Article

The 3D Printing of Biomass–Fungi Composites: Effects of

Waiting Time after Mixture Preparation on Mechanical
Properties, Rheological Properties, Minimum Extrusion
Pressure, and Print Quality of the Prepared Mixture
Al Mazedur Rahman 1 , Abhinav Bhardwaj 1 , Zhijian Pei 1, * , Chukwuzubelu Ufodike 2
and Elena Castell-Perez 3

1 Department of Industrial and Systems Engineering, Texas A&M University, College Station, TX 77843, USA
2 Department of Engineering Technology and Industrial Distribution, Texas A&M University,
College Station, TX 77843, USA
3 Department of Biological and Agricultural Engineering, Texas A&M University,
College Station, TX 77843, USA
* Correspondence: zjpei@tamu.edu

Abstract: Biomass–fungi composites, an emerging class of sustainable materials, have potential

Citation: Rahman, A.M.; Bhardwaj,
A.; Pei, Z.; Ufodike, C.; Castell-Perez,
E. The 3D Printing of Biomass–Fungi
Composites: Effects of Waiting Time
after Mixture Preparation on
Mechanical Properties, Rheological
Properties, Minimum Extrusion
Pressure, and Print Quality of the
Prepared Mixture. J. Compos. Sci.
2022, 6, 237. https://doi.org/
10.3390/jcs6080237
Academic Editors: Ahmed Koubaa,
Mohamed Ragoubi and
Frédéric Becquart
applications in the construction and packaging industries. Molding-based manufacturing methods
are typically employed to make products from these composites. Recently, a 3D printing-based
method was developed for biomass–fungi composites to eliminate the need for making molds and to
facilitate customized product design compared with manufacturing methods based on molding and
hot-pressing. This method has six stages: biomass–fungi material preparation; primary colonization;
mixture preparation; printing; secondary colonization; and drying. This paper reports a study about
the effects of waiting time between the mixture preparation and 3D printing using biomass–fungi
composites. As the waiting time increased from 0.25 to 3 h, the hardness and compressibility of
the prepared mixture increased. As the waiting time increased from 0.25 to 8 h, the shear viscosity
showed a decreasing trend; the yield stress of the prepared mixture increased at the beginning,
then significantly decreased until the waiting time reached 3 h, and then did not significantly vary
after 3 h. As the waiting time increased, the storage modulus and loss modulus decreased, the loss
tangent delta increased, and the minimum required printing pressure for continuous extrusion during
extrusion-based 3D printing increased. The print quality (in terms of layer-height shrinkage and
filament-width uniformity) was reasonably good when the waiting time did not exceed 4.5 h.

Keywords: biomass-fungi; 3D printing; mycelium; rheology; biocomposite; texture analysis

Received: 22 July 2022
Accepted: 11 August 2022
Published: 12 August 2022

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1. Introduction
Both the construction and packaging industries have a substantial negative impact on
the environment [1]. The production of cement (widely used in the construction industry)
and petroleum-derived plastics (widely used in the packaging industry) emits a huge
amount of CO2 . In 2015, the U.S. construction sector generated 565.8 million metric tons of
CO2 emissions, making it the fourth highest CO2 -emitting sector in the U.S. [2]. The CO2
emissions from fossil fuel extraction and transportation attributed to plastic production are
about 9.5 to 10.5 million metric tons, respectively, per year in the U.S. [3]. The packaging
industry consumes 38% of the petroleum-based plastics produced globally [4]. Based on the
current trends in production and waste management, it is estimated that roughly 12 billion
metric tons of plastic waste will be in the natural environment or landfills by 2050 [5].
Sustainable materials that do not emit CO2 in their production and have biodegradable
characteristics are desirable in the construction and packaging industries.

J. Compos. Sci. 2022, 6, 237. https://doi.org/10.3390/jcs6080237 https://www.mdpi.com/journal/jcs

 landfills by 2050 [5]. Sustainable materials that do not emit CO2 in their production and
J. Compos. Sci. 2022, 6, 237 have biodegradable characteristics are desirable in the construction and packaging 2 of 17
industries.
Biomass–fungi composites, as an alternative to petroleum-based plastics, are an
emerging class of sustainable
Biomass–fungi composites,materials for the construction
as an alternative and packaging
to petroleum-based plastics,industries
are an emerg-[6].
Lignocellulosic biomass (such as corn stalks, wheat straws,
ing class of sustainable materials for the construction and packaging industries [6]. Lig- sugarcane leaves, and
sorghum stalks)
nocellulosic [7–18],
biomass used
(such as for
cornproducing
stalks, wheat biomass–fungi composites
straws, sugarcane [19–21],
leaves, is one of
and sorghum
the most abundant sustainable raw materials on earth. In
stalks) [7–18], used for producing biomass–fungi composites [19–21], is one of the these composites, the biomass
most
serves as asustainable
abundant nutrition source for fungi;on
raw materials the fungiIngrow
earth. theseinto a networkthe
composites, of fine white
biomass filaments
serves as a
(mycelium)
nutrition sourceandfor
bind thethe
fungi; biomass together
fungi grow into[22]. These of
a network composites
fine white generally have a low
filaments (mycelium)
density,
and bindlow the cost, and together
biomass tailorable[22].biodegradability.
These composites generally have a low density, low
There are publications
cost, and tailorable biodegradability. on biomass–fungi composites [22–27] and their applications
[28–31]. These publications report the
There are publications on biomass–fungi composites effects of fungal
[22–27]species,
and theirnutrient substrates,
applications [28–31].
environmental
These publications growth
report conditions
the effects of (e.g.,
fungallight or dark
species, andsubstrates,
nutrient CO2 concentration)
environmental on
mycelium-based
growth biocomposites,
conditions (e.g., light or darkbiocomposite fabrication
and CO2 concentration) methods [25,32,33],
on mycelium-based and their
biocomposites,
effects on thefabrication
biocomposite mechanical properties
methods of fabricated
[25,32,33], and theirsamples
effects [34]. Currently,
on the mechanical molding-based
properties of
methods are
fabricated used[34].
samples to manufacture parts using biomass–fungi
Currently, molding-based methods are used composites. Another
to manufacture method
parts using
is based on hot-pressing. This method includes three steps:
biomass–fungi composites. Another method is based on hot-pressing. This method includes (1) fungal or enzymatic
incubation;
three (2)fungal
steps: (1) preparation for hot-pressing;
or enzymatic incubation; and
(2)(3) hot-pressing
preparation [35]. These two
for hot-pressing; andmethods
(3) hot-
can produce
pressing denser
[35]. These twoproducts,
methods can butproduce
consume more
denser energybut
products, and offer less
consume moreflexibility
energy and in
offer less flexibility
designing complexinshapes
designing complex
in small shapes in
quantities. Bothsmall quantities.
fabrication Both fabrication
methods methods
restrict the access
restrict
to oxygenthe access
needed to for
oxygen needed
the fungi to for
grow thethrough
fungi to grow throughThe
the biomass. the biomass.
3D printing Theof 3Dbiomass–
printing
of biomass–fungi
fungi compositescomposites could the
could facilitate facilitate the manufacturing
manufacturing of partsofwith
partscomplex
with complex
shapesshapes
(such
(such as sandwich and topology-optimized structures) in
as sandwich and topology-optimized structures) in art, architecture, interior art, architecture, interior design, and
and
construction [6,27,36–40] that cannot be easily produced using
construction [6,27,36–40] that cannot be easily produced using molding-based or hot- molding-based or hot-pressing
based
pressingmethods.
based methods.
The
The effects ofthe
effects of themixture
mixturecomposition
composition onon
thethe rheological
rheological properties
properties of a of a mixture
mixture have
have
alreadyalready
beenbeen reported
reported [22].[22]. Using
Using biomass–fungicomposites,
biomass–fungi composites,aa 3D 3D printing-based
printing-based
manufacturing
manufacturing method method consisting
consisting of of six
six stages
stages (Figure
(Figure 1) 1) has
has been
been reported
reported [24].
[24]. The
The red
red
boxes in Figure 1 represent the outcome
boxes in Figure 1 represent the outcome of each stage. of each stage.

