3.

Results

3.1 Extraction Experiments in batch cultivation

Explicar porque? As a first approach and to understand the potential accumulation of diterpenes by
C. reinhardtii B4, it was performed a five-day extraction experiment in flasks with an overlay
of 5% of dodecane. The average diterpene productivity results at the end of the experiment
were 73.6 mg L-1 d-1 of sclareol and 2.9 mg L-1 d-1 of manoyl oxide (Figure 12). Meanwhile, it
was observed a biomass concentration at the fifth day of 1.2 g L-1 (Figure 13); which resulted
in a sclareol biomass-equivalent values of 30.7 % and manoyl oxide yield of 1.2 %.

Figure 12 Diterpenoid concentration: sclareol and manoyl oxide (mg L -1) in culture during
batch cultivation in flasks over the time of the experiment. (linea del eje x)

Figure 13. Biomass concentration (g L-1) in culture during batch cultivation in flasks over the
time of the experiment. Unificar las unidades.
3.2 In-stream extraction experiments by pertraction in continuous cultivation in
photobioreactors

3.2.1 Pertraction using a PTFE hollow fiber membrane filter at 50 cycles per day

Te falta Antonio dar un pequeña introducción de lo que vas a hacer en cada resultado. Algo
así como, poner en situación al lector de lo que vas a hacer y por qué. “In order to optimize
the extraction of diterpenes, we use 2 different type of filters.... “ simplemente un parrafito de
2-3 lineas para; Y aqyuí contar que primero vas a centrarte en un tiempo alto de contacto (50
ciclos/dia) y luego 13 ciclos dia The first experiment performed was a pertraction system
using a PTFE hollow fiber membrane filter, in which the total volume of the reactor (400
mL) circulated 50 cycles per day. The biomass productivity was 0.21 g L -1 d-1, while the
quantum yield was 0.64. This experiment showed a sclareol productivity of 18.93 mg L-1 d-1,
and manoyl oxide productivity of 8.02 mg L -1 d-1 (Figure 14). These values represent a
sclareol yield of 9.16 % and a manoyl oxide yield of 3.74 %. Unificar puntos y comas en
graficas.

Table 3. Pertraction using PTFE hollow fiber membrane filter at 50 cycles per day. Mejorar
detalle titulo de tablas. Colocar desviaciones.

Biomass productivity (g L-1 d-1) 0.21

Quantum yield 0.64
Sclareol productivity (mg L-1 d-1) 18.93
Sclareol yield (%) 9.16
Manoyl productivity (mg L-1 d-1) 8.02
Manoyl oxide yield (%) 3.74
Figure 14. Diterpenoid accumulation in the solvent layer (dodecane) over time of pertraction
using PTFE hollow fiber membrane filter at 50 cycles per day.

The solvent (dodecane) was recycled for X days until the end of the experiment; however, it
was noticed the formation of an aqueous solution (denser than dodecane) in the bottom part
of the extraction vessel that looks like an emulsion. The aqueous phase/emulsion formation
increased the volume with every cycle of the pertraction, which resulted in pure dodecane
losses every day (on average 75 mL daily). It was possible to identify two layers in the
solvent vessel at the end of each experiment, an upper layer that resembles the pure solvent;
and the bottom layer that comes across as a turbid yellowish emulsion (Figure 15). This
aqueous phase was sampled to diterpene analysis in the same way as the pure dodecane, but
no traces of sclareol or manoyl oxide were found.

A B

Figure 15 A. Dodecane vessel at the first day of the experiment of pertraction using PTFE
hollow fiber membrane filter at 50 cycles per day. B. Dodecane vessel at the end of the
experiment of pertraction using PTFE hollow fiber membrane filter at 50 cycles per day.
Cambiar orden de la letra en la imagen, superior izquierda.

