3. Results and discussion
3.1 Short-term BSA adhesion test
The effect of different coatings and materials on short-term BSA
adhesion was evaluated as described in Section 2.3. The maximum adhesion
(0.03 μg mm-2) was measured in HX
(p <0.05, one-way ANOVA). The BSA adhesion values
relative to HX are shown in Figure 1. The minimum adhesion was
determined for H-X3, being one order of magnitude lower than that in the
HX sample (p <0.05, one-way ANOVA) (Fig. 1A). There were
no statistically significant differences between PMMA, PETG and GS
(p <0.05, one-way ANOVA).
Protein adsorption on surfaces is the primary event in biofouling.
Consequently, low protein adsorption should be recognized as the most
important prerequisite for a surface’s resistance to biofouling. As
PBR-grown microalgae are known to secrete proteins to the supernatant
(Xiao and Zheng, 2016), protein adhesion is an expected process. The
importance of this phenomenon in microalgae cultures was revealed in a
recent study on N. gaditana adhesion dynamics (Zeriouh et al.,
2017a). The surface of N. gaditana was clearly hydrophilic
throughout the culture. However, the substrate surface (glass), which
was initially hydrophilic, turned almost amphiphilic after two days of
culture and strongly hydrophobic a week later. This behavior could only
be compatible with the additional adhesion of other materials with
hydrophobic properties such as proteins, most likely excreted byN. gaditana .
Consequently, PBR surfaces should be designed or selected to resist
protein adsorption. However, the control of this multifactorial process
is so complex that there is no known surface with complete protein
resistance. The hydrophobicity and
hydrophilicity of a surface, generally referred to as wettability, are
important factors affecting protein adsorption (Firkowska‐Boden et al.,
2018). The static water contact
angle θw is commonly used as a descriptor of
wettability. Figure 1B shows the relative BSA adhesion versusθw for each surface used in this study. A cutoff
of θw ≈ 65° was inserted to distinguish between
hydrophilic (θw <65°) and hydrophobic
(θw >65°) surfaces, as previously
reported (Vogler, 2012). The data did not present a clear pattern. The
highest BSA adhesions were observed in the most hydrophobic surfaces (HX
and NW). This is consistent with the literature since it is expected
that proteins undergo partial unfolding and spreading on strongly
hydrophobic surfaces (Firkowska‐Boden et al., 2018). In contrast, the
glass surface (GS), which is strongly hydrophilic, did not show a marked
efficacy in reducing BSA attachment compared to the other surfaces. It
is supposed that hydrophilic surfaces promote easier protein desorption
because the proteins are shielded from the surface by a high-density
water layer (Firkowska‐Boden et al., 2018). Therefore, GS and PMMA
failed as hydrophilic surfaces to inhibit or mitigate the protein
adhesion. Surprisingly, BSA adhesion on PS, a moderately hydrophobic
solid material, was lower than the hydrophilic surfaces (GS and PMMA).
This odd result has so far remained a controversial issue in the field
of protein adsorption. Recent studies have revealed that the extent of
protein adsorption can be governed by the synergistic effect of the
surface hydrophobicity and the relative charge state of the protein and
surface (Attwood et al., 2019); so, just because a surface is
hydrophilic does not mean it is protein resistant. In fact, globular
protein adsorption onto hydrophobic solid surfaces occurs regardless of
the protein charge state. Conversely, the adsorption charge of
hydrophilic surfaces might be very high under charge-favorable
conditions, or very low under unfavorable charge conditions (Attwood et
al., 2019). Notwithstanding, in our study, the adhesion propensity of
BSA molecules to both hydrophobic and hydrophilic surfaces was
significant, the interaction being much stronger on the hydrophobic
surfaces. Indeed, this property is exploited in immunology where BSA is
used as a blocking agent on both hydrophobic and hydrophilic surfaces
(Jeyachandran et al., 2009). Additionally, phosphate groups can be bound
with the BSA molecules to form
BSA-phosphate surface complexes,
the preferred conformation of adsorbed BSA on surfaces (Jeyachandran et
al., 2009). Phosphate is the P-source used in the N. gaditanaculture medium.
Interestingly, H-X3 (which is hydrophobic) had the lowest BSA adherence
propensity. H-X3 is a fouling release coating based on silicone
hydrogel. According to recent studies, comparing the protein adsorption
results of hydrogels and other non-hydrogel materials, based on a scale
of hydrophobicity and hydrophilicity, could be elusive because hydrogels
absorb water and create a deformable surface (i.e. water-swollen
coatings) (Vogler, 2012).
