of 6
Microstructure provides insights into evolutionary
design and resilience of
Coscinodiscus
sp. frustule
Zachary H. Aitken
a,1,2
, Shi Luo
b
, Stephanie N. Reynolds
c,3
, Christian Thaulow
d
, and Julia R. Greer
b
a
Department of Mechanical and Civil Engineering, California Institute of Technology, Pasadena, CA 91125;
b
Division of Engineering and Applied Science,
California Institute of Technology, Pasadena, CA 91125;
c
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA
91125; and
d
Department of Engineering Design and Materials, Norwegian University of Science and Technology, NO-7491 Trondheim, Norway
Edited by David A. Weitz, Harvard University, Cambridge, MA, and approved January 5, 2016 (received for review October 6, 2015)
We conducted in situ three-point be
nding experiments on beams with
roughly square cross-sections, which we fabricated from the frustule
of
Coscinodiscus
sp. We observe failure by brittle fracture at an aver-
age stress of 1.1 GPa. Analysis of crack propagation and shell mor-
phology reveals a differentiation in the function of the frustule layers
with the basal layer pores, which deflect crack propagation. We cal-
culated the relative density of the frustule to be
30% and show that
at this density the frustule has the highest strength-to-density ratio of
1,702 kN
·
m/kg, a significant departure from all reported biologic ma-
terials. We also performed nanoi
ndentation on both the single basal
layer of the frustule as well as the girdle band and show that these
components display similar mechanical properties that also agree well
with bending tests. Transmission electron microscopy analysis reveals
that the frustule is made almost entirely of amorphous silica with a
nanocrystalline proximal layer. No f
laws are observed within the frus-
tule material down to 2 nm. Finite element simulations of the three-
point bending experiments show t
hat the basal layer carries most of
the applied load whereas stresses within the cribrum and areolae
layer are an order of magnitude lo
wer. These results demonstrate
the natural development of architecture in live organisms to simulta-
neously achieve light weight, strength, and exceptional structural in-
tegrity and may provide insight into evolutionary design.
biomaterials
|
diatoms
|
nanoarchitecture
|
lightweight nanostructure
|
organic
inorganic composite
D
iatoms are single-cell algae that form a hard cell wall made of a
silica/organic composite. The ability to produce a functional
biosilica shell presents several natural precedents that fascinate and
inspire scientists and engineers. One fascinating aspect of such silica
glass shells is their intricate, varied, and detailed architecture. Dia-
toms are generally classified based on the symmetry of their shells:
Centric diatoms display radial symmetry whereas pennate diatoms
have bilateral symmetry. Fig. 1
A
shows a schematic of a typical centric
diatom and reveals that the shells are composed of two halves, called
frustules, that fit together in a Petri-dish configuration. The frustules
are attached to each other around the perimeter of the shell by one or
several girdle bands. The frustules are usually porous with pore size
and density varying between species. The frustule shell can also be
composed of multiple layers with a cellular structure within the shell.
The proposed evolutionary functions for these intricate shell designs
include nutrient acquisition, control of diatom sinking rate, control of
turbulent flow around the cell, and protection from grazing and viral
attack (1). Evidence in favor of a protective function is that the degree
of shell silification depends on the environment, with greater amounts
of silica found in shells grown in a predatory environment (2). As a
deterrent to predation, the frustule makes use of an inherently brittle
glass as a structurally protective material while balancing other evo-
lutionary pressures. A denser shell may provide greater protection but
will cause the diatom to sink beyond depths suitable for photosynthesis.
A solid shell might also prevent exchange of resources and waste be-
tween the diatom cell and its environment. This requires adaption
through control of the frustule micromorphology or modification of the
constituent silica/organic composite material (3). The protective as-
pects of the frustule shell are clear; what remains an open question is
how much the intricate pore structure and cellular design contribute to
the amplified structural resilience vs. biological function.
The size of most diatom species ranges from 2 to 200
μ
m (4, 5),
which renders most of the traditional mechanical testing methods
inadequate to characterize such complex materials; a few mechanical
studies on diatoms have been reported (6
11). The majority of
studies perform atomic force microscopy (AFM) indentation
(6
9) on a full frustule of centric or pennate diatoms. Reported
values of hardness ranged from 0.06
12 GPa and values of elastic
modulus from 0.35
22.4 GPa. Differences in local pore structure and
the nonplanar geometry of the frustule were often cited for the
variance in mechanical properties. Three-point bending tests on
beams that were extracted from the diatom frustule reported failure
strengths of 336
±
73 MPa but were complicated by local penetration
of the indenter tip and tilting of the frustule during testing (10, 11).
