the highest extraction efficiency (Lauersen et
al, 2018; Vieira, 2019). Nonetheless, some technical problems have arisen now in the
different approaches tested: emulsion formation and extraction membrane damage are
difficulties that must be solved to make an efficient scalable process possible (Awad,
2021; Camacho, 2020).

Aim

In this thesis, the main objective is to assess two different approaches to improve the
efficiency of a continuous in-stream extraction of sclareol produced by genetically
modified microalgae Chlamydomonas reinhardtii (B4 strain) to achieve a technically
feasible scale-up process to a 10L photobioreactor.

Project description

Genetically modified microalgae Chlamydomonas reinhardtii (B4 strain) will be cultivated

in an Algaemist photobioreactor (400ml) in a chemostat mode. Then, two different
approaches to continuous in-stream extraction of sclareol would be carried on, in which
dodecane (organic solvent) will not have direct contact with the broth (media + algae +
sclareol).

In both cases, it would be carried on a pertraction system to transfer organic compounds

from the broth to the extractant or solvent (dodecane). It is a two aqueous phases
extraction, that works with the aid of a porous hollow fiber membrane, which increases
the surface area per volume of broth. In the inner side of the porous hollow fiber
membrane flows the extraction liquid, meanwhile, the liquid phase flows in the outer side
of the membrane. Advantages as low energy consumption, the ability to regulate
independently the flows of both phases to optimize the process, and the possibility of
using small quantities of solvent are important to mention. Furthermore, it prevents two
phases from mixing, and allows the reusage of solvent, which makes it an attractive
option and economically feasible to scale up linearly.

The difference between both approaches is the material of the membrane that will
conform the extraction systems. In the first approach a Polyether-sulfone membrane
(PES) will be used, which has a hydrophilic affinity, and asymmetrical pore structure that
allows the water phase to flow through the pores, acting like capillaries. It is expected
that a direct contact between the liquid phase and the solvent allow diffusion of sclareol
from broth to dodecane. In the second approach Polytetrafluoroethylene membrane
(PTFE) will be used, which has a hydrophobic behaviour, that shows compatibility with
organic-based liquids. Because PTFE acts as a barrier to water flows, it is expected that
the low-pressure compounds fill the porous and diffuse directly into the dodecane.

Basically, in the two set-ups, the broth and the dodecane will be pumped through the
hollow fiber membrane during the day-light periods. The broth will return to the
Algaemist after crossing the membrane, the extraction solution (dodecane + sclareol) will
be stored in a different vessel for further analysis. The measurements considered for
these experiments is residence time and sclareol content after the extraction process.
 Figure 1. Schematic set-up for the experiments

References

Camacho Morales, A.S. 2020. In-situ diterpenoid extraction from a GMO Chlamydomonas
reinhardtii. Wageningen university and research.

Caniard, Anne et al. 2012. “Discovery and Functional Characterization of Two Diterpene
Synthases for Sclareol Biosynthesis in Salvia Sclarea (L.) and Their Relevance for
Perfume Manufacture.” BMC Plant Biology 12: 1–13.

Davies, F. K., Jinkerson, R. E., & Posewitz, M. C. (2015). Toward a photosynthetic

microbial platform for terpenoid engineering. Photosynthesis Research, Vol. 123, pp.
265–284. https://doi.org/10.1007/s11120-014-9979-6

Dimas, Konstantinos et al. 2007. “Sclareol Induces Apoptosis in Human HCT116 Colon
Cancer Cells in Vitro and Suppression of HCT116 Tumor Growth in Immunodeficient
Mice.” Apoptosis 12(4): 685–94.

Khan, M. I., Shin, J. H., & Kim, J. D. (2018, March 5). The promising future of
microalgae: Current status, challenges, and optimization of a sustainable and
renewable industry for biofuels, feed, and other products. Microbial Cell Factories,
Vol. 17, p. 36. https://doi.org/10.1186/s12934-018-0879-x

Kovács, Marie. 2020. Towards a scalable, continuous in-situ diterpenoid extraction from
genetically modified C. reinhardtii. Wageningen University & Research.

Lauersen, Kyle J. 2018. Phototrophic production of heterologous diterpenoids and a

hydroxy-functionalized derivative from Chlamydomonas reinhardtii. Metabolic
Engineering, 49, 116-127. https://doi.org/10.1016/j.ymben.2018.07.005.

Papaefthimiou, Dimitra et al. 2019. “Heterologous Production of Labdane-Type

Diterpenes in the Green Alga Chlamydomonas Reinhardtii.” Phytochemistry
167(November 2018): 112082. https://doi.org/10.1016/j.phytochem.2019.112082.

Vieira, Sara. 2019. Continuous Manoyl Oxide production by a genetically modified strain
of C. reinhardtii. Wageningen Univerisity & Research.

Wang, C., Liwei, M., Park, J.-B., Jeong, S.-H., Wei, G., Wang, Y., & Kim, S.-W. (2018).
Microbial Platform for Terpenoid Production: Escherichia coli and Yeast. Frontiers in
Microbiology, 9(OCT), 12. https://doi.org/10.3389/fmicb.2018.02460