Another way to quantitatively represent the surface-thermodynamic
hydrophobicity/hydrophilicity scale of condensed-phase materials is the
interfacial free energy of
interaction between two surfaces, i, immersed in water,
ΔGiwi (commonly referred to as the free energy of
cohesion) (Van Oss, 2008). Figure 1C shows the relative BSA adhesion
values as a function of the corresponding ΔGiwi values
for each assayed surface. Although the distribution of the experimental
points was different to that of θw (Fig. 1B), the
conclusions are similar. Only GS presented a clearly positive
ΔGiwi value (ΔGiwi > 0),
indicating that it is strongly hydrophilic. PMMA presented an
ΔGiwi value close to 0, but as its \(\gamma_{s}^{-}\) value (27.7 mJ m-2) was not significantly
greater than the 30 mJ m-2 cut-off, it is most likely
hydrophobic, as suggested elsewhere (Van Oss, 2008).
From Figures 1B and 1C, one can conclude that criteria based on
hydrophobicity and hydrophilicity are not enough to completely explain
the protein adhesion on the surfaces. Protein adsorption behavior is a
poorly understood and very complex process that can be influenced by
diverse factors, acting either synergistically or antagonistically, such
as protein molecular properties, surface chemistry and charge,
wettability, and topography (Firkowska‐Boden et al., 2018). For example,
polymers exhibiting similar surface hydrophobicity and roughness,
differing only in the chemical end groups, can have divergent protein
adsorption profiles (Vijaya Bhaskar et al., 2015). What is even more
disconcerting is observing how the same polymer provided by different
commercial suppliers can present very different protein adhesion values
(Vijaya Bhaskar et al., 2015), likely attributed to minor differences in
one or more of the above-mentioned factors, or to a variety of chemical
residues generated during manufacturing, which cause the protein to
interact differently with the groups on the surface (Contreras-Naranjo
and Aguilar, 2019). Interestingly, it is well-known that protein
adsorption increases along with the water’s ionic strength. Studies have
reported that BSA adsorption on glass was markedly higher in seawater
than in low ionic-strength buffer (Kirchman et al., 1989). The most
common explanation is based on the double layer theory: the surface
charge of BSA increases in seawater (i.e. a higher ionic strength),
causing a reduced repulsive double-layer interaction and a more globular
BSA configuration (Kirchman et al., 1989). The results from Figure 1B
are consistent with others obtained at ionic strengths as high as
seawater (Ruckenstein and Berim, 2019): the greatest BSA adsorption took
place on the hydrophobic surfaces, was substantial on glass and
significantly lower on the hydrogels. Recent observations support the
hypothesis that BSA in seawater forms a multilayer structure involving
Mg2+ cations as bridges between the BSA molecules
(Pradier et al., 2002). Furthermore, it has been demonstrated that the
salts dissolved in seawater produce micrometer-size surface spots
containing mainly metal species, these spots being preferential
adsorption sites for BSA (Poleunis et al., 2002).
Figure 2 summarizes in an idealized way how the interaction of both the
protein and the surface properties may tune the adsorption process, as
conceptualized elsewhere (Contreras-Naranjo and Aguilar, 2019). One can
observe how the proteins’ versatile nature is related to their primary
structure, ultimately represented by the sequence of amino acids and
functional groups available for bonding. This hinders protein adsorption
prevention and leads to experimental data spreading, generated from
different laboratories studying the same protein-surface system but from
different manufacturers.
3.2 Long-term biofouling tests
3.2.1 Culture experiments in the vessels
The biofouling tests consisted of exposing the coupons to the N.
gaditana cultures for a long period (50 days), as explained in Section
2.4. Characterizing these cultures is essential for analyzing the
adhesion results based on the interaction between the coupon surface and
the broth that surrounds it. The representative culture kinetics for all
the vessels are displayed in Figure 3. The experiments started as batch
cultures (Stage S1) to acclimate the cells to the new set conditions in
the vessels. After 18 days, the fed-batch mode was established. Two
nutrient pulses were performed - on days 18 (Stage S2) and 33 (Stage
S3). Given that the differences between the experiments that had an
initial N/P from 45 to 90 were not significant compared to those with an
N/P of 5 and 15, Figure 3 shows the mean values together of the three
N/P 45 to 90. The highest growth rates and biomass concentrations were
obtained at an N/P≥15 in all of the S1 to S3 stages (Fig. 3A).