This overview demonstrates a wide range in the reported hard-
nesses and elastic moduli for biosilica shells. Most of these experi-
ments were performed on full diatom shells, which in some instances
contained organic cellular material; it is unclear whether the mea-
sured mechanical data represent the deformation of the constituent
biosilica or the overall deformation of the shell through bending,
local twisting, pivoting, and so on. Indentation using AFM can in-
troduce inaccuracies such as tip sliding, and the resulting uncertainty
in compliance within a single set of experiments, as well as among
the data obtained with different instruments, makes it challenging to
compare mechanical properties of the diatoms across the reported
experiments. Within a single species, these mechanical data may
provide qualitative trends in the structural response of the diatom
shells; it is difficult to make any conclusions on the mechanical
properties of the constituent biosilica. The mechanisms of silica
Significance
Diatoms are unicellular algae that form an intricate silica cell
wall. A protective shell that is light enough to prevent sinking
while simultaneously offering strength against predators is of
interest to the design of lightweight structural materials. Using
three-point bending experiments, we show that the diatom shell
has the highest specific strength of all previously reported bi-
ological materials. Fracture analysis and finite element simula-
tions also suggest functional differentiation between the shell
layers and features to mitigate fracture. These results demon-
strate the natural development of architecture in live organisms
to simultaneously achieve light weight, strength, and structural
integrity and may provide insight into evolutionary design.
Author contributions: Z.H.A., S.L., C.T., and J.R.G. designed research; Z.H.A., S.L., and S.N.R.
performed research; C.T. provided diatom samples; Z.H.A., S.L., and S.N.R. analyzed data;
and Z.H.A., S.L., S.N.R., and J.R.G. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1
To whom correspondence should be addressed. Email: zach-aitken@ihpc.a-star.edu.sg.
2
Present address: Institute of High Performance Computing, A*STAR, Singapore 138632.
3
Present address: School of Chemical and Biomolecular Engineering, Georgia Institute of
Technology, Atlanta, GA 30332.
This article contains supporting information online at
www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1519790113/-/DCSupplemental
.
www.pnas.org/cgi/doi/10.1073/pnas.1519790113
PNAS
|
February 23, 2016
|
vol. 113
|
no. 8
|
2017
2022
ENGINEERING
biogenesis likely varies among the species (12), but it is unclear to
what extent these differences reflect the variation in elastic modulus
and hardness between species and within an individual frustule.
To investigate the mechanical properties of the diatom frustule
and constituent biosilica as decoupled from the full-shell structural
response, we conducted in situ three-point bending experiments on
beams with roughly 3.5-
μ
m-square cross-sections fabricated from the
frustule of
Coscinodiscus
sp. performed in a scanning electron micro-
scope (SEM) equipped with a nanoindenter, as well as ex situ nano-
indentation on an individual basal plate that had been isolated from a
frustule and the girdle band. We dete
rmined the elastic modulus to be
36.4
±
8.3GPaandthefailurestrengthtobe1.1
±
0.3 GPa. We discuss
these results, as well as deformation and failure mode of the diatoms,
in the context of their atomic-level microstructure obtained from
transmission electron microscopy
(TEM) and finite element method
(FEM) simulations of the three-point bending tests.
Frustule Morphology and Microstructure
The cross-section of the frustule of a
Coscinodiscus
sp. is shown in Fig.
1
B
. The cribrum and cribellum constitute the distal surface of the shell,
and the basal plate composes the proximal surface. Areolae walls span
these two layers and approximate a honeycomb lattice configuration.
Fig. 1
C
shows the hexagonal arrangement of pores of the cribrum
and cribellum. The cribrum is composed of clusters of hexagonally
arranged, elliptical pores with average dimensions of 364 nm for
the major axis and 283 nm for the minor axis, and the clusters
are arranged in a hexagonal pattern. The cribellum layer is com-
posed of 50-nm-diameter pores that are hexagonally arranged across the
entirety of the surface and is laid atop the cribrum. Fig. 1
D
shows the
basal plate and areolae cells. Each areolae cell has five or six side walls
and contains a single foramen with an average inner diameter of 822 nm.
Fig. 2
A
shows a TEM micrograph of the frustule of
Coscinodiscus
sp.
Fig. 2
B
D
provide site-specific energy di
spersive spectroscopy (EDS)
data with diffraction patterns in the insets and convey that the material in
the areolae wall is made nearly exclusively of silicon. Smaller copper
peaks correspond to signal contamination from the copper TEM grid.
