24-well plate cultivator for photoautotrophic organisms
One of the microbes that we used for our proof of concept is Synechocystis PCC6803, which is
one of the most studied strains of cyanobacteria since it was initially isolated from a
freshwater lake in 1968 (Yu et al., 2013). Cyanobacteria convert solar energy into
biomass-based chemical energy by means of photosynthesis (Yu et al., 2013); they are
photoautotrophic organisms capable of harnessing CO2 fixation and free energy (i.e. photons)
for their growth, which is why they are a widely studied chassis for production of carbon
compounds (Branco dos Santos et al., 2014). The specific growth rate of any photoautotrophic
organism depends mainly on light irradiance (LI), temperature, pH, and concentrations of
CO2, nitrogen, and phosphorus (Kim et al. 2011).
Development of new strains for the purpose of creating sustainable green cell factories
(e.g. by implementing genetic engineering strategies from Forbidden FRUITS) requires a
prioritisation of the understanding of the physiology of cyanobacteria after genetic
modifications are successfully implemented. Cell factories often do not perform optimally
and can be tweaked to gain further increases in productivity (e.g. adjust promoter
strength). This can be achieved through adaptive laboratory evolution (ALE) (Godara and Kao,
2020), which requires screening of obtained mutants. Well plates are suitable for this
process, but unfortunately, current incubators are not able to support photoautotrophic
growth.
We have worked on the development of a cultivation setup that allows the selection of
individual light settings for a large set of samples in a well plate. This addresses the
need for a high-throughput platform for the culture of photoautotrophs to explore the full
range of their biological diversity, tweak cell factories and evaluate their commercial
potential (Ojo et al., 2015). A rough concept can be observed in Figure 1.
Figure 1. Concept of a system to provide light individually to the wells of a 24-well plate.
The main purpose of the system is to make a controlled feedback system that supports the
growth of photoautotrophic microorganisms. Using LEDs coupled to optical fibers, we can
provide light of various colors to 48 individual wells independently. This allows us to
decouple most of the electronics from the incubator. Light is also captured from the bottom
of each well and transported back to the electronic board via optical fibers. Using
photodiodes we can then measure the optical density (OD) of each well.
Using this system we can scale advanced feedback systems, currently only found in large
bioreactors, to well plates. For example, we can control the light intensity of each well
depending on real-time optical density (OD) measurements of the culture in the well.
Ensuring that all cells in the culture receive a constant amount of photons per cell.
By varying the settings of each individual well, any (modified) cyanobacteria culture can be
subjected to a range of light regimes allowing for simultaneous testing of different
conditions, which would provide valuable information to understand the limiting effects of
light (i.e. of photosynthesis) on the growth of genetically engineered cyanobacteria, like
the ones we have used for our proof of concept. Likewise, having a large number of
independent wells makes possible testing these new strains in many different conditions and
in parallel with many replicates.
While each of the proposed elements already exists in one form or another, this system
combines incubation, illumination and measurement equipment into one centralized construct,
removing the need to perturbe the experiment for sampling.
Figure 2. Light-providing and OD730 measuring system for an individual well. (courtesy of Joeri A. Jongbloets)
Figure 3. Design of the 24-well plate unit (from Favie, B., 2016).
Figure 4. Figure 2.8: Optical fibers connected
to individual LEDs. By attaching the fibers on top of each individual LED the light
produced by the LED is mostly funneled into the optical fibers, which are used to
illuminate the well. This image shows how the light of each well is variated, allowing
for multiple cultivation conditions within a single microplate (from Favie, B.,
2016).
As it is shown in figures 2 and 3, the 24-well plate bottom and top holders isolate the
24-well plate from external light while the glass marbles embedded in both the top and
bottom holders collimate the light entering through the optical fibers. The optical fibers
are used to guide light from the LEDs into the wells, as well as return the light to the
photodiodes.
Figure 5. 24-well plate used in the setup (VisiPlate-24 white).