As can be seen in Figure 3B, the dissolved phosphate was rapidly taken
up soon after being added to the cultures with N/P>15
compared to N/P=5. At the end of each stage, the phosphate was almost
completely consumed at N/P values above 5 (Fig. 3B) whereas the
dissolved nitrate continued to be present in excess in all experiments
(Fig. 3C). The culture dynamics presented above describe a growth
pattern that is clearly controlled by the phosphate availability in the
culture medium for the experiments with an N/P>15.
Conversely, growth in the culture medium with an N/P of 5 seemed to be
inhibited by phosphate. The Fv/Fmdid not change significantly throughout the culture period, the average
value of all vessels being 0.549± 0.035, which is indicative of healthy
cells; the one exception was the value of 0.4 with N/P 5 during S1 and
S2.
The coupons were detached from the vessels at the end of stage S3 and
the cell adhesion intensity was analyzed as a proxy for microalgal
biofouling, as indicated in Section 2.4.2. The factors that could
potentially affect the fluid dynamics of biofouling formation on the
coupon surface are: the coupon position in the culture vessel (bottom or
wall), the coupon surface properties represented by the coupon type, and
the culture properties to which the coupons are exposed, represented by
the N/P ratio. Therefore, to analyze the effect of these factors, and
their interactions on the variability of the cell adhesion intensity
(AI), a multifactor ANOVA was carried out. All factors and interactions
had a statistically significant effect on AI at the 95.0% confidence
level (p < 0.05). However, the most significant contributions
to AI variability corresponded to the N/P (27.31%), the coupon type
(23.78%) and the N/P interaction with the coupon position (12.71%).
The N/P factor is a vehicle to combine into one variable the different
alterations affecting the environment that directly interacts with the
coupons (cells and supernatant) due to the effect of N on cell
metabolism. Nutrient availability, biomass concentration and EOS
concentration in the culture were taken into account as cellular
responses. Figure 4 shows the effect of the above-mentioned factors on
AI, EOS, and cell biomass concentrations at the end of the culture
experiments.
The cell adhesion intensity and the secreted protein concentration
varied inversely to N/P (see Fig. 4A), with no significant effect from
N/P ratios above 15 (p <0.05), while an opposite effect
on the final biomass concentration was observed. This implies that cell
adhesion correlated positively with the concentration of proteins, but
negatively with the concentration of biomass in the culture. The latter
seems to contradict previous observations, where it was suggested that
an increased cell flow density reaching a coupon surface, governed by
cell concentration gradients, increased the cell adhesion rate (Zeriouh
et al., 2019b). These seemingly contradictory results are easily
reconciled if the protein adhesion that occurred during the formation of
the conditioning film preceded cell adhesion.
Considering the results observed in the short-term BSA adhesion tests,
the above assumption is highly plausible. A recent study supports this
point, in which proteins secreted by the cyanobacterium Annabaenasp were first adsorbed onto suitable PBR materials during the formation
of the conditioning film (Talluri et al., 2020). Figure 4A demonstrates
that protein adsorption on the coupon surface increased as protein
concentration increased in the supernatant, as reported for BSA on
surfaces immersed in seawater (Kirchman et al., 1989).
The stickiness of proteins on the different surfaces tested was
previously evidenced in Section 3.1, even on hydrophilic surfaces.
Therefore, it is expected that N. gaditana will adhere to the
coupons in proportion to the amount of proteins adhered to them, even on
hydrophilic surface (Zeriouh et al., 2017a). This means that N.
gaditana attachment on the coupon surface is mediated by prior protein
adsorption, which occurs at a rate faster than N. gaditanaadhesion; that is to say, cells grown in media with N/P ratios of 5 and
15 interacted firstly with the coupon surfaces coated with a
conditioning film composed of sticky proteins. In this scenario, the
importance of the flow densities of nutrients reaching the cells that
were attached to the coupon surface was greater in cultures with the
lowest N/P ratios, where nitrates and phosphates were in excess (see
Fig. 2B), while in cultures with high N/P ratios (above 15), phosphate
was present in the supernatant at very low concentrations. As a result,
in cultures with an N/P above 15, the microalgae adhering to the coupon
surfaces (or embedded in the formed biofilm) continuously experienced
significantly reduced phosphate flux densities, which limited their
growth. The role played by the excess phosphate present in cultures with
an N/P below 15 may also have been relevant during protein adsorption if
the proteins secreted by N. gaditana had the capacity to bind
with the phosphate groups to form protein-phosphate, as mentioned above
for BSA. These explanations are consistent with Figure 4B, where the
effect of the coupon surface properties (i.e. the type of coupon) on the
cell adhesion intensity is observed. The resulting pattern resembled
that of BSA adsorption on the same type of coupon represented in Figure
1A.