The inset diffraction pattern indica
tes that the region is entirely amor-
phous. Fig. 2
B
shows nearby regions that correspond to the interior
surfaces of the areolae cell; the am
orphous/nanocrystalline Pt peaks
come from the Pt needle that was used during ion-beam assisted de-
position and not from the biological sample. Fig. 2
C
provides EDS
data for a 275-nm-thick region of the basal plate and shows strong Si
and Ga peaks, with the latter caused by the Ga
+
-ion milling during
thinning. It seems that Ga was localized within this basal plate region
even though the entire sample was exposed to ion milling. We be-
lieve that the porosity observed within the nanocrystalline region
(Fig. 2
C
,
Inset
) facilitated Ga sequestration into it; Ga content is
lower in the amorphous regions (Fig. 2
B
,
Inset
). The difference in
the microstructure between the nanocrystalline and amorphous silica
is reflected in the difference in the diffraction contrast between the
two regions (Fig. 2
A
,
Inset
) and shows that a sharp interface exists
between the two microstructures.
Fig. S1
shows this contrast dif-
ference within the basal plate was also observed in SEM imaging.
The vast majority of the frustule, including the areolae walls, shows
a smooth, amorphous microstructure (labeled region A in Fig. 2
A
).
The only place where microstructure deviated from amorphous was
within the basal plate, which shows several bands between 50
80 nm in
thickness, oriented parallel to the frustule surface, and separated by
striated patterns of darker contrast (region B in Fig. 2
A
). There is a
10-nm-thick band that displays rough contrast that is adjacent to the
smooth material in region A. The nanocrystalline diffraction pattern
in Fig. 2
C
confirms the presence of these regions of local order. Re-
gion B has a total thickness of 275 nm, about 47% of the thickness of
the basal plate, and displays a darker contrast than the material in
region A, which suggests that it is either more densely packed or
contains a higher silica/organics ratio than material in region A.
Previous work has uncovered that the microstructure of the frustule
silica can vary between species and across stages of frustule development.
Reported microstructures include compacted spheres, networks of fused
spheres, silica microfibrils, and sm
ooth homogeneous silica (13, 14).
Schmid et al. (3) and Rogerson et al. (15) investigated wall morpho-
genesis in centric diatoms
Thalassiosira eccentrica
and
Coscinodiscus
asteromphalus
and identified
growing zones
that are loose aggregates
of 12-nm-diameter silica spheres, whe
re new frustule growth occurred,
and
compacting zones
that display a homogenous morphology where
more mature growth had occurred. In the centric diatoms studied by
these authors, the frustule growth occurred in a distal direction such that
the growing zones were oriented outward relative to the compacting
zones. This orientation suggests that the differences in microstructure
observed here likely do not correspond to growing and compacting
zones. Observations of wall morphogenesis by Schmid and Volcani (16)
and Hildebrand et al. (17) reported the presence of clustered spheres
in
Coscinodiscus wailesii
and
Thalassiosira pseudonana
. These authors
reportedthatfrustulegrowthbeganinthecenterandextendedcom-
pacted silica sphere strings radially
to the margin. Later growth stages of
the basal plate occurred by cross extensions and compaction, forming an
85- to 100-nm-thick template for subseq
uent distal growth. The proximal
nanocrystalline microstructures obse
rved in this work may correspond to
this initial basal template. It has been suggested that following radial
growth, sintering plays an important part in the morphogenesis of the
frustule by filling in the spaces of th
e compacted spheres with monomeric
silica, flattening the deposition surface, increasing its radius of curvature,
and promoting adherence (14, 18). This type of sintering would result in
the amorphous material observed in region A shown in Fig. 2
A
and
B
.
Fig. 1.
(
A
) Schematic of the diatom frustule shell.
(
B
) Cross-section of shell demonstrating the honey-
comb sandwich plate configure of the silica shell.
(
C
) Cribrum, the outer layer of the frustule shell,
displays hexagonal arrangements of pores. (
D
)The
basal plate, the inner layer of the frustule shell, is
punctuated by reinforced pores called foramen.
2018
|
www.pnas.org/cgi/doi/10.1073/pnas.1519790113
Aitken et al.
Mechanical Response in Bending and Nanoindentation
Fig. 3
B
shows the stress strain data for representative three-point
bending tests. The data indicate linear elastic loading with no plastic
deformation up to failure. Failure stress varied between 850
1,460 MPa
and failure strain varied between 2.2
4.0%. The average elastic
modulus was calculated to be 36.4
±
8.3 GPa based on a linear fit to
the slope. The experiments were performed in displacement-con-
trolled mode, and if a settling event resulted in a displacement rate
greater than the prescribed one the instrument controller adjusted
the indenter head to maintain the prescribed rate. This feedback
response occasionally manifested itself as a drop in stress when the
feedback loop adjustment caused a short local unloading. Failure
was catastrophic and occurred faster than the imaging scan rate of
the SEM. Upon failure, most samples released the accumulated
strain energy by launching from the substrate and could not be re-
covered. Fig. 4 shows one half of such a bending sample that was
recovered and shows that failure occurred by propagation of a crack
through the center of the beam.