We studied the growth of the photoautotrophic bacteria Synechocystis PCC6803 in the 24-well
plate cultivator system. Four 7-day long experiments were carried out to observe the growth
characteristics of Synechocystis in this new apparatus. We focussed on studying the effect
of different light and carbon conditions, see table 1.
Table 1. Overview of the four growth experiments carried out in the 24-well plate cultivator system.
| Experiment |
Light intensity (µE*m-2*s-1) |
Bicarbonate concentration (mM) |
| 1 |
0 - 30 - 60 - 90 |
0 |
| 2 |
60 |
0 - 50 - 100 |
| 3 |
30 - 60 - 90 |
50 |
| 4 |
60 - 90 |
25 - 50 |
For each experiment, the initial cultures were prepared with an initial OD730 of 0.1. For
every condition within each experiment control flasks were kept in the incubator (see figure
5). The 24-well plates were sealed with a sterile membrane (BreathEasy Membrane, Diversified
Biotech) and wells were sampled each day using a randomized experimental design. The control
flasks were sampled each time the plates were sampled.
For further details on the experimental designs click here.
To view the protocol click here.
Figure 6. General overview of experimental subjects.
Experiment 1
In the first experiment we set out to investigate the effect of light intensity. We expected
to observe different growth rates depending on the light intensity provided to the culture,
where the wells that received more light would grow faster than the ones cultivated under a
lower light intensity.
Figure 7. Mean OD730 values of replicates in the 24-well plates and control flasks vs time (h).
We can see from figure 7 that the conditions in the 24-well plate are far from optimal.
Hardly any growth was achieved in comparison with the control flasks. Since light seemed to
have little effect we considered other potential limitations. Besides light another common
limitation for cyanobacteria is the atmospheric CO2 concentration. We hypothesized the
membrane used to seal the 24-well plates was not allowing for enough flow of CO2. Thus we
went to test if supplementing CO2 in the form of bicarbonate would improve growth.
Experiment 2
In the second experiment we studied the effect of bicarbonate concentration using the same
intensity value (60 µE*m-2*s-1 of white light) on all wells. One control flask per bicarbonate
condition was kept in the incubator.
Figure 8. Mean OD730 values per bicarbonate concentration vs. time (h).
Figure 8 clearly shows that growth in the wells is limited by carbon availability. Without
bicarbonate no exponential growth is achieved, whereas in the two other bicarbonate
conditions, exponential growth is observed.
We can also observe that the effect of bicarbonate is mostly noticeable around day 4 (98h),
when cells reach the stationary phase (i.e. we observed a similar behaviour in control
flasks). Before this time point, no noticeable difference is visible in the growth rate
between 50mM or 100mM, this can indicate that cells have a limited capacity to absorb
available carbon and for this reason higher concentrations in the beginning do not
necessarily lead to increased growth. Knowing this, we decided to repeat our first
experiment to study the effects of different light intensities on the cultures when carbon
availability is not a limiting factor.
Experiment 3
In the third experiment, the effect of light intensity was again studied, but this time 50 mM
of bicarbonate was used to relieve the carbon limitation.
Figure 9. Measured OD730 values per light condition vs. time (h).
Figure 9 shows the effect of light intensity on growth. The first two time points seem to
indicate that the growth rates increase at higher light intensities; however, it is
interesting to note that no effect is seen in the stationary phase. Although, differences
can be observed between the different light conditions, the variance in the data from this
experiment makes it difficult to draw a conclusion on this. For this reason, the last
experiment was also designed to study the effects of light intensity under bicarbonate
supplementation.
Experiment 4
And lastly in the fourth experiment the two limitations were combined. We created 4
conditions (shown in Table 2) by combining two different intensities along with two
different bicarbonate concentrations. The goal of this experiment was to investigate the
lower bicarbonate limit under which carbon limitation would occur; as well as to further
study the effect of light limitation. For the latter, a comparison between conditions 2 and
4 can be analysed in parallel with the results from experiment 3.