The effect of the coupon position on cell adhesion intensity is shown in
Figure 4C. One can observe that the slight variability in the cell
adhesion intensity associated with the coupons’ position (bottom or
wall) mainly occurred in the experiments with an NP = 5, where the
highest EOS excretion took place.
It has been suggested that wall shear influences cell attachment to the
bottom well plates when agitated on a shaking table (Saleck et al.,
2011).The strain rate values were always higher on the coupons attached
to the flask wall than those on the bottom (Table 2).
All the wall values were almost twice those on the bottom. The
differences were higher for the maximum (∼2.7 times) and minimum (∼6.2
times) values. It has been posited that the initial adhesion might be
reversible if the kinetic energy of the microalgae cells in the boundary
layer of the solid were superior to the total interaction energy between
the solid surface and the microalgae cell. The strain rate (γ) 5 µm from
the coupons is indicative of the drag force exerted by the liquid flow
on the boundary layer of the solid surface (Zeriouh et al., 2017b).
However, it has been suggested that the shear stress factor alone is not
sufficient to explain what was observed occurring in the different
attachment experiments. Nonetheless, when linked to flow field topology,
it helps in understanding the underlying mechanisms of the different
biological processes (Salek et al., 2011). Hence, it is necessary to
consider both the strain rate values and the different flow structures
developed on the bottom and walls of the vessel (Chakraborty et al.,
2011). Usually, in orbitally agitated devices, the liquid height is low
and a thin liquid film periodically sweeps the bottom, provoking the
formation of a radial shear gradient (Salek et al., 2011). The trailing
wave formed at the liquid surface forms a recirculation cell that
increases the interaction of the cells with the vertical wall of the
vessel (Salek et al., 2011). The liquid wetting the coupons on the walls
and the bottom of the vessel does not have the flow influence associated
with the liquid-gas interphase (Fig. 5B); therefore. the liquid velocity
at the bottom is practically constant across the entire cross section
(Fig. 5C). The coupons on the vertical wall, however, are closer to the
liquid free surface and are subjected to a periodic velocity radial
gradient, which generates higher strain rates (Fig. 5D and Table 2),
very similar to that occurring in shallow agitated wells (Salek et al.,
2011, Berson et al., 2008). In addition, the trailing wave of the liquid
surface generates a recirculation cell that repeatedly throws the cells
against the wall (Salek et al., 2011, Kim and Kizito, 2009), to some
degree centrifuging the cells onto their surface (Kim and Kizito, 2009),
which would promote cell adhesion. The different liquid flows developed
in the different parts of the flasks apparently counteracted the
different strain rate values and, as a consequence, cell attachment was
similar on all the coupons.
When the protein concentration in the culture is very high (N/P = 5)
(Fig. 4A), the fluid flow also seems to carry more proteins towards the
wall coupons; therefore, interaction with the cells is higher, as is the
cell attachment, compared to the coupons on the bottom (Fig. 4C).
3.2.2 Nitrogen balance
The synthesis of intracellular components and the release of
extracellular organic substances is mediated by nitrogen availability to
the cells. As the nitrogen added to a culture is not only fixed in
proteins, the nitrogen balance needs to be acceptably closed. The
nitrogen taken up by the cells can be distributed amongst the main
extracellular and intracellular proteinaceous and non-proteinaceous
nitrogenous compounds, namely proteins, amino acids, chlorophylls,
nitrate, nitrite and ammonium.
The analyses carried out to determine the total nitrogen present in both
the biomass and the supernatant have revealed that, in general, this
element has been taken up in a similar way for all the N/P ratios:
10-13% has been used to form part of the biomass (both cells in
suspension and those attached to the vessel), while 87-90% was used in
the extracellular medium. Furthermore, one should point out that only
25-35% of the nitrogen was released into the medium as nitrate (not
consumed), nitrite and ammonium; that is to say, the rest of the
nitrogen (65-75%) went to forming part of the organic compounds
(proteins, ammonia acids, chlorophylls, DNA fragments and lipids, etc.).
As for organic nitrogen, this element has also been distributed
similarly for all the N/P ratios. The most appreciable difference is
that at the N/P 5 ratio, 50% of the intracellular nitrogen is present
in the adhered cells; this is because there are twice as many adhered
cells as there are in suspension. This can be explained by the fact that
in this culture, there was 10% more extracellular protein than in the
other cultures and, as previously mentioned, this substance has a great
influence on cell adhesion.