Figure1.
Figure 1. Six
Six stages
stages of
of the
the 3D
3D printing-based
printing-basedmethod
methodfor
forbiomass–fungi
biomass–fungicomposites.
composites.

There are
There are nonoreported
reported studies
studies regarding
regarding the
the effects
effects of
ofwaiting
waiting time
timebetween
between mixing
mixing
(mixturepreparation
(mixture preparationfor for3D
3Dprinting)
printing)(Stage
(Stage3)3)and
and 3D
3D printing
printing with
with the
the prepared
prepared mixture
mixture
(Stage 4) in the
(Stage the 3D3Dprinting
printingofofbiomass–fungi
biomass–fungicomposites.
composites. TheThe
purpose
purposeof this paper
of this was
paper
to fill
was thisthis
to fill gapgap in inthe
theliterature.
literature.For
Forextrusion-based
extrusion-based3D 3D printing biomass–fungi
printing of biomass–fungi
composites,
composites, itit is
is necessary
necessary to to know
know the
the effects
effects of
of waiting
waiting time
time between
between mixing
mixing (Stage 3)3)
and
and 3D
3D printing (Stage
(Stage 4)4) on
onthe
theprint
printquality.
quality.InIn this
this paper,
paper, wewe report
report a study
a study about
about the
the effects of waiting time on the mechanical properties and rheological properties of a
prepared mixture (at Stage 3), the minimum printing pressure required for continuous
extrusion during extrusion-based 3D printing, and the quality of printed samples (in terms
of layer-height shrinkage and filament-width uniformity). A texture profile analysis (TPA)
was employed to measure the mechanical properties of the prepared mixture. Oscillatory
J. Compos. Sci. 2022, 6, 237 3 of 17

and steady shear experiments were conducted to evaluate the rheological properties of the
prepared mixture, including viscosity, viscoelasticity, and yield stress [41–43]. These results
were helpful in explaining the changes in print quality and minimum extrusion pressure of
the prepared mixture.

2. Methodology
2.1. Materials
Biomass–fungi-mix material, wheat flour, water, and psyllium husk powder were
used to prepare the biomass–fungi mixture. The biomass–fungi-mix material (Grow-it-
Yourself: GROW.bio, Green Island, NY, USA) was received in a polypropylene bag with a
filter (1.5 inch square in size) with 0.2 µm pores. The feedstock material underwent three
steps in the factory. First, the biomass material was pasteurized to eliminate any harmful
organisms. The pasteurized biomass material was then inoculated with fungi that belong
to the Basidiomycete group and the fungi spread and multiplied in the biomass material.
In the third step, this biomass–fungi-mix material was dehydrated before packaging [24].
Wheat flour (all-purpose flour: Great Value, Walmart, Bentonville, AR, USA) and
psyllium husk (NOW Supplements, Bloomingdale, IL, USA) were procured from a local
Walmart store.
The mixture was prepared batch by batch and each batch had 50 g of biomass–fungi-
mix material. Every batch had the same composition as shown in Table 1. This composition
was successfully used in a previous study [22].

Table 1. Mixture composition for each batch of 50 g biomass–fungi-mix material.

Material Amount
Biomass–fungi-mix material 50 g ± 0.02 g
Wheat flour 20 g ± 0.02 g
Water 200 mL ± 0.02 mL
Psyllium husk powder 10 g ± 0.02 g

2.2. Experimental Procedures

The procedures (the six stages shown in Figure 1) described in a recent paper [22] were
followed in this study. The biomass–fungi-mix material from Stage 1 underwent primary
colonization in Stage 2. During Stage 3 (mixing), the primary colonized biomass–fungi
material was mixed with wheat flour and water in a commercial mixer (NutriBullet PRO:
Capital Brands, Los Angeles, CA, USA) for 15 s and turned into a liquid slurry. Psyllium
husk powder was then added to the liquid slurry. The liquid slurry was manually mixed
using a spoon for 1 to 2 min. The resultant biomass–fungi mixture was then ready for 3D
printing (Stage 4).
Figure 2 shows the setup for the 3D printing experiments. A Delta 2040 (Delta 2040:
WASP, Massa Lombarda RA, Italy) 3D printer was equipped with a storage container (vol-
ume: 3 L) to hold the mixture prepared in Stage 3. It had a pneumatically operated plastic
piston that pushed the mixture through a custom-built nozzle assembly. An air compressor
(Kobalt 4.3 gallon Electric Twin Stack Quiet Air Compressor: Kobalt, Mooresville, NC,
USA) was used to generate the printing pressure on the piston in the storage container. A
pressure gauge was used to measure the printing pressure. The nozzle assembly had a
screw extruder and a custom-designed casing with a 6 mm × 6 mm nozzle opening. Stages
2 to 5 (primary colonizing, mixing, 3D printing, and secondary colonizing) were conducted
at room temperature. All the apparatus was cleaned before and after every use with a 70%
ethyl alcohol solution to avoid any contamination.
 nozzle opening. Stages 2 to 5 (primary colonizing, mixing, 3D printing, and secondary
J. Compos. Sci. 2022, 6, 237 4 of 17
colonizing) were conducted at room temperature. All the apparatus was cleaned before
and after every use with a 70% ethyl alcohol solution to avoid any contamination.