Figure 9. Measured OD730 values per light condition vs. time (h).
Figure 9 shows the effect of light intensity on growth. The first two time points seem to
indicate that the growth rates increase at higher light intensities; however, it is
interesting to note that no effect is seen in the stationary phase. Although, differences
can be observed between the different light conditions, the variance in the data from this
experiment makes it difficult to draw a conclusion on this. For this reason, the last
experiment was also designed to study the effects of light intensity under bicarbonate
supplementation.
Table 2. Experimental conditions observed in experiment 4.
| Experiment |
Light intensity (µE*m-2*s-1) |
Bicarbonate concentration (mM) |
| 1 |
60 |
25 |
| 2 |
60 |
50 |
| 3 |
90 |
25 |
| 4 |
90 |
50 |
Figure 10. Measured OD730 values per bicarbonate concentration vs. time (h).
With this last experiment we could clearly observe the effect of light limitation. Figure 10
shows how the effect of light limitation can only be observed when the microbes are not
carbon limited, which occurs in the 25mM bicarbonate condition, where no difference between
the two light conditions is occurring because growth is being limited by carbon
availability. In the exponential growth phase (until 52h) in the 50mM bicarbonate condition,
light limitation is clearly visible, since the OD730 achieved with 90 µE*m-2*s-1 is more
than two times higher than the one achieved with 60 µE*m-2*s-1.
It is noteworthy to observe that carbon availability seems to determine the OD730 obtained at
the stationary growth phase (after 52h), since a higher OD730 is achieved in stationary
phase under higher bicarbonate concentrations. This can be explained because more biomass
requires more carbon(also shown in figure 8).
On the other hand, the found carbon limited effect in exponential phase can be explained by
the inability of the cells to capture carbon at a higher rate when the bicarbonate
concentrations drop. Light intensity only limits the achievable growth rate during the
exponential phase. This means that cultures provided with a higher light intensity will
reach the stationary phase faster than those under a lower light intensity condition but
both will eventually reach the same stationary OD730.
References
[1]Branco Dos Santos F., Du, W., & Hellingwerf, K.J. (2014) Synechocystis: Not Just a Plug-Bug for CO2, but a Green E. coli. Front. Bioeng. Biotechnol. 2: 36.
[2]Favie, B. (2016) Towards a 24-well microplate incubator for phototrophic organisms. Software development for control and calibration of light sources (MSc thesis). University of Amsterdam/Vrije University, Amsterdam, Netherlands.
[3]Godara, A., Kao, K.C. Adaptive laboratory evolution for growth coupled microbial production. World J Microbiol Biotechnol 36, 175 (2020).
[4]Kim, H.W., Vannela, R., Zhou, C. and Rittmann, B.E. (2011), Nutrient acquisition and limitation for the photoautotrophic growth of Synechocystis sp. PCC6803 as a renewable biomass source. Biotechnol. Bioeng.
[5]Ojo, E. O., Auta, H., Baganz, F., & Lye, G. J. (2015). Design and parallelisation of a miniature photobioreactor platform for microalgal culture evaluation and optimisation. Biochemical Engineering Journal, 103, 93–102.
[6]Pereira, H., Barreira, L., Mozes, A., Florindo, C., Polo, C., Duarte, C. V., Custódio, L., & Varela, J. (2011). Microplate-based high throughput screening procedure for the isolation of lipid-rich marine microalgae. Biotechnology for biofuels, 4(1), 61.
[7]Tillich, U.M., Wolter, N., Schulze, K. et al. High-throughput cultivation and screening platform for unicellular phototrophs. BMC Microbiol 14, 239 (2014).
[8]Yu, Y., You, L., Liu, D., Hollinshead, W., Tang, Y., & Zhang, F. (2013). Development of Synechocystis sp. PCC 6803 as a Phototrophic Cell Factory. Marine Drugs, 11(8), 2894–2916.