3.2.3 Analysis in the context of the Baier and Vogler biocompatibility
theories
A work recently carried out on N. gaditana has demonstrated that
the surface biocompatibility theories of Baier and Vogler may offer
interesting insights into developing antifouling surfaces for microalgae
photobioreactors (Zeriouh et al., 2019a). Figure 6 shows the relative
adhesion values as a function of the water adhesion tension (Vogler’s
theory) and the critical surface tension (Baier’s theory). The
microalgae adhesion was calculated based on the maximum obtained at the
minimum N/P ratio of 5 (4.06 ·105 cells
cm2). For clarity’s sake, only the results
corresponding to the N/P ratios that led to the highest (N/P=5) and the
lowest (N/P=90) cell adhesion and secreted protein concentration values
were represented. For the BSA protein model, the values represented in
Figure 1 were used. One can observe that the Baier and Vogler theories
are only reconciled at the Baier curve minimum attained by the H-X3
coating, based on FRCs‐Hydrogel. The exceptional antibiofouling
properties of H-X3 have recently been demonstrated in N. gaditanacultures (Zeriouh et al., 2019a). H-X3, although initially having low
energy surface properties (γc = 22 mJ
m-2 and τ0 = −21.2 mJ
m-2), can acquire amphiphilic properties after
exposure to seawater; these properties are close to Vogler’s minimum
(τ0 of around 35 mJ m-2), as
reported by Zeriouh et al. (2019a) (see Figure 6). The BSA and cell
adhesion intensity on the rest of the coupons in the vicinity of
Vogler’s minimum were clearly several orders of magnitude higher than
that of H-X3. As mentioned above, these analyses, which are based
exclusively on surface wettability, are not universal. Even though there
is a substantial body of literature dealing with the topic of protein
adsorption on surfaces, in a recent excellent review, Vogler confirmed
the huge lack of consensus and the impossibility of extracting general
conclusions beyond a few general rules of thumb based on the biophysical
chemistry of protein adsorption (Vogler, 2012).
Likewise, two further observations from Figure 6 are of particular
importance: (i) the pattern of relative BSA adsorption intensity clearly
resembles those of the different N/P ratios assayed; and (ii) the higher
the concentration of proteins in the supernatant (high for 5 and low for
90, see Fig. 4), the higher the cell adhesion.
These observations support the assumption that the initial event that
initiates the biological response of an N. gaditana culture to
artificial materials and coatings is the adsorption of proteins secreted
by the microalgae into the culture medium. This is in accordance with
results from studies on photobioreactor materials used in the
cultivation of the cyanobacterium Anabaena sp., where proteins
were present on the conditioning films formed on glass and PMMA (Talluri
et al., 2020).
3.2.4 Short-term BSA adhesion is indicative of the microalgal biofouling
propensity
Given that protein-resistant materials and coatings are also likely to
be resistant to microalgae adhesion, two experimental approaches were
addressed in this study: the short-term batch adhesion of the sticky BSA
model protein under dynamic conditions and the long-term microalgal
adhesion under flow conditions that are representative of practical
operation. Relative BSA adhesion tended to approximate the Baier curve
and successfully mimic the long-term biofouling test results (Fig. 6).
These results demonstrate that the short-term batch adhesion test of a
sticky model protein such as BSA is a proxy for predicting biofouling in
microalgae cultures. Therefore, long-term representative studies may not
be mandatory as a preliminary step in assessing potential biofouling
control in strategies applied to the massive screening of surfaces.
3.3 Future prospects
The work reported here indicates that, to effectively design or select
materials and coatings for use in PBR construction, protein adsorption
is a more critical event to consider than that of the cell/clean surface
interaction. When PBR surfaces are exposed to cultures containing EOS,
including proteins, they are likely to be rapidly coated by a thin layer
comprised mainly of proteins dissolved in the supernatant which adsorb
to the PBR surface as a monolayer or multilayer. The process of cell
adhesion to the surface is indirect because the cells do not bind
directly to the PBR surface but instead bind to the adsorbed proteins.
According to Vogler (Vogler, 2012), adsorbed protein on water-surface
interphases displaced an equivalent volume of interphase water
(interphase dehydration), causing adsorbed proteins to concentrate into
closely-packed arrangements. Microalgae make contact with, and anchor
to, the adsorbed proteins in certain patches and domains.
Although BSA represents an interesting laboratory