Figure 2.
Figure2.
Figure The
2. The 3D
The3D printing
3Dprinting setup
printingsetup with
setupwith custom-built
withcustom-built nozzle
custom-builtnozzle assembly.
nozzleassembly.
assembly.

2.3.
2.3. Measurement
Measurement of of the
the Mechanical
Mechanical Properties
Propertiesofofthe
thePrepared
PreparedMixture
Mixture
AA texture
textureanalyzer
analyzer(TA.XT.Plus,
(TA.XT.Plus,Texture
TextureTechnologies, Hamilton,
Technologies, MA, MA,
Hamilton, USA)USA)was used
was
to perform a texture profile analysis. This method has been extensively used
used to perform a texture profile analysis. This method has been extensively used to to characterize
biological
characterizematerials, including
biological food including
materials, materials (such
food asmaterials
fruit, whipped
(such toppings,
as fruit, gels, and
whipped
pudding
toppings,desserts)
gels, and [44–47].
puddingFigure 3a shows
desserts) the solid
[44–47]. probe
Figure 3a of the analyzer
shows the solidand a mixture
probe of the
sample.
analyzerTheand cylindrical mixtureThe
a mixture sample. samples were prepared
cylindrical using a were
mixture samples cylindrical
prepared cupusing
and aa
plunger, as cup
cylindrical shown andina Figure 3b.asThe
plunger, cup in
shown had an inner
Figure diameter
3b. The of 15
cup had an mm
innerand a height
diameter of
of 10 mm. The plunger had an outer diameter of 14.5 mm. The prepared
15 mm and a height of 10 mm. The plunger had an outer diameter of 14.5 mm. The biomass fungi
mixture
preparedwas filled in
biomass the mixture
fungi cup and was
thenfilled
the plunger
in the cupwasand
used to ensure
then there was
the plunger wereused
no air
to
pockets in the sample.
ensure there were no air pockets in the sample.

Figure
Figure3.3.
Figure Texture
3. Texture analysis
Textureanalysis (a)
analysis (a) measurement
(a)measurement setup
measurement setup using
setup using TA.XT.Plus
using TA.XT.Plus Texture
TA.XT.Plus Texture Analyzer
TextureAnalyzer and
Analyzer and (b)
and (b) cup
(b)cup
cup
and plunger
andplunger used
plungerused to
usedto prepare
toprepare mixture
preparemixture samples.
mixturesamples.
samples.
and

Duringthe
During themeasurement,
measurement,the thesample
samplewaswascompressed
compressedby byaasolid
solidprobe
probefromfroman aninitial
initial
height of 10 mm to 2 mm (80% strain) at a constant rate of 0.5 mm/s (during
height of 10 mm to 2 mm (80% strain) at a constant rate of 0.5 mm/s (during the pre-test, the pre-test,
post-test, and
post-test, and test)
test) and
and aa trigger
trigger force
force of
of 5.0
5.0 g.
g. The
The instrument
instrument beganbegan toto record
record thethe data
data
whenthe
when theforce
forcereached
reachedthethevalue
valueofofthe
thetrigger
triggerforce.
force. After
After the
the first
first compression
compression cyclecycle was
was
completed, and
completed, and after
after aa 10
10 ss delay,
delay,the
thesample
samplewas wascompressed
compressedagain. again. After
After each
each test,
test, aa
force-time(or
force-time (orforce-distance)
force-distance)curvecurvewas
wasobtained,
obtained,as asshown
shownin inFigure
Figure4.4. From
From thethe resultant
resultant
force-distance plot, the following mechanical properties could be
force-distance plot, the following mechanical properties could be obtained [48,49]. obtained [48,49].
(1) Hardness (the maximum force recorded
(1) recorded during
during thethe first
first compression
compressioncycle);
cycle);
(2) Compressibility (the work required to deform the sample during the first compression
and calculated as the area under the force-distance curve for the first compression cycle).
 (2) Compressibility (the work required to deform the sample during the first
J. Compos. Sci. 2022, 6, 237 5 of 17
compression and calculated as the area under the force-distance curve for the first
compression cycle).

Figure4.
Figure 4. Force-time
Force-time curve
curveobtained
obtainedfrom
fromthe
thetexture
textureanalyzer
analyzerfor
foraawaiting
waitingtime
timeof
of11min.
min.

2.4.
2.4. Measurement
Measurement of of the
the Rheological
Rheological Properties
Propertiesofofthe
thePrepared
PreparedMixture
Mixture
AA rheometer (ARES G2, TA Instruments, New Castle, DE,USA)
rheometer (ARES G2, TA Instruments, New Castle, DE, USA)was
wasused
used to
to measure
measure
the rheological properties of the prepared mixture. It had a pair of parallel
the rheological properties of the prepared mixture. It had a pair of parallel plates plates 25 25
mmmm in
diameter and a gap of 1 mm between the two plates (Figure 5). A forced
in diameter and a gap of 1 mm between the two plates (Figure 5). A forced convection convection oven
was
ovenused
wastoused
ensure that thethat
to ensure temperature of the test
the temperature of environment remainedremained
the test environment at 25 ◦ C.
constant constant
All the
at 25 experiments
°C. were performed
All the experiments three times.
were performed threeTo determine
times. the shear
To determine theviscosity of the
shear viscosity
prepared mixture, the shear rate was increased from 0.01 to 100 s −1 and a linear viscoelastic
of the prepared mixture, the shear rate was increased from 0.01 to 100 s and a linear −1
range was observed
viscoelastic range was with a strainwith
observed sweep (0.1 tosweep
a strain 100%) (0.1
at ato
fixed angular
100%) frequency
at a fixed of
angular
10 rad/s. A dynamic frequency sweep was performed at a constant strain of 1%. To obtain
frequency of 10 rad/s. A dynamic frequency sweep was performed at a constant strain
J. Compos. Sci. 2022, 6, x FOR PEER REVIEW 6 of of
18
the yield stress (yield stress in rheology is defined as the applied stress at which irreversible
1%. To obtain the yield stress (yield stress in rheology is defined as the applied stress at
plastic deformation is first observed across the sample), an oscillatory measurement was
which irreversible plastic deformation is first observed across the sample), an oscillatory
conducted at a constant frequency of 1 Hz and the stress was increased from 0.1 to 104 Pa.
measurement was conducted at a constant frequency of 1 Hz and the stress was increased
from 0.1 to 104 Pa.