Fig. 3
E
gives the calculated modulus against the contact depth ac-
quired during nanoindentation of the basal plate and the girdle band.
In both materials, the modulus decreased with contact depth. The
modulus calculated from indentation on the basal plate varied from
Fig. 2.
(
A
) TEM micrographs of the frustule shell.
(
Inset
) A zoomed in view of the basal plate. EDS data
and spot patterns taken from (
B
) region A, (
C
) region
B, and (
D
) region C show that the majority of the
frustule shell is amorphous silica.
Fig. 3.
(
A
) Snapshot from a video of three-point bending of the frustule shell. Circles show the locations that were used to fit a circle for strain calculation.
(
B
) Stress-strain data collected from bending experiments that has been corrected for rigid-body displacements. Nanoindentation was performed on (
C
)an
isolated basal plate and (
D
) girdle band. (
E
) The reduced modulus as measured from nanoindentation on both an isolated basal plate and girdle band plot
against indentation depth. For comparison the elastic modulus as determined from bending experiments is also shown.
Aitken et al.
PNAS
|
February 23, 2016
|
vol. 113
|
no. 8
|
2019
ENGINEERING
21.0
±
7.7 GPa at 28 nm to 39.7
±
8.3 GPa at 217 nm and in the girdle
band from 19.9
±
2.1 GPa at 53 nm to 40.2
±
4.0 GPa at 267 nm.
Differentiation of Frustule Layers in Mechanical Response
Fig. 4 shows the fractured surface of a representative bending test sample.
Fig. 4
A
and
B
show the path of crack propagation across the basal plate
and the cribrum. Bending induces a compressive stress along the inner
edge of the beam, abutting the indenter head, and a tensile stress along
the outer edge, which is likely where the crack initiated. It seems that a
central pore present in the basal plate (indicated by the blue arrow) and
the cribrum (indicated by the red arrow) served as a stress concentrator
and the crack nucleation site because it is close to the location of maxi-
mum bending moment. Within the cribrum, the crack traveled upward
toward a cluster of pores beneath the applied load, and its trajectory
continued through a series of these pores upward, tracing a path between
stress concentrations (Fig. 4
A
). The postdeformation crack surface shown
in Fig. 4
A
,
Inset
illustrates that the pores acted as perforations for the
crack propagation. In the basal plate (Fig. 4
B
) the crack propagated up-
ward, bending slightly to follow the path of the crack within the cribrum.
These paths diverge when the crack propagating through the basal plate
encounters a foramen near the top edge of the beam. Instead of traveling
through the pore, the crack is deflected around the pore. The foramina
differ from the cribrum pores in that they have a raised rim around the
circumference of the pore. Our experiments revealed that the pores in the
cribrum act as stress concentrators and fail by crack propagation whereas
the rim reinforcements in the foramina seem to shield them against
failure. This may shed light on the diffe
rentiation in function between the
basal plate and the cribrum: The res
ilient pores in the basal plate may
have a primarily structural function, whereas the pores of the cribrum and
cribellum may serve more in the ca
pacity of resource acquisition.
Elastic Properties of Diatom Shell Components
Fig. 3
B
shows that the stress strain data from bending tests has a
signature of linear elastic loading ending in brittle failure. No plasticity
or controlled crack nucleation or growth was observed before failure.
Any nonlinearity in the data can be correlated to a rigid body move-
ment of the beam captured in the video. We expect some variation in
the measured load due to a small misorientation (
2°) of the indenter
head relative to the sample surface normal between tests as well as
nonideal contact between the beam and the testing platform and
indenter head. The measured elastic modulus from bending tests was
consistent among all four reported samples, at 36.4
±
8.3 GPa.
Nanoindentation response of the isolated basal plate and the girdle
band shows remarkable similarity. Average elastic moduli varied with
increasing contact depth from 21.0
±
7.7 at 28 nm to 39.7
±
8.3 GPa
at 217 nm in the basal plate, and from 19.9
±
2.1 at 53 nm to 40.2
±
4.0 GPa at 267 nm in the girdle band. The submicron thickness of the
samples and the nanomechanical experiments in general render it
challenging to eliminate all source
s of experimental error. For shallow
contact depths, some measurement error likely stems from the nonideal
geometry of the tip, and at greater contact depths we encounter effects
from the stiff substrate. Despite the uncertainty, these extreme cases set a
range that matches well with the modulus calculated from bending tests.
