1 Towards a feasible and sustainable scale-up of continuous

2 diterpenoid extraction from microalgae

3
Student Ivo Adriaan Tjalma
Study programme MSc Biotechnology
Reg. number 970817836040
Start date 31-08-2020
End date 28-02-2021
Duration 26 weeks (36 ECTS)
Supervisor(s) Iago Dominguez Teles

4 Introduction
5 Terpenoids are an extremely diverse class of secondary metabolites commonly found in
6 plants, in which they fulfil a wide range of photosynthetic, regulatory, defensive and
7 metabolic functions (Cheng et al., 2007; Langenheim, 1994). Besides their natural
8 functions, they can also serve in many high value applications, such as in the
9 pharmaceutical- and cosmetic industries (Bohlmann & Keeling, 2008).
10
11 A particularly interesting (di)terpenoid is Forskolin. It can be isolated from the plant
12 Coleus forskohlii and is known to activate the human regulatory enzyme adenylate
13 cyclase. This compound has been used to treat various illnesses such as eczema,
14 hypertension and asthma. The extract is also consumed across the globe as a health
15 supplement to promote lean body mass, fat loss and weight loss. (Alasbahi & Melzig,
16 2012; Daly, 1984; Lieberman, 2004)
17
18 Although terpenoids can serve in high value applications, the productivities of these
19 substances in plants is low. This is mainly because plants grow slow, seasonal and often
20 result in a low product content (Wang et al., 2018). For these reasons, alternative
21 terpenoid production methods have been explored. At present, terpenoids can be
22 produced by different hosts such as the traditional biotechnological platforms,
23 Escherichia coli and Saccharomyces cerevisiae (Wang et al., 2018). Alternatively,
24 terpenoids may also be produced by genetically modified microalgae by exploiting their
25 native terpenoid biosynthesis pathways (Davies, Jinkerson, & Posewitz, 2015; Lauersen
26 et al., 2018).
27
28 In contrast to the low terpenoid productivities in plants, micro-organisms grow relatively
29 fast (multiply daily or faster), can be cultivated continuously, are less reliant of water and
30 land resources while they could also achieve high terpenoid product yields due to tailored
31 metabolic pathways (Wang et al., 2018). On top of that, microalgae can grow
32 phototrophically, eliminating the dependency on food-competing feedstocks. Therefore,
33 these light driven microbial factories have gained interest over the past few decades for
34 the sustainable production of value-added products (Davies et al., 2015; Khan, Shin, &
35 Kim, 2018).
36
37 Previous work during this project, “MicroalgaE as Renewable Innovative green cell
38 facTories” (MERIT), showed that product inhibition can be a major obstacle for the
39 production of diterpenoids using microalgae (Lauersen et al., 2018). A solution to this
40 problem could be to implement continuous product removal during the cultivation. This
41 can be done by means of extraction with a biocompatible organic solvent (Lauersen et
42 al., 2018). Experiments from de Boeck (2019) showed that dodecane is the least toxic
43 organic solvent to algae, and therefore most suitable for this purpose. Experiments from
44 Kovács (2020) showed that application of a continuous extraction method is technically
45 difficult, as the solvent can form viscous emulsions with the broth, causing stirring
46 problems and gas-transfer limitations during cultivation. This thesis will use the marine
47 microalgae Phaeodactylum tricornutum which is engineered to produce manoyl oxide
48 (MO), a diterpenoid precursor of Forskolin. It will be investigated whether this strain is
49 affected by product inhibition. Furthermore, it will be assessed if a MO production process
50 using this strain could be feasible.
51
52 Aim
53 The first goal of this thesis is to develop a model that describes the growth and MO
54 productivity of a novel genetically engineered P. tricornutum strain under presumed
55 optimal conditions. The second goal is to design a scalable and sustainable process in
56 which MO is continuously extracted from the algal cells and to assess its feasibility and
57 sustainability.
58
59 Project description
60 To develop the kinetic model for the novel MO-producing P. tricornutum strain, small
61 scale batch cultivation experiments will be performed in shake flasks with or without a
62 dodecane overlay. Biomass accumulation, cell viability and the product concentration in
63 biomass, water-phase and solvent-overlay will be measured. These data should give
64 insights into the maximum growth-rate, maximum MO-production rate and the probable
65 inhibition dynamics of MO on growth and productivity of the strain. Furthermore, the data