Figure 5.
Figure 5. The
The pair
pair of
of parallel
parallel plates
plates of
of 25
25 mm
mm diameter
diameter with
withaa11mm
mmgap
gapin
inbetween.
between.

2.5. Determination of the Minimum Printing Pressure Required for Continuous Extrusion
For all 3D printing experiments, the same design file was used to print the samples
shown in Figure 5. The printing speed was 30 mm/s and layer height was 6 mm. The
experiment at each level of waiting time was started at a printing pressure of 100 kPa. If
J. Compos. Sci. 2022, 6, 237 6 of 17
Figure 5. The pair of parallel plates of 25 mm diameter with a 1 mm gap in between.

2.5. Determination of the Minimum Printing Pressure Required for Continuous Extrusion
2.5. Determination of the Minimum Printing Pressure Required for Continuous Extrusion
For all 3D printing experiments, the same design file was used to print the samples
For all 3D printing experiments, the same design file was used to print the samples
shownininFigure
shown Figure5. 5.The
The printing
printing speed
speed waswas 30 mm/s
30 mm/s and and
layerlayer
heightheight
was 6wasmm.6 The
mm. The
experiment at each level of waiting time was started at a printing pressure of 100 kPa.kPa. If
experiment at each level of waiting time was started at a printing pressure of 100
Ifcontinuous
continuousextrusion
extrusionwaswas not
not obtained (as shown
obtained (as shown in inFigure
Figure6a),
6a),the
theprinting
printingpressure
pressure was
increased
was by an
increased by increment
an incrementof 50 kPa.
of 50 This
kPa. continued
This continueduntil
untilcontinuous
continuousextrusion
extrusion(as
(asshown
in Figure
shown 6b) was
in Figure 6b)obtained. The printing
was obtained. pressure
The printing waswas
pressure recorded as the
recorded minimum
as the minimum printing
printing pressure at this level of waiting time. If continuous extrusion was not
pressure at this level of waiting time. If continuous extrusion was not obtained when the obtained
when the printing
printing pressurepressure was raised
was raised to 300
to 300 kPa,kPa,
thethe printingpressure
printing pressure was
wasincreased
increasedby by an
an increment of 20 or 40 kPa (instead of 50 kPa) for the subsequent
increment of 20 or 40 kPa (instead of 50 kPa) for the subsequent printing. printing.

Figure6.6.Printed
Figure Printedsamples
samples
byby
(a)(a) discontinuous
discontinuous extrusion
extrusion andcontinuous
and (b) (b) continuous extrusion.
extrusion.

2.6. Measurement of Print Quality

2.6. Measurement of Print Quality
The print quality was evaluated using layer-height shrinkage and filament-width
The print quality was evaluated using layer-height shrinkage and filament-width
uniformity. A square-shaped sample, as shown in Figure 7a, was printed at each level of
waiting time.AThe
uniformity. square-shaped
width and heightsample, as shown
of each in Figure
of the four 7a, was
segments wereprinted at each
measured usinglevel of
awaiting time. (Absolute
slide caliper The widthDigimatic,
and height of each of
Mitutoyo, the four
Japan) segments
at five differentwere measured
locations of eachusing a
slide caliper (Absolute Digimatic, Mitutoyo, Japan) at five different
segment (as shown in Figure 7). The second layer of the square-shaped segments was then locations of each
segment
printed on(as
topshown in Figure
of the first layer, 7). The second
as shown layer
in Figure ofThe
7b. the height
square-shaped
of the foursegments
segmentswasin then
printed
the on top
first layer wasofmeasured
the first layer,
again.asThe
shown in Figure
layer-height 7b. The height
shrinkage of the four
was calculated segments
using the in
following equation:
H1 − H2
 
Height shrinkage = ∗ 100% (1)
H1
where H1 and H2 are the average layer height of the four segments in the first layer before
and after the second layer was printed, respectively. Each sample (Figure 7a) had four
segments and five measurements were taken from each segment using a slide caliper
(Absolute Digimatic, Mitutoyo, Japan). Filament-width uniformity was evaluated by a 95%
confidence interval of the measured filament width data (a total of 20 data points at each
level of waiting time).
 1

where H1 and H2 are the average layer height of the four segments in the first layer before
and after the second layer was printed, respectively. Each sample (Figure 7a) had four
segments and five measurements were taken from each segment using a slide caliper
(Absolute Digimatic, Mitutoyo, Japan). Filament-width uniformity was evaluated by a
J. Compos. Sci. 2022, 6, 237 7 of 17
95% confidence interval of the measured filament width data (a total of 20 data points at
each level of waiting time).

Figure7.
Figure 7. Printed
Printed samples
samples for
for measurements
measurementsof
ofprint
printquality:
quality: (a)
(a)one
onelayer
layerand
and(b)
(b)two
twolayers.
layers.