The elastic moduli obta
ined in this work are in contrast to previously
reported elastic moduli for
Coscinodiscus
sp. between 0.06
0.53 GPa (9)
obtained from AFM indentation as well as results reporting differences
in mechanical properties between d
ifferent components of the diatom
shell. Beyond inherent differences between three-point bending, nano-
indentation, and AFM indentation, the boundary conditions of the
previous AFM indentation tests are the most significant difference from
the results reported here. Indentation into the full diatom frustule shell
or even into half of the frustule
in either a concave or convex config-
uration
can result in a deformation response of the entire structure,
which would not be indicative of its material mechanical properties. This
can include pivoting of the shell or lo
calized shell buckling if the shell is
immobilized. Such movement and loca
lized deformation has been veri-
fied in situ during previous mechanical testing (10, 11). The low values
and variance reported in previous studies are most likely due to move-
ment or deflection of the frustule during testing and differences in
compliance between loading the diatom axially and radially. By using the
in situ bending tests on an extracted lamella representative of the frustule
and nanoindentation on an isolated basal plate and girdle band we are
able to significantly reduce these possible adverse displacements and to
characterize the material more precisely.
The similarity in the elastic propert
ies between the basal plate and the
girdle band observed in this study s
uggests that they are mechanically
equivalent composite materials. Swi
ft and Wheeler (19) estimated that
20
40% of the dry weight inside the diatom silica valves is protein and
carbohydrate. Kröger et al. (12) repo
rted that the silica precipitated from
in vitro studies of silica-depositing
long-chain polyami
nes extracted from
a diatom frustule had 1.25
μ
gofSiO
2
per 1
μ
g of polyamine. Using the
density of 2 g/cm
3
for silica and 0.8 g/cm
3
for the organic material in the
diatoms studied here (14), we estimat
e the volumetric fraction of silica to
be between 37.45 and 66.67%.
The rule of mixtures can provide an upper bound for the elastic
modulus of the composite material. The expression for the com-
posite modulus is given by
E
c
=
fE
s
+
ð
1
f
Þ
E
o
,
[1]
where
f
is the volume fraction of silica and
E
s
and
E
o
are the
elastic moduli of the silica and organic component, respectively. It
is reasonable to assume that the elastic modulus of the organic
component is significantly lower than that of the silica; applying
Eq.
1
to the estimated range of volume fractions predicts the
composite modulus to be between 26.2
46.7 GPa, consistent with
the values reported here and similar to synthetic bio-silica com-
posites such as Bioglass45S5 (45% SiO
2
by weight) at 35 GPa (20).
Strength vs. Relative Density
The average failure stress was 1.1
±
0.3 GPa at a strain of 3.5
±
0.7%,
with variations up to 250 MPa, which seem to be partly related to the
distribution of pores in the beam. The highest stresses occurred in
samples that lacked foramen segments along the bottom edge of the
testing beam. Samples that failed at lower stresses had foramen seg-
ments, which suggests that their role in strengthening the diatom
against stress concentration does not prevent them from serving as the
weak points for failure along the free edge when the beam is subjected
to bending. The average relative density of the beam samples used in
this study, as calculated from direct volume measurements taken in
Fig. 4.
(
A
) The path of crack propagation through the cribrum. The crack
follows the stress concentrations surrounding the cribrum pores. (
B
) The
path of crack propagation through the basal plate. The crack is deflected by
the reinforced foramen. (
Insets
) The fractured beam. The red arrow corre-
sponds to the location where the crack intersects the bottom edge of the
cribrum layer and the blue arrow corresponds to the location where the
crack intersects the bottom of the basal layer.
2020
|
www.pnas.org/cgi/doi/10.1073/pnas.1519790113
Aitken et al.
the SEM, is 30.1%. A schematic of the frustule and list of measured
geometries and relative densities is provided in
Fig. S2
and
Tables S1
and
S2
. Using the density of bulk silica at 2,210 kg/m
3
, which provides
the upper bound for the constituent material density, gives the frustule
material a specific strength of 1702 kN
·
m/kg, a value well above those
of other natural cellular, composite and silk materials including
bamboo (693 kN
·
m/kg), mollusk shell (127 kN
·
m/kg) (21, 22), and
spider silk (1,000 kN
·
m/kg) (23).
Fig. 5 shows an Ashby plot of strength vs. density for several natural
biomaterials (22). We find that the diatom frustule occupies a pre-
viously untapped space above the upper limit of natural cellular ma-
terials and has strengths comparable to the strongest natural polymers
but at a lower density (22). We attribute this high specific strength to
the honeycomb architecture combined with a low density of flaws in
the constituent material. The TEM image in Fig. 2
A
shows no visible
defects or flaws down to the estimated contrast roughness of
2nmin
the nanocrystalline layer within the basal plate.