3.2.2 Pertraction using a PTFE hollow fiber membrane filter at 13 cycles per day

In this case, the pertraction experiments were performed using a PTFE hollow fiber
membrane filter, in which the total volume of the reactor (400 mL) circulated 13 cycles per
day. This scenario showed biomass productivity of 0.10 g L -1 d-1, and a quantum yield of 0.70.
Besides this, the diterpene productivity showed results of 17.11 mg L-1 d-1 and 8.33 mg L-1 d-1
of sclareol and manoyl oxide, respectively (Figure 16). The final yield for each diterpene is
17.37% for sclareol, and 8.52 % for manoyl oxide. Explicar que se busca con cada uno de los
experimentos

Table 4 Pertraction using PTFE hollow fiber membrane filter at 13 cycles per day.
 Biomass productivity (g L-1 d-1) 0.10
Quantum yield 0.70
Sclareol productivity (mg L-1 d-1) 17.11
Sclareol yield (%) 17.37
Manoyl productivity (mg L-1 d-1) 8.33
Manoyl oxide yield (%) 8.52

Figure 16. Diterpenoid accumulation over time of pertraction using PTFE hollow fiber
membrane filter at 13 cycles per day.

Qué explicación tienes para justificar que perdamos en el segundo día diterpenos? Acaso
volvía al interior celular? Se lo vuelve a comer el alga? No puede ser un error de pipiteo? Por
que mi duda en estas gráficas es: es acumulativo ese número o no? Porque debería ser algo
asi:
1dia: 34,4
2dia: 34,4+17.7=52,1
3dia: 52,1+24,5=76,5

Estas de acuerdo conmigo en que no se ha podido perder los diterpenos verdad?

At this culture flow, to obtain 13 extraction cycles per day for the total volume of the
photobioreactor was also noticed the formation of the aqueous phase at the bottom of the
solvent vessel (Figure 17). The losses of dodecane by the formation of this aqueous phase
was on average 20 mL of solvent daily, which is lower in comparison with the same filter at
50 cycles per day.
 A B

Figure 17 A. Dodecane vessel at the first day of the experiment of pertraction using PTFE
hollow fiber membrane filter at 13 cycles per day. B. Dodecane Vessel at the end of the
experiment of pertraction using PTFE hollow fiber membrane filter at 13 cycles per day.

In addition, it was monitored the microalgae cell morphology during each experiment. A
relevant point is that reduction of biomass concentration can be observed between the
beginning and final day of each experiment (Figure 18).

A B

Figure 18 A. Chlamydomonas reinhardtii B4 at the beginning of the experiments. B.

Microalgae at the end of the experiments of pertraction using PTFE hollow fiber membrane
filter.
Si antes has empezado por 50, no cambies ahora y lo hagas por 13. Se consistente con tu
estructur

3.2.3 Pertraction using a PES hollow fiber membrane filter at 13 cycles per day

It was performed a pertraction system based on a PES hollow fiber membrane filter, with a
culture flow of 13 cycles (total volume of the reactor) per day passing throw the filter. The
biomass productivity for this scenario was 0.12 g L -1 d-1, while the quantum yield was 0.71.
The result of sclareol productivity was 5.56 mg L-1 d-1 (yield of 4.66 %,) and for manoyl oxide
productivity of 5.09 mg L-1 d-1 (yield of 4.21 %.) (Figure 19).

Table 5 Pertraction using PES hollow fiber membrane filter at 13 cycles per day.

Biomass productivity (g L-1 d-1) 0.12

Quantum yield 0.71
Sclareol productivity (mg L-1 d-1) 5.56
Sclareol yield (%) 4.66
Manoyl productivity (mg L-1 d-1) 5.09
Manoyl oxide yield (%) 4.21

Figure 19. Diterpenoid accumulation in the solvent (dodecane) layer over time of pertraction
using PES hollow fiber membrane filter at 13 cycles per day.