2.7.
2.7. Statistical
Statistical Analysis
Analysis
The
The differences
differences inin the
the variables
variables (such
(such as
as hardness
hardness and
and compressibility)
compressibility) caused
caused by
by aa
change in the waiting time were examined by a t-test using Minitab software
change in the waiting time were examined by a t-test using Minitab software (Minitab, (Minitab, USA).
The
USA).significance level (α)level
The significance used(α)in these
used statistical analyses was
in these statistical 0.05. Therefore,
analyses when the
was 0.05. Therefore,
p-value was smaller than 0.05, the difference was considered to be statistically
when the p-value was smaller than 0.05, the difference was considered to be statistically significant.
significant.
3. Results and Discussion
3.1. Mechanical
3. Results and Properties
Discussionof the Prepared Mixture
Table 2 shows the measured results on the hardness and compressibility of the pre-
3.1. Mechanical Properties of the Prepared Mixture
pared mixture at each level of waiting time. The presented values of the mean and 95%
Table interval
confidence 2 showsatthe eachmeasured results time
level of waiting on the hardness
were andfrom
calculated compressibility
the measuredofdata the
prepared
of at least mixture at each level
four samples. Each of waiting time.
experiment wasThe presentedwith
performed values of the mean
a different and 95%
sample. As
confidence interval at each level of waiting time were calculated from the
the waiting time increased from 0.25 to 3 h, the hardness significantly increased (p < 0.05) measured data
of at least
from four
3.53 to 6N samples.
and theEach experiment was
compressibility performed
significantly with a different
increased (p < 0.05)sample. As the
from 6.70 to
waiting time increased from 0.25 to 3 h, the hardness significantly
10.89 N.mm. The hardness values obtained for the prepared mixtures were comparableincreased (p < 0.05) from
3.53 the
with to 6hardness
N and the compressibility
values significantly
of other hydrogels increased
reported (p < 0.05)
in different fromHowever,
studies. 6.70 to 10.89
we
N.mm.
noted The
that thehardness values
other studies didobtained
not havefor thethe
sameprepared mixturescompression
probe diameter, were comparable
speed, with
and
the hardnessheight
deformation values[50–52].
of otherChanges
hydrogels inreported in different
the hardness studies. However,
and compressibility as thewe noted
waiting
that the other studies did not have the same probe diameter, compression
time increased may have been caused by the swelling behavior of psyllium husk [53], speed, and
deformation height [50–52]. Changes in the hardness and compressibility
water evaporation from the samples, and fungal growth inside the samples. A further as the waiting
time increased
investigation may have
is needed beenunderstand
to fully caused by the the causes.
swelling behavior of psyllium husk [53],
water evaporation from the samples, and fungal growth inside the samples. A further
investigation
Table 2. Hardnessis needed to fully understand
and compressibility the causes.
of the prepared mixture at different levels of waiting time.

Waiting Time (h) Hardness (N) Compressibility (N.mm)

0.25 3.53 ± 0.58 6.70 ± 0.82
0.5 3.83 ± 0.41 6.90 ± 0.78
1 4.98 ± 1.05 9.05 ± 1.51
2 5.55 ± 0.32 9.64 ± 0.50
3 6.00 ± 0.81 10.89 ± 1.37
4 5.09 ± 0.63 9.15 ± 0.93
5 5.53 ± 0.34 10.24 ± 0.4
6.5 5.75 ± 0.86 10.25 ± 1.55
8 5.42 ± 0.63 9.82 ± 0.83

3.2. Rheological Properties of the Prepared Mixture

To study the effects of the waiting time on the rheological properties, nine levels of
waiting time were selected: 0.25; 1; 2; 3; 4; 5; 6; 7; and 8 h. Figure 8 shows the viscosity vs.
shear rate graphs for the nine levels of waiting time. In the figure, each data point (the
mean and 5% standard deviation) was calculated from the measured data of three samples
at each level of waiting time. It can be seen that the viscosity had a general decreasing
 3.2. Rheological Properties of the Prepared Mixture
To study the effects of the waiting time on the rheological properties, nine levels of
waiting time were selected: 0.25; 1; 2; 3; 4; 5; 6; 7; and 8 h. Figure 8 shows the viscosity vs.
J. Compos. Sci. 2022, 6, 237 shear rate graphs for the nine levels of waiting time. In the figure, each data point 8 of 17(the
mean and 5% standard deviation) was calculated from the measured data of three samples
at each level of waiting time. It can be seen that the viscosity had a general decreasing
trend
trend when the waiting
when the waitingtime
timechanged
changedfrom from0.25 0.25toto 8 h.
8 h. The
The prepared
prepared mixture
mixture should
should have have
exhibited shear-thinning behavior to enable a continuous flow through
exhibited shear-thinning behavior to enable a continuous flow through the nozzle [54,55]. the nozzle [54,55].
This
This prepared biomass–fungimixture
prepared biomass–fungi mixtureshowed
showed pronounced
pronounced shear-thinning
shear-thinning behavior
behavior at allat all
levels
levels of
of waiting time.
time. Its
Itsapparent
apparentviscosity
viscositydecreased
decreased byby several
several orders
orders of magnitude
of magnitude as as
the
the shear
shear rate increasedfrom
rate increased from0.01
0.01toto 100
100 s−s1−1. .These
Theseshear
shearrate
rate values
values were
were close
close to the
to the shear shear
rate experienced by extruded material during extrusion-based 3D printing
rate experienced by extruded material during extrusion-based 3D printing [56]. Similar [56]. Similar
shear-thinning properties
shear-thinning propertieswerewereobserved
observedininanother another reported
reported study on on
study thethe
printability of of
printability
psyllium husk
psyllium husk gel/gelatin
gel/gelatin blends
blends[57].
[57].

Figure
Figure 8.
8. Viscosity vs. shear
Viscosity vs. shearrate
rategraphs
graphsfor
for9 9levels
levels
ofof waiting
waiting time.
time.

The yield
The yieldstress
stressof of
thethe
prepared mixture
prepared shouldshould
mixture be highbe
enough
high to maintain
enough to the overall the
maintain
structure after 3D printing and support the weight of the extruded material [58,59].
overall structure after 3D printing and support the weight of the extruded material [58,59]. Figure 9
shows the yield stress data obtained from the stress sweep from 0.1 Pa to 10 4 Pa at different
Figure 9 shows the yield stress data obtained from the stress sweep from 0.1 Pa to 104 Pa
levels
at of waiting
different levelstime. The yield
of waiting stress
time. Thesignificantly
yield stresschanged (p < 0.05)
significantly when(p
changed the< waiting
0.05) when
time increased until it reached 3 h and did not considerably change (p > 0.05) as the waiting
the waiting time increased until it reached 3 h and did not considerably change (p > 0.05)
time increased from 3 to 8 h. The obtained yield stress values of the prepared mixture were
comparable with the reported values of psyllium gels in the literature [60].
Figure 10 shows the storage modulus (Pa) vs. oscillation strain (%) graph obtained
from the amplitude sweep. Viscoelastic properties such as the storage modulus, loss modu-
lus, and loss tangent delta become strain-dependent at the end of the linear viscoelastic
region. From the amplitude sweep, the end of the linear viscoelastic region was identified
so that quality data could be obtained from the linear region. Initially, when the waiting
time was 0.25 h, the linear viscoelastic region ended close to a 1% oscillation strain and
then started to increase to around 10% as the waiting time increased to 8 h. A comparable
behavior has been reported for psyllium husk gels [61].
 as the waiting time increased from 3 to 8 h. The obtained yield stress values of the
J. Compos. Sci. 2022, 6, 237 9 of 17
prepared mixture were comparable with the reported values of psyllium gels in the
literature [60].