This strength is still well below theoretical, in contrast to the strengths
of synthesized high-purity silica na
nowires that attain near-theoretical
tensile strengths between 10
25 GPa (24). This is likely because the
frustule is a silica composite rather than pure silica. Within the last
decade, much research has been dedicated to elucidate the composite
nature of the frustule, with some studies revealing the presence and
function of species-specific biopolyme
rs within the frustule (25). In vitro
studies have shown that these biopolymers aid in polymerization and
flocculation of silica particles (
26). Among the studied biopolymers,
Coscinodiscus
seems to exclusively contain long-chain polyamines
(LCPAs) with reported molecular masses ranging between 600
1,500 Da
(12, 25). The exact distribution of silica and organic material within the
frustule has not been experimentally
verified, but it seems to be tightly
bound to the silica within the frustule because it survived the cleaning
process to remove the organic cellular material. Further evidence for
these tight, likely chemical bonds is that active LCPAs were recovered
from fossilized diatomaceous material (27, 28). A proposed mechanism
for the promotion of silica precipitation by LCPAs is the presence of
alternating protonated and nonprotonated tertiary amine groups in the
polyamine chains, which form strong
hydrogen bonds to silicic acid and
facilitate the Si
O bond formation (29). Such a mechanism suggests that
interactions in the composite material are through hydrogen bonding
between silica and organic phases and through covalent bonding within
the silica. Failure in the biosilic
a composite most likely occurs at the
weaker hydrogen bonds between the silica and organic material.
Stress Distribution Within Frustule Layers
The presence of an architecture within the diatom design results in a
heterogeneous stress distribution within the frustule. Fig. 6 shows
the Mises stress distribution at the maximum bending strain of the
simulated FEM beam (Fig. 6
B
and
E
) along the bottom edge of the
cribrum and basal plates (Fig. 6
C
and
F
). The Mises stress of a solid
beam at similar strain is shown as a black line for comparison. Within
the cribrum, some thin sections surrounding the pores show local
stress concentrations, manifested as two symmetric peaks
±
0.12
away from the center and one peak
2.2 away from the center. In the
rest of the sample, the stresses remain below those in the solid beam.
This is likely because the multiple pores present along the bottom
edge are unable to sustain high stresses along this edge. Within the
basal plate, the stress distribution closely follows that in the solid
beam and increases toward the center of the beam. Near the center,
the presence of a reinforced foramen results in a local reduction of
stress. Local fluctuations in the stress distribution within both layers
Fig. 5.
Ashby plot of strength vs. density for naturally occurring biological
materials (22). Diatom frustule samples show specific strengths above other
reported cellular materials and are comparable to the strongest natural
polymers. The theoretical maximum is determined by extrapolation of the
strength and density of diamond.
Fig. 6.
(
A
and
D
) SEM imaged of the cribrum layer and the basal plate of a bending sample. (
B
and
E
) The corresponding von Mises stress distribution from
FEM simulations. (
C
and
F
) Variation in stress along the bottom edge, with the stress of a solid beam at equivalent strain shown in black for comparison. It
appears that the variation in stress in the basal plate follows that of the solid beam more closely than in the cribrum.
Aitken et al.
PNAS
|
February 23, 2016
|
vol. 113
|
no. 8
|
2021
ENGINEERING
likely stem from the presence of pores or locations where the areolae
walls intersect the cribrum or the basal plate.
Fig. S3
shows the Mises stress distribution within the areolae layer.
This distribution shows that the stresses attained in the areola walls are
less than those in the cribrum and in the basal plate. Within the center
of the areolae walls, stresses are up to an order of magnitude lower than
those in the cribrum and in the basal plate, and near the intersection
with either outer layer the stresses in the areolae are approximately half
of the maximum stresses observed in the cribrum and in the basal plate.
At the locations where the areolae walls intersect the outer layers, the
major contribution to Mises stress comes from the shear stresses, which
suggests that under bending the areolae
is not contributing significantly
to the mechanical response, and max
imum Mises stresses occur around
the pores in the cribrum and the foramen within the basal plate.
Conclusion
We used in situ three-point bending
and nanoindentation experiments
to investigate the mechanical properties and fracture behavior of the
diatom
Coscinodiscus
sp. frustule. These experiments disclosed similar-
ities in the elastic properties of the
biosilica found in the frustule and in
the girdle band, with average modulus
from three-point bending tests of
36.4
±
8.3 GPa. We discovered that the frustule has an unprecedentedly
high specific strength, exceeding that of all other reported natural bio-
materials, which we attribute to the combination of the honeycomb
sandwich plate architecture and extremely low flaw density in the con-
stituent biosilica. TEM analysis of th
e frustule revealed that it is almost
entirely an amorphous, solid material
, with some local regions displaying
a nanocrystalline microstructure. Ana
lysis of crack propagation at failure
provides strong evidence for the biof
unctional differentiation between
the frustule layers, with the foramen deflecting crack propagation and
the cribrum layer seen to fail along its pores. FEM simulations convey
that most of the applied stress is supported by the basal plate and that
the areolae walls do not contribute si
gnificantly to bearing load. These
results provide useful insights to
ward understanding the extreme resil-
ience of hard biological materials to failure and aid in efficient design of
new classes of bioinspired, low-density, and high-strength materials.