66 should elucidate the effect of dodecane on the growth, viability and MO-productivity of
67 the cells. The obtained parameters will be used to develop a model (in MathCad15) that
68 can predict the growth and productivity of the cells in a process.
69
70 After the establishment of the kinetic model, efforts will be shifted towards selecting the
71 most promising process design. Based on previous work by M. Kovacs (2020), the
72 following process designs will be compared in terms of technical feasibility and
73 scalability:
74 - A two-phase photobioreactor: In which an illuminated, continuous fermenter will
75 be cultivated with the microalgae whereby the product will be continuously
76 extracted into the dodecane overlay.
77 - A continuous process with an in-stream extraction vessel: In this design, a part of
78 the broth is continuously transported from a photobioreactor (any type) towards a
79 separate extraction vessel, in which the cells release the product to the solvent
80 and are subsequently transported back into the photobioreactor.
81 - A two-phase photobioreactor in which a semi-permeable silicone membrane
82 separates the solvent from the broth to allow efficient product extraction but
83 prevent the emulsification.
84
85 The most promising design will be modelled (in SuperPro Designer v.11) to assess its
86 economic feasibility and its sustainability in terms of energy, material and water
87 consumption. The findings will be compared to alternative MO production processes,
88 including the process described in previous work, which used a genetically modified
89 Chlamydomonas reinhardtii strain and a silicone membrane (Kovács & Teles, 2020;
90 Meijs, Teles, & Vermue, 2020). Finally, a sensitivity analysis will be done to identify the
91 parameters most affecting the economics and sustainability of the process.
 92 References
93 Alasbahi, R. H., & Melzig, M. F. (2012, January). Forskolin and derivatives as tools for
94 studying the role of cAMP. Pharmazie, Vol. 67, pp. 5–13.
95 https://doi.org/10.1691/ph.2012.1642
96 Bohlmann, J., & Keeling, C. I. (2008, May). Terpenoid biomaterials. Plant Journal, Vol.
97 54, pp. 656–669. https://doi.org/10.1111/j.1365-313X.2008.03449.x
98 Cheng, A. X., Lou, Y. G., Mao, Y. B., Lu, S., Wang, L. J., & Chen, X. Y. (2007, February).
99 Plant terpenoids: Biosynthesis and ecological functions. Journal of Integrative Plant
100 Biology, Vol. 49, pp. 179–186. https://doi.org/10.1111/j.1744-7909.2007.00395.x
101 Daly, J. W. (1984, January 1). Forskolin, adenylate cyclase, and cell physiology: an
102 overview. Advances in Cyclic Nucleotide and Protein Phosphorylation Research, Vol.
103 17, pp. 81–89. Retrieved from https://europepmc.org/article/med/6328947
104 Davies, F. K., Jinkerson, R. E., & Posewitz, M. C. (2015). Toward a photosynthetic
105 microbial platform for terpenoid engineering. Photosynthesis Research, Vol. 123, pp.
106 265–284. https://doi.org/10.1007/s11120-014-9979-6
107 de Boeck, R. (2019). Designing an in-situ extraction of manoyl oxide produced by
108 Chlamydomonas reinhardtii. - Wageningen University and Research.
109 Khan, M. I., Shin, J. H., & Kim, J. D. (2018, March 5). The promising future of
110 microalgae: Current status, challenges, and optimization of a sustainable and
111 renewable industry for biofuels, feed, and other products. Microbial Cell Factories,
112 Vol. 17, p. 36. https://doi.org/10.1186/s12934-018-0879-x
113 Kovács, M., & Teles, I. D. (2020). Towards a scalable , continuous in-situ diterpenoid
114 extraction from genetically modified C . reinhardtii. Wageningen University and
115 Research.
116 Langenheim, J. H. (1994). Higher plant terpenoids: A phytocentric overview of their
117 ecological roles. Journal of Chemical Ecology, 20(6), 1223–1280.
118 https://doi.org/10.1007/BF02059809
119 Lauersen, K. J., A , ⁎, , Wichmann, J., Baier, T., Kampranis, C., Pateraki, I., … Kruse, O.
120 (2018). Phototrophic production of heterologous diterpenoids and a hydroxy-
121 functionalized derivative from Chlamydomonas reinhardtii.
122 https://doi.org/10.1016/j.ymben.2018.07.005
123 Lieberman, S. (2004). A new potential weapon for fighting obesity: Forskolin - The active
124 diterpene in Coleus. Alternative and Complementary Therapies, Vol. 10, pp. 330–
125 333. https://doi.org/10.1089/act.2004.10.330
126 Meijs, V., Teles, I. D., & Vermue, M. (2020). Modelling large - scale production and
127 extraction of manoyl oxide from Chlamydomonas Reinhardtii. Wageningen University
128 and Research.
129 Wang, C., Liwei, M., Park, J.-B., Jeong, S.-H., Wei, G., Wang, Y., & Kim, S.-W. (2018).
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132