J. Compos. Sci. 2022, 6, x FOR PEER REVIEW 10 of 18

Figure 9.
Figure 9. Yield
Yield stress
stress data
data obtained
obtained from
fromthe
thestress
stresssweep
sweepfrom
from0.1
0.1Pa
Pato
to10
1044 Pa
Pa at
at different
different levels
levels of
of
waiting time.
waiting time.

Figure 10 shows the storage modulus (Pa) vs. oscillation strain (%) graph obtained
from the amplitude sweep. Viscoelastic properties such as the storage modulus, loss
modulus, and loss tangent delta become strain-dependent at the end of the linear
viscoelastic region. From the amplitude sweep, the end of the linear viscoelastic region
was identified so that quality data could be obtained from the linear region. Initially, when
the waiting time was 0.25 h, the linear viscoelastic region ended close to a 1% oscillation
strain and then started to increase to around 10% as the waiting time increased to 8 h. A
comparable behavior has been reported for psyllium husk gels [61].

Figure10.
Figure 10.Storage
Storagemodulus
modulusvs.
vs.oscillation
oscillation strain
strain graphs
graphs obtained
obtained from
from the the amplitude
amplitude sweep
sweep at 25at◦ C.
25
°C.
Figures 11–13 show the storage modulus (G’), loss modulus (G”), and loss tangent
delta Figures
vs. angular11–13 show the
frequency storage
graphs, modulus At
respectively. (G’),
allloss
levelsmodulus (G’’),
of waiting and
time, asloss tangent
the angular
delta vs. angular
frequency increased, frequency
both the graphs, respectively.
storage modulus and At
lossallmodulus
levels ofincreased,
waiting time,
but the asloss
the
tangent
angular delta decreased.
frequency As the
increased, waiting
both time increased,
the storage modulus and both themodulus
loss storage modulus
increased,and but
loss modulus
the loss tangent decreased, but the As
delta decreased. lossthe
tangent
waitingdelta
timeincreased.
increased,Printing
both thematerials should
storage modulus
be
andnotloss
onlymodulus
viscoelastic, but alsobut
decreased, elasticity-dominant
the loss tangent(loss deltatangent deltaPrinting
increased. < 1) to ensure the
materials
continuous
should be not extrusion of strands [62].
only viscoelastic, butThe
alsoprepared mixture should
elasticity-dominant have
(loss rapidly
tangent recovered
delta < 1) to
to a solid-like
ensure response with
the continuous a sufficiently
extrusion high storage
of strands modulus
[62]. The and yield
prepared stressshould
mixture to maintain
have
its strand shape after 3D printing [54,55]. All the loss tangent delta
rapidly recovered to a solid-like response with a sufficiently high storage modulus values were less thanand1
(Figure 13), which
yield stress is required
to maintain in order
its strand shapetoafter
ensure3Dthe continuous
printing extrusion
[54,55]. of strands
All the loss tangentindelta
3D
printing [62]. less
values were If thethan
prepared mixture
1 (Figure 13),had shown
which predominantly
is required in orderviscous behavior
to ensure (G” > G’),
the continuous
extrusion of strands in 3D printing [62]. If the prepared mixture had shown
predominantly viscous behavior (G” > G’), it would have suggested that the mixture was
not printable. The mixture at different levels of waiting time should have been printable,
according to Figures 11 and 12. However, their ability to retain their filamentous shape
 J. Compos. Sci. 2022, 6, 237 10 of 17

it would have suggested that the mixture was not printable. The mixture at different levels
of waiting time should have been printable, according to Figures 11 and 12. However, their
ability to retain their filamentous shape deteriorated with an increase in waiting time as
J. Compos. Sci. 2022, 6, x FOR PEER REVIEW 11 of 18
the storage modulus (Figure 11) significantly decreased (p < 0.05) [55]. When the waiting
time reached 3 h, the changes in storage modulus and loss modulus became insignificant
(p > 0.05); overlaps of a few graphs were observed, as shown in Figures 11 and 12.

Figure11.
Figure 11.Storage
Storagemodulus
modulusvs.vs. angular
angular frequency
frequency graphs
graphs at 9at 9 different
different levels
levels of waiting
of waiting time.time.

Figure12.
Figure 12.Loss
Lossmodulus
modulusvs.vs.angular
angular frequency
frequency graphs
graphs at 9atdifferent
9 different levels
levels of waiting
of waiting time.time.
J. Compos. Sci. 2022, 6, x FOR PEER REVIEW 12 of 18
J. Compos. Sci. 2022, 6, 237 11 of 17

Figure13.
Figure 13.Loss
Losstangent
tangentdelta
deltavs.
vs. angular
angular frequency
frequencygraphs
graphsat
at99different
differentlevels
levelsof
ofwaiting
waitingtime.
time.