Under the conditions of this set-up, using PES hollow fiber membrane filter was also
observed the formation of an aqueous phase at the bottom of the solvent vessel. The dodecane
losses by the formation of the emulsion in the aqueous phase were on average 20 mL daily. It
could be possible to notice two different layers in the vessel, one that looks like pure
dodecane and one that resembles a turbid yellowish color emulsion/solution (Figure 20).
 A B

Figure 20 A. Dodecane vessel at the first day of the experiment of pertraction using PES
hollow fiber membrane filter at 13 cycles per day. B. Dodecane Vessel at the end of the
experiment of pertraction using PES hollow fiber membrane filter at 13 cycles per day.

3.2.4 Pertraction using a PES hollow fiber membrane filter at 50 cycles per day

Last, an extraction based on a set-up for pertraction using PES hollow fiber membrane filter
with 50 cycles per day of the total volume of the reactor throw the filter rendered a d biomass
productivity of 0.11 g L-1 d-1 and a quantum yield of 0.72. The final sclareol productivity was
9.40 mg L-1 d-1 (yield 9.35%) and manoyl productivity of 6.41 mg L-1 d-1 (yield 6.36%)
(Figure 21).

Table 6. Pertraction using PES hollow fiber membrane filter at 50 cycles per day.

Biomass productivity (g L-1 d-1) 0.11

Quantum yield 0.72
Sclareol productivity (mg L-1 d-1) 9.40
Sclareol yield (%) 9.35
Manoyl productivity (mg L-1 d-1) 6.41
Manoyl oxide yield (%) 6.36
Figure 21. Diterpenoid accumulation over time of pertraction using PES hollow fiber
membrane filter at 50 cycles per day.
De nuevo Antonio, esto hay que darle una vuelta... no tiene explicación que la concentración
vaya cayendo... en todo caso debería o quedarse igual (es decir, no ha habido extracción ese
día) o ir incrementandose... pero con esos datos parece que el alga reabsorbe los diterpenos...

As well as the other experiments, it was noticed the same kind of aqueous phase, the
dodecane losses ratio in this scenario was 75 mL on average per day. It was possible to
observe the two layers of solvent at the top, and a turbid yellowish color solution at the
bottom of the vessel (Figure 22).

A B

Figure 22 A. Dodecane vessel at the first day of the experiment of pertraction using PES
hollow fiber membrane filter at 50 cycles per day. B. Dodecane Vessel at the end of the
experiment of pertraction using PES hollow fiber membrane filter at 50 cycles per day.
3.3 Summary

Pero este summary ...por qué aquí? Es decir, no haces ninguna referencia a él en los resultados. No puedes poner una tabla sin que venga de un
contexto en el texto... Quizás puedas usarla en la discusión. Empezando con el summary de todos los resultados y que te sirva de apoyo para
montar la discusión y teorizar y comparar con otros papers
Table 7 Details of each performed experiment showing their diterpenoid productivities and yield results at end of extraction.

Pore size N-cycles Reynolds Contact SC-Np SC - Y MO-Np MO - Y

Experiment Type of Filter
(µm) (d-1) number Time (s) (mg L-1 d-1) (%) (mg L-1 d-1) (%)

F1 _ PTFE 50 Cycles PTFE Hydrophobic 0.08 49.5 18.8 840 14.49 9.47 4.81 3.14

F2 _ PTFE 13 Cycles PTFE Hydrophobic 0.08 12.6 4.8 3300 8.15 12.94 4.55 7.22

P1 _ PES 13 Cycles PES Hydrophilic 0.02 12.6 4.8 3300 3.30 4.39 2.87 3.83

P2 _ PES 50 Cycles PES Hydrophilic 0.02 49.5 18.8 840 4.64 4.42 3.50 3.33

SC: Sclareol, MO: Manoyl Oxide.

Np: Diterpenoid Productivity, Y: Yield.
3.4 Yield comparison between different experiment set-up for pertraction systems using hydrophobic and hydrophilic hollow fiber
membrane filters at two different culture flows.

Figure 26 Diterpenoid yield at the end of each performed experiment. Cambiar orden de table 50 y 13 y 50 y 13