Methods
Diatoms.
Coscinodiscus
sp. used in this study were obtained from the Biological
Institute at Norwegian University o
f Science and Technology and had been
previously collected from the Trondheimsfjord inlet of the Norwegian Sea. These
samples were washed in Milli-Q (MQ) water, centrifuged in a solution of 3 mL
H
2
O
2
and 1 mL HCl, and finally rinsed with MQ water and ethanol. This treat-
ment was intended to remove the cellula
r organic material and to separate the
frustule half-shells and girdle band fro
m one another; this type of treatment also
unintentionally fractured many of th
e components. Samples were stored in
methanol before nanomechanical expe
riments. SEM samples were prepared by
applying a drop of this methanol solution containing the cleaned diatoms onto a
silicon chip that was coated with 100 nm of gold and then allowing the meth-
anol to evaporate in air. Despite the absence of a conductive coating, we found
that the diatoms could be successfully imaged in SEM with no excessive charge
accumulation. Fig. 1
B
D
show SEM images of an example frustule sample fol-
lowing cleaning and mounting.
Fig. S4
provides a graphical description of
the fabrication procedure for samples f
or three-point bending experiments,
nanoindentation, and TEM analysis.
Three-Point Bending Experiments.
Bending tests were performed in a custom-
made in situ SEM (FEI Quanta) with a n
anomechanical module, InSEM (Nano-
mechanics, Inc.). A wedge-shape
d diamond tip with a radius of 5
μ
m was used to
indent the beam at a constant nominal displacement rate of 5 nm/s to failure.
Load and displacement were continuous
ly measured at a rate of 500 Hz with a
simultaneous video capture of the deformation process. Stresses and strains
were calculated along the bottom edge of the beam directly under the point of
applied load, which corresponds to the point of maximum tensile stress. Ex-
panded discussion of the stress calculation is included in
Supporting Information
.
TEM Analysis.
Microstructural analysis was performed via transmission elec-
tron microscopy (FEI Tecnai F30) at an accelerating voltage of 300 kV.
Standard-less energy dispersive spectroscopy (INCA EDX; Oxford Instruments)
was used to investigate the local elemental composition of the sample.
Nanoindentation.
Nanoindentation was performed ex situ in a Hysitron
nanoindenter using a diamond Berkovich tip under a constant nominal
displacement rate. The thickness of the basal plate and girdle used here was
observed to vary between 600
700 nm, so indentation was performed up to
a total displacement between 50
300 nm. Elastic moduli were calculated
using the method of Oliver and Pharr (30).
ACKNOWLEDGMENTS.
We thank the Kavli Nanoscience Institute at Caltech
for the availability of cleanroom facilities, Carol Garland for assistance with
TEM analysis, and David Z. Chen for assistance with in situ mechanical
testing. We also thank Hilde Skogen Chatou at the Norwegian University of
Science and Technology (NTNU) for aid in diatom preparation and the NTNU
Nanolab for access to the focused ion beam. This work was supported by the
Institute for Collaborative Biotechnologies through Grant W911NF-09-0001
from the US Army Research Office.
1. Finkel ZV, Kotrc B (2010) Silica use through time: Macroevolutionary change in the
morphology of the diatom fustule.
Geomicrobiol J
27(6-7):596
608.
2. Pondaven P, et al. (2007) Grazing-induced changes in cell wall silicification in a marine
diatom.
Protist
158(1):21
28.
3. Schmid A-MM, Schulz D (1979) Wall morphogenesis in diatoms: Deposition of silica by
cytoplasmic vesicles.
Protoplasma
100(3-4):267
288.
4. Stoermer EF, Julius ML (2003) Centric diatoms.
Freshwater Algae of North America:
Ecology and Classification
, eds Wehr JD, Sheath RG (Elsevier, San Diego), pp 559
594.
5. Werner D (1977)
The Biology of Diatoms
(Blackwell, Oxford).
6. Almqvist N, et al. (2001) Micromechanical and structural properties of a pennate di-
atom investigated by atomic force microscopy.
J Microsc
202(3):518
532.
