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Ghosh and Yi
ii. As needle diameter increased, so did the number of of the bioink and the nature of the printing substrate
algae cells in printed samples; this pattern was observed both play important roles in preserving the viability
3 and 6 days after printing. and functionality of cells in the resulting bioprinted
materials, as well as the overall 3D structure. When a
bioink composed of sodium alginate and microalgae is
5.2.3. Photosynthetic living components printed onto a substrate composed of bacterial cellulose
The hydrogel was developed by encapsulating an and calcium chloride, an alginate hydrogel can form
alginate hydrogel matrix onto non-living bacterial wherein microalgal cells are immobilized.
cellulose for 3D bioprinting of photosynthetically The bacterial cellulose is placed on top of a
active microalgae (C. reinhardtii) capable of generating microalgal nutrient medium (minimal medium or carbon-
energy and O 2 [118] . The calcium alginate-based hydrogel supplemented medium) to ensure that microalgae can
enhances nutrient permeability, light transmission, and grow inside the bioprints on the bacterial cellulose.
gas (O and CO ) emission, all of which are important The bacterial cellulose supporting the living bioprinted
2
2
for microalgal growth [119] . Bacterial cellulose, in general, microalgae can then be peeled off and used in different
is a flexible, cell-friendly, and durable biopolymer ways. Microalgal cells in bioprints can also be regenerated
with exceptional properties such as toughness (2 – 25 and used as new bioinks in subsequent bioprinting
MJ m ) and tensile strength (73 – 194 MPa) [120,121] . The processes.
−3
fermentation of bacteria such as Komagataeibacter 5.2.4. Hydrogel filters for copper removal
rhaeticus and Gluconacetobacter hansenii produces
bacterial cellulose [120,122,123] . Bacterial cellulose The bioink in this study was prepared with sodium alginate
possesses a nano-fibrous architecture and absorbent containing the C. reinhardtii algae strain cc125 [125] .
properties that, if further employed as a substrate for
microalgal bioprint, could allow nutrients to circulate
and penetrate into microalgal cells, thus promoting the
growth of algae [124] . In addition, these hydrogels are
perfectly biodegradable and biocompatible, and are
good encapsulators as they do not interfere with cell–
cell interactions while still allowing the transport of
water . This hydrogel can be utilized to fabricate living
[61]
microalgal materials as well as for the development of
low-cost microalgal bioprinters [118] . Microalgae have
been printed onto agar and bacterial cellulose substrates
using home-built bioprinters (Figure 14). Microalgae
can be bioprinted into various sizes and pre-defined
geometries as mono- or multi-layered constructs using
this technology. Surprisingly, the bioprints may be
removed from the bacterial cellulose and reattached
to new bacterial cellulose surfaces while maintaining
adhesion. The resistance of these bioprinted microalgal Figure 14. Regenerative bioprinting of photosynthetic living
structures to physical deformation and immersion components (from ref. [118] licensed under Creative Commons
in water demonstrates their physical stability. The Attribution license).
bioprinted microalgal cells retain good viability for at
least a month. The patterned microalgae in bioprints can A B
also be regenerated to make new bioinks. After removal
from the nutrient broth, the microalgal cells bioprinted
onto bacterial cellulose could live for at least 3 days, with
their longevity being extended further when placed onto
fresh agar. Overall, these regenerative photosynthetic
living materials composed of microalgae bioprinted on
bacterial cellulose have a wide range of potential uses,
including adhesive labels, photosynthetic bio-garments,
and artificial leaves. Finally, living materials can be 3D Figure 15. (A) Design of the filter; (B) Custom filtration setup
printed by depositing living cells (bioink) layer by layer developed for the experiment (from ref. [125] licensed under Creative
onto a non-living matrix (substrate). The composition Commons Attribution license).

International Journal of Bioprinting (2022)–Volume 8, Issue 4 187