Page 178 - IJB-10-5

International Journal of Bioprinting Control nutrients to manipulate fungal growth

stirring for 7–10 days before they were used for subsequent were used. Samples were pre-sheared using a shear rate
experiments. of 10 s for 30 s before being allowed to equilibrate for
−1
60 s. For steady-state viscosity tests, a shear rate range of
2.3. Effect of malt and peptone on mycelium growth 0.01–1000 s was used. The viscosity as a function of shear
−1
A two-level full factorial design of experiment was adopted rate was then recorded. For amplitude sweep tests, using
to study the effect of malt and peptone on mycelium growth. a frequency of 0.5 Hz, the shear stress was increased from
Malt agar plates were made as described above, except 0.1 to 500 Pa. The storage and loss moduli were recorded.
that the malt and peptone concentrations were varied at
two different levels, as shown in Table 1. The growth of All samples were tested at 25°C. Triplicates of each sample
were analyzed.
mycelium on each set was monitored over 14 days. Digital
images of the agar plates were taken and processed using 2.6. 3D printing of multi-material constructs
ImageJ, and the growth rate was quantified by measuring 3D-printed constructs were fabricated using DIW with
30
the area of mycelium on each set over 14 days. To determine an Allevi 2 bioprinter (3D Systems, USA). The structures
the dry weight of biomass produced, the mycelium films were designed using FreeCAD and the gcode files were
were peeled from each agar plate after 14 days of culture. generated using PrusaSlicer. The inks were loaded
They were then dried in an oven at 50°C for 3 days before into a 10 mL syringe (BD, USA). 22G tapered nozzles
they were subsequently weighed. Triplicates were analyzed (Nordson, USA) were used. Pneumatic pressure ranging
to ensure reproducibility of results. from 20 to 30 psi was applied along with a print speed of
2.4. Inks preparation for 3D printing 20 mm/s. After printing, constructs were sprayed with a
The inks for DIW were made by dissolving alginate (1.2% solution of deionized water containing calcium chloride
w/v) and CMC (3.6% w/v) in a solution of malt and (3% w/v) supplemented with gentamicin sulfate (50 μg/
peptone at 45°C. The concentration of malt and peptone mL) to crosslink the alginate. The growth of mycelium
was adjusted according to Table 1. In addition, another on the multi-material constructs was recorded over a
set of inks that would be mixed with liquid mycelium period of 10 days. Triplicates of each design were printed
was made using malt (2% w/v) and peptone (0.1% w/v) and observed.
and were denoted as the medium level of both malt
and peptone, respectively. The inks were stirred using 2.7. Scanning electron microscopy
a magnetic stirrer until all the alginate and CMC had The microstructure of pure mycelium sheets and fabricated
completely dissolved. Agar (3% w/v) was then added, and fungal-based ELMs were imaged by means of scanning
the temperature was increased to facilitate the dissolution electron microscopy (SEM; JEOL-5600LV, Japan) on
of agar. The mixture was then autoclaved at 121°C for 20 square samples each with a length of roughly 5 mm. Prior
min, and once removed from the autoclave, it was stirred to imaging via SEM, all samples were placed in a −20°C
until it had set. Liquid mycelium was then mixed into the fridge overnight and subsequently lyophilized for 24 h.
ink at a ratio of 1:5 (v/v) and was stirred until a fine paste Samples were cut into small pieces and mounted onto the
was formed. For acellular inks, malt and peptone broths SEM stage using carbon tape. All samples were coated with
of the corresponding malt and peptone concentrations, gold for 75 s at 15 mA, which is equivalent to about 11 nm
respectively, that were devoid of mycelium were added coating thickness. For the SEM, an acceleration voltage of
instead at the same volumetric ratio. 10 kV was used. SEM images were processed using ImageJ
to measure the surface porosity and hyphae diameter. The
2.5. Rheology surface porosity was calculated by obtaining the average of
The rheological properties of the inks were evaluated using the percentage of areas occupied by pores in the electron
a Bohlin Gemini HR Nano rheometer (Malvern, UK). micrograph at three random points of each sample. The
A 15 mm serrated plate and a measuring gap of 0.5 mm hyphae diameter was obtained by measuring the projected
width of hyphae at 15 different points in an electron
Table 1. Concentrations of malt and peptone associated with micrograph of the sample.
high and low levels, respectively, as used in the two-level full
factorial design of experiment 2.8. Statistical analysis
Statistical analysis was conducted using Microsoft Excel. A
Nutrient component Concentration (% [w/v])
Low level (−1) High level (+1) two-way analysis of variance (ANOVA) with a significance
level of 0.05, followed by a post hoc Tukey’s HSD test, was
Malt 0.1 5 performed for data comparison. Data are presented as
Peptone 0.01 1 mean ± standard deviation.

Volume 10 Issue 5 (2024) 170 doi: 10.36922/ijb.3939