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Colony development of laser printed eukaryotic (yeast and microalga) microorganisms in co-culture

−1
−1
500 the two microorganisms (0.344 d and 0.325 d re-
S. bayanus spectively for S. bayanus and C. vulgaris) and far be-
C. vulgaris low the values usually encountered for these microor-
400 –1 –1
ganisms (1.19 d and 8.32 d measured for the two
organisms in liquid batch cultures). The authors pro-
Radius (µm) 300 pose that the apparent “unrestricted growth” observed

in this experiment is related to only growth in a part of
the colonies such as the cells in contact with the sub-
200
strate and/or close to the external radius of the colony.
Indeed, if one (almost) constant part of the colony
100 grew without restriction, the apparent growth of the
0 2 4 6 8 10
entire colony would still present itself as exponential
Time (day) growth, but with a smaller growth rate. This was ob-

Figure 5. Microcolony growth measured by image analysis. served in this study.
Average radial growth of 10 colonies, error bars represent the
standard deviation over these 10 values. 4. Discussion and Conclusion

Laser printing and separate observation of the micro-
100 S. bayanus colonies in two geographically separate laboratories
C. vulgaris presented a number of technical uncertainties. Despite
90 these difficulties, both organisms were robust enough
Surface fraction (%) 80 micro-colonies were monitored. The results suggested
to survive the treatment, and the growth rates of their

that for both organisms, the cells across the entire
70
colony grew.
To the best of our knowledge, this is the first time
60 that laser printing has been applied to print microbial
micro-colonies of single cell eukaryotes. This tech-
50
0 2 4 6 8 10 nique has recently been used to print earth samples in
[1]
order to aid in the isolation of new soil organisms .
Time (day)
However, the technique has not been applied to the
Figure 6. Fraction of the total droplet surface filled with mi- printing of pure culture of eukaryotic microorganisms.
croorganisms. Average values for 10 colonies, the error bars are From the results presented here, it is difficult to see
the standard deviation over the 10 values.
whether the two organisms exhibit a symbiotic or a
1.5x10 5 competitive relationship. In any case, in the presence
S. bayanus of a heterotrophic carbon substrate and complete ex-
C. vulgaris posure to air, there is no need for the two organisms to
y = 4740 × exp(0.344 t)
cooperate.
1.0x10 5 This protocol is a first step in a series of studies that
Surface (µm 2 ) will aim to study and model the development of these

model-organisms in the presence of each other. The
5
interaction of the two organisms in the moments before
0.5x10
and after the colonies touch is of particular interest.
y = 4064 × exp(0.325 t) This work and laser printing could be applied to
0 strain selection, optimisation of growth and target
0 2 4 6 8 10 molecule production through factorial experiments,
Time (day) and even the development of engineered symbioses

for better production of target molecules. Additionally,
Figure 7. Microcolonies’ development as followed by the in-
crease in surface area. Average of 10 colonies, the error bar is the techniques reported here could be used to study
the standard deviation over these 10 values. the reaction of organisms to one another and quorum
42 International Journal of Bioprinting (2016)–Volume 2, Issue 2