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

lows the deposition of, statistically speaking, prede- starts to metabolise internal storage compounds “het-
termined number of cells on the growth surface with erotrophically”, releasing CO 2. The study of such
micrometre positional-precision, and with droplets phenomenon with a single organism would be very
that can be smaller than 10 pL. This level of precision difficult in liquid culture but with two organisms, the
opens the way for exact growth, selection, and inter- phenomenon of heterotrophic/autotrophic growth co-
action studies with the advantage that replication of uld be simulated in low cell density cultures. Before
the experiment would be only limited by the power of this possibility can be explored, the interaction be-
the observation and data analysis techniques. Another tween the two organisms could be studied with the aid
advantage of this technique is that by printing micro- of laser bioprinting.
organisms in grids, the technique can very easily lend Different printing techniques, mainly inkjet printing,
itself to developmental models based on grid arrange- extrusion printing (also called bio-plotting or syringe-
ments such as lattice automata or agent-based mod- based printing technique), and laser bioprinting have
els [2–5] . The work presented here is the result of collab- been applied for two and three-dimensional assembly
orative research between the Department of Biopro- of biological materials including proteins, DNA, micro-
cess Engineering and Biomaterials of Centralesupélec organisms, and living mammalian cells [12] . Laser prin-
Paris in France and the Department of Nanotechnol- ting offers the capability to combine high resolution
ogy of the Laser Zentrum Hannover in Germany, two and ultra-small droplet volumes (below 10 pL) with
laboratories that are almost 800 km apart. high sample viscosities and high cell densities due to
Two robust microorganisms were used for this work, the absence of a nozzle. In this paper, laser printing is
namely S. cerevisiae var. bayanus (hereafter referred used to print droplets of alginate solution with embed-
to as S. bayanus)—a wine yeast—and C. vulgaris, a ded yeast or microalgae cells in predefined patterns.
well-studied green microalga of the Chlorophyceae In brief, bioprinting of microorganisms paves the
group. Both organisms are rather spherical in shape way for the development of new and precise methods
(2–10 µm in diameter), showing good resistance to that could be used to study: (i) the development of
harsh environmental conditions. C. vulgaris has a hard microorganisms in solid matrices in the presence of
[6]
cellulosic cell wall containing some chitin and S. nutrient gradients, (ii) the interaction of the same and
[7]
bayanus has a hard glucan-based cell wall . Both different organism-colonies next to each other, (iii) the
organisms can grow heterotrophically with glucose as response to stress and resistance to inhibitors, and (iv)
[8]
the sole carbon source , but C. vulgaris is addition- cell communication or quorum sensing. This method
ally capable of autotrophic (photosynthetic) growth on provides a relatively simple way to perform experi-
[9]
inorganic carbon in the presence of light . Mixotro- ments with a large number of replicas and could be
phic growth of C. vulgaris based on a mixture of aut- even applied to strain selection in the future. Printing
[9]
otrophic and heterotrophic growth is also possible . S. could also provide the means to perform multifactorial
bayanus can metabolise glucose either respiratively experiments.
[10]
with O 2 or fermentatively in the absence of O 2 . The To the best of our knowledge, the two eukaryotic
metabolism mode chosen by the organism depends on microorganisms mentioned above are printed for the
the conditions but in general, Saccharomyces yeasts first time in order to observe and study these microor-
are Crabtree-positive [10] and “prefer” the fermentative ganisms in close proximity and potentially different
mode of metabolism. Despite this, even when fer- topologies. Here, we report on how S. bayanus and C.
menting sugars, yeast requires a small amount of vulgaris can be printed in specific patterns. Their de-
oxygen that is utilised for biosynthesis of cell mem- velopment is observed and measured via confocal mi-
brane components [11] . croscopy.
A form of symbiosis could be imagined where an
autotrophic organism uses the CO 2 released by a het- 2. Materials and Methods
erotrophic organism and vice versa; the latter uses the 2.1. Strains, Media and Growth Conditions
O 2 released by the former. This symbiosis of course
already exists on a planetary scale with an additional Chlorella vulgaris (211-11b Göttingen) and Saccharo-
input of CO 2 from geothermic activity. Even under myces cerevisiae var. bayanus (DSMZ 3774—referred
autotrophic conditions, once a culture becomes dense to as S. bayanus hereafter) were used for this work.
enough to be light-limited, a part of the population Bristol medium [13] was used to grow C. vulgaris in

38 International Journal of Bioprinting (2016)–Volume 2, Issue 2