3.3.
3.3. Minimum
Minimum Printing
Printing Pressure
PressureRequired
Requiredfor
forContinuous
ContinuousExtrusion
Extrusion
Table 3 presents the experimental data of the minimum
Table 3 presents the experimental data of the minimum printing pressure
printing required
pressure for
required
continuous extrusion at each level of waiting time. As the waiting time increased
for continuous extrusion at each level of waiting time. As the waiting time increased to 3to 3 h, the
minimum printingprinting
h, the minimum pressure requiredrequired
pressure for continuous extrusionextrusion
for continuous increasedincreased
rapidly torapidly
360 kPa.to
When the When
360 kPa. waiting thetime was 3.5
waiting timeh was
or longer,
3.5 h orthelonger,
minimumthe printing
minimumpressure
printingremained
pressure
approximately constant atconstant
remained approximately 360 kPa.atThis phenomenon
360 kPa. could be could
This phenomenon explained using texture
be explained using
analyzer data and rheological data. As the yield stress and hardness
texture analyzer data and rheological data. As the yield stress and hardness did not drastically
did not
change afterchange
drastically the 3 hafter
waiting
the time, a similar
3 h waiting minimum
time, required
a similar minimum extrusion pressure
required was
extrusion
expected for the prepared mixture after 3.5 h of waiting time.
pressure was expected for the prepared mixture after 3.5 h of waiting time.

Table
Table3. Experimentaldata
3. Experimental dataonon
thethe minimum
minimum required
required printing
printing pressure
pressure at different
at different levels
levels of of
waiting
waiting
time. time.

Pressure (kPa) Pressure (kPa)

Waiting Time (h) Waiting Time (h)
100 150 200 250 100 150
300 200 250320
300 320 360
360 400
400
0.25 0 1
0.25 1 1
0 11 1 1 1 1 1 11 11
0.5 0 0 0.5 1 1 0 01 1 1 1 1 1 11 11
0.75 0 0 0.75 0 1 0 01 0 1 1 1 1 1 11
1 0 0 1 0 0 0 01 0 0 1 1 1 11 11
1.5 0 0 0 0 1 1 1 1
1.5 0 0 0 0 1 1 1 1
2 0 0 0 0 1 1 1 1
2.5 0 0 2 0 0 0 01 0 0 1 1 1 11 11
3 0 0 2.5 0 0 0 00 0 0 1 1 1 1 11
3.5 0 0 3 0 0 0 00 0 0 0 0 1 11 11
4 0 0 3.5 0 0 0 00 0 0 0 0 0 11 11
4.5 0 0 0 0 0 0 1 1
5 0 0 4 0 0 0 00 0 0 0 0 0 11 11
5.5 0 0 4.5 0 0 0 00 0 0 0 0 0 11 11
6.5 0 0 5 0 0 0 00 0 0 0 0 0 1 11
7.5 0 0 5.5 0 0 0 00 0 0 0 0 0 11 11
9 0 0 0 0 0 0 1 1
6.5 0 0 0 0 0 0 1 1
0 = Discontinuous, 1 = Continuous
7.5 0 0 0 0 0 0 1 1
9 0 0 0 0 0 0 1 1
0 = Discontinuous 1 = Continuous
 J. Compos. Sci. 2022, 6, x FOR PEER REVIEW 13 of 18

J. Compos. Sci. 2022, 6, 237 12 of 17

3.4.Print
3.4. PrintQuality
Quality
3.4.1.
3.4.1.Layer-Height
Layer-HeightShrinkage
Shrinkage
To
Tostudy
studythetheeffects
effectsofofthe
thewaiting
waitingtime
timeon onthe
theprint
printquality,
quality,six
sixlevels
levelsof
ofwaiting
waitingtimetime
were
wereselected:
selected:0.5;0.5;1.5;
1.5;2.5;
2.5;4.5;
4.5;6.5;
6.5;and
and8.5
8.5h.h.AAminimum
minimumprinting
printingpressure
pressurewas wasused
usedat at
each level of waiting time. Figure 14 shows the change in layer-height
each level of waiting time. Figure 14 shows the change in layer-height shrinkage at each shrinkage at each
level
levelof
ofwaiting
waitingtime.
time.TheTheerror
errorbars
barsrepresent
representthe the95%
95%confidence
confidenceinterval.
interval.ItItcan
canbebeseen
seen
from
fromthethefigure
figure that thethe
that layer-height
layer-heightshrinkage
shrinkageincreased as the
increased aswaiting time increased
the waiting from
time increased
0.5 to 4.5
from 0.5hto(p4.5
< 0.05);
h (p <no0.05);
significant changes were
no significant changesobserved after 4.5 hafter
were observed of waiting
4.5 h oftime. The
waiting
storage
time. Themodulus
storagedata (Figuredata
modulus 11) (Figure
showed11) thatshowed
the storage modulus
that the storageexhibited
modulus a decreasing
exhibited a
trend as the waiting
decreasing trend astime theincreased.
waiting timeChanges in the storage
increased. Changes modulus
in the at different
storage levels of
modulus at
waiting time might have been a cause for the changes in layer-height shrinkage
different levels of waiting time might have been a cause for the changes in layer-height when the
waiting
shrinkagetime increased.
when the waiting time increased.

Figure14.
Figure 14.Effects
Effectsof
ofwaiting
waitingtime
timeon
onlayer-height
layer-heightshrinkage.
shrinkage.

3.4.2.
3.4.2.Filament-Width
Filament-WidthUniformity
Uniformity
Figure
Figure 15 shows theeffects
15 shows the effectsof
ofthe
thewaiting
waitingtime
timeon
onthe
thefilament-width
filament-widthuniformity
uniformity(the
(the
95%
95% confidence interval of the measured data on the filament width). Figure16
confidence interval of the measured data on the filament width). Figure 16shows
shows
typical
typicalexamples
examplesof ofthe
theprinted
printedsamples
samplesatatdifferent
differentlevels
levelsofofwaiting
waitingtime.
time.
As the waiting time increased from 0.5 to 2.5 h, the filament-width uniformity was
relatively good (the 95% confidence interval of the measured data on the filament width
was relatively small) and did not considerably change. As the waiting time increased to
4.5 h and longer, increased inconsistencies were observed (the 95% confidence interval of
the measured data on the filament width became significantly larger). Figure 16f shows
that the extruded filament became discontinuous.
The prepared mixture was used for 3D printing and secondary colonization at different
levels of waiting time. During Stage 4 (secondary colonization), the printed samples were
kept in filter patch bags and a box with an open lid for seven days. After three days, the
samples were flipped so that an even fungal growth could be obtained throughout the
whole printed material. The samples did not show any visible effect of different waiting
times on the fungal growth (Figure 17). Future in-depth studies are required to confirm
this initial observation.
J. Compos. Sci. 2022, 6, x FOR PEER REVIEW 14 of 18
J. Compos. Sci. 2022, 6, x FOR PEER REVIEW 14 of 18
J. Compos. Sci. 2022, 6, 237 13 of 17

Figure15.
15. Effectsof
ofwaiting
waitingtime
timeon
onfilament
filamentwidth
widthuniformity.
uniformity.
Figure 15.Effects
Figure Effects of waiting time on filament width uniformity.