7. Subhash G, Yao S, Bellinger B, Gretz MR (2005) Investigation of mechanical properties
of diatom frustules using nanoindentation.
J Nanosci Nanotechnol
5(1):50
56.
8. Hamm CE, et al. (2003) Architecture and material properties of diatom shells provide
effective mechanical protection.
Nature
421(6925):841
843.
9. Losic D, Short K, Mitchell JG, Lal R, Voelcker NH (2007) AFM nanoindentations of
diatom biosilica surfaces.
Langmuir
23(9):5014
5021.
10. Bjørnøy SH (2012) Nanomechanical testing of diatoms. Master thesis (Norwegian Univ
of Science and Technology, Trondheim, Norway).
11. Vebner MJ (2013) Nanomechanical testing of diatoms. Master thesis (Norwegian Univ of
Science and Technology, Trondheim, Norway). Available at hdl.handle.net/11250/241821.
12. Kröger N, Deutzmann R, Bergsdorf C, Sumper M (2000) Species-specific polyamines
from diatoms control silica morphology.
Proc Natl Acad Sci USA
97(26):14133
14138.
13. Li C-W, Volcani BE (1984) Aspects of silicification in wall morphogenesis of diatoms.
Philos Trans R Soc B Biol Sci
304(1121):519
528.
14. Gordon R, Drum RW (1994) The chemical basis of diatom morphogenesis.
Int Rev
Cytol
150:243
372.
15. Rogerson A, Defreitas ASW, Mcinnes AG (1986) Observations on wall morphogenesis in
Coscinodiscus asteromphalus (Bacillariophyceae).
Trans Am Microsc Soc
105(1):59
67.
16. Schmid A-MM, Volcani BE (1983) Wall morphogenesis in Coscinodiscus Wailesii Gran and
Angst. I. Valve morphology and development of its architecture.
JPhycol
19(4):387
402.
17. Hildebrand M, et al. (2011) Nanoscale control of silica morphology and three-
dimensional structure during diatom cell wall formation.
J Mater Res
21(10):2689
2698.
18. Iler RK (1979)
The Chemistry of Silica: Solubility, Polymerization, Colloid and Surface
Properties and Biochemistry
(Wiley, New York).
19. Swift DM, Wheeler AP (1992) Evidence of an organic matrix from diatom biosilica.
J Phycol
28(2):202
209.
20. Hench LL (2005) Bioceramics.
J Am Ceram Soc
81(7):1705
1728.
21. Meyers MA, Chen P-Y, Lin AY-M, Seki Y (2008) Biological materials: Structure and
mechanical properties.
Prog Mater Sci
53(1):1
206.
22. Wegst UGK, Ashby MF (2004) The mechanical efficiency of natural materials.
Philos
Mag
84(21):2167
2186.
23. Vollrath F, Madsen B, Shao Z (2001) The effect of spinning conditions on the me-
chanics of a spider
s dragline silk.
Proc Biol Sci
268(1483):2339
2346.
24. Brambilla G, Payne DN (2009) The ultimate strength of glass silica nanowires.
Nano
Lett
9(2):831
835.
25. Sumper M, Kröger N (2004) Silica formation in diatoms: The function of long-chain
polyamines and silaffins.
J Mater Chem
14(14):2059.
26. Poulsen N, Sumper M, Kröger N (2003) Biosilica formation in diatoms: Characteriza-
tion of native silaffin-2 and its role in silica morphogenesis.
Proc Natl Acad Sci USA
100(21):12075
12080.
27. Bridoux MC, Annenkov VV, Keil RG, Ingalls AE (2012) Widespread distribution and
molecular diversity of diatom frustule bound aliphatic long chain polyamines (LCPAs)
in marine sediments.
Org Geochem
48:9
20.
28. Bridoux MC, Ingalls AE (2010) Structural id
entification of long-chain polyamines
associated with diatom biosilica in a Southern Ocean sediment core.
Geochim
Cosmochim Acta
74(14):4044
4057.
29. Pohnert G (2002) Biomineralization in diatoms mediated through peptide- and
polyamine-assisted condensation of silica.
Angew Chem Int Ed Engl
41(17):3167
3169.
30. Oliver WC, Pharr GM (2011) Measurement of hardness and elastic modulus by in-
strumented indentation: Advances in understanding and refinements to methodol-
ogy.
J Mater Res
19(1):3
20.
31. Craig RR (2000)
Mechanics of Materials
(Wiley, New York), pp 338
447.
32. Zaykova-Feldman L, Moore T (2005) The total release method for FIB in-situ TEM
sample preparation.
Microscopy and Microanalysis
11(S02):848
849.
2022
|
www.pnas.org/cgi/doi/10.1073/pnas.1519790113
Aitken et al.