Figure 16. Samples printed at different levels of waiting time: (a) 0.5 h; (b) 1.5 h; (c) 2.5 h; (d) 4.5 h;
(e) 6.5 h; (f) 8.5 h.
Figure16.
Figure 16.Samples
Samplesprinted
printed
at at different
different levels
levels of waiting
of waiting time:
time: (a) h;
(a) 0.5 0.5(b)
h; 1.5
(b)h;1.5
(c)h;2.5
(c)h;2.5
(d)h;4.5
(d)
h;4.5 h;
(e) 6.5
Ash; (f)
the 8.5
(e) 6.5 h; (f) 8.5 h.h.
waiting time increased from 0.5 to 2.5 h, the filament-width uniformity was
relatively good (the 95% confidence interval of the measured data on the filament width
As the waiting
was relatively small) time increased
and did from 0.5 to
not considerably 2.5 h, As
change. thethe
filament-width uniformity
waiting time increased to was
relatively
4.5 good (the
h and longer, 95% confidence
increased interval
inconsistencies wereof the measured
observed (the 95%data on the filament
confidence width
interval of
was relatively small) and did not considerably change. As the waiting time increased to
4.5 h and longer, increased inconsistencies were observed (the 95% confidence interval of
 The prepared mixture was used for 3D printing and secondary colonization at
different levels of waiting time. During Stage 4 (secondary colonization), the printed
samples were kept in filter patch bags and a box with an open lid for seven days. After
three days, the samples were flipped so that an even fungal growth could be obtained
throughout the whole printed material. The samples did not show any visible effect of
J. Compos. Sci. 2022, 6, 237 14 of 17
different waiting times on the fungal growth (Figure 17). Future in-depth studies are
required to confirm this initial observation.

Figure17.
Figure 17.The
The3D3D printing
printing (Stage
(Stage 3) and
3) and secondary
secondary colonization
colonization (Stage (Stage
4) using4)the
using the prepared
prepared mixture
mixture from
from Stage 3. Stage 3.

4.
4. Conclusions
Conclusions
In
In this paper,
paper, wewereport
reporta astudy
studyonon
thethe extrusion-based
extrusion-based 3D printing
3D printing of biomass–
of biomass–fungi
fungi composites. The input variable investigated was the waiting
composites. The input variable investigated was the waiting time between the mixture time between the
mixture preparation and the 3D printing. The output variables
preparation and the 3D printing. The output variables were the hardness and were the hardness and
compressibility
compressibility ofof the
the prepared
prepared mixture
mixture as
as well
well as
asthe
therheological
rheologicalproperties,
properties, minimum
minimum
printing
printingpressure
pressurerequired
required forfor
continuous extrusion,
continuous and and
extrusion, printing quality
printing (in terms
quality of layer-
(in terms of
height shrinkage
layer-height and filament-width
shrinkage uniformity).
and filament-width The main
uniformity). Theconclusions are stated
main conclusions arebelow.
stated
The hardness and compressibility of the prepared mixture significantly increased as
below.
the waiting time increased
The hardness to 3 h, but did
and compressibility not
of the significantly
prepared change
mixture as the waiting
significantly increasedtimeas
increased
the waiting time increased to 3 h, but did not significantly change as the waiting 0.25
further. The shear viscosity decreased as the waiting time increased from time
to 8 h. Thefurther.
increased yield stress significantly
The shear viscositychanged
decreased as as
thethe
waiting
waitingtime
timeincreased
increasedtofrom
3 h and
0.25
did
to 8 h. The yield stress significantly changed as the waiting time increased to 3 h waiting
not considerably vary when the waiting time increased beyond 3 h. As the and did
time increased, both
not considerably varythe storage
when modulustime
the waiting andincreased
the loss modulus
beyond 3decreased, but the time
h. As the waiting loss
tangent delta increased. The minimum printing pressure required for continuous
increased, both the storage modulus and the loss modulus decreased, but the loss tangent extrusion
increased as the waiting time increased to 3 h and remained at 360 kPa as the waiting
delta increased. The minimum printing pressure required for continuous extrusion
time increased further. When the waiting time increased from 2.5 to 4.5 h, the layer-height
increased as the waiting time increased to 3 h and remained at 360 kPa as the waiting time
shrinkage increased, and the filament-width uniformity deteriorated. Changes in the yield
increased further. When the waiting time increased from 2.5 to 4.5 h, the layer-height
stress, storage modulus, loss modulus, hardness, and compressibility significantly affected
shrinkage increased, and the filament-width uniformity deteriorated. Changes in the yield
the quality of the printed samples. The results showed that after preparing the mixture at
stress, storage modulus, loss modulus, hardness, and compressibility significantly
Stage 3, the waiting time should not be longer than 4.5 h to obtain a good print quality. In
affected the quality of the printed samples. The results showed that after preparing the
the future, research could be conducted to understand how different additives, biomass
mixture at Stage 3, the waiting time should not be longer than 4.5 h to obtain a good print
substrates, and fungi species could change the effects of the waiting time. The rheological
quality. In the future, research could be conducted to understand how different additives,
and mechanical properties of the prepared biomass–fungi mixture presented in this paper
biomass substrates, and fungi species could change the effects of the waiting time. The
can be used as a reference for developing new printable biomass–fungi mixtures for 3D
printing in the future.

Author Contributions: Methodology, A.M.R.; formal analysis, A.M.R. and A.B.; investigation,
A.M.R., A.B. and E.C.-P.; writing—original draft preparation, A.M.R.; writing—review and editing,
A.M.R., Z.P., C.U. and E.C.-P.; supervision, Z.P. All authors have read and agreed to the published
version of the manuscript.
Funding: This research received no external funding.
Data Availability Statement: The authors confirm that the data to support the findings of this study
are available within the article or upon request to the corresponding author.
J. Compos. Sci. 2022, 6, 237 15 of 17

Conflicts of Interest: The authors declare that they have no known competing financial interests or
personal relationships that could have appeared to influence the work reported in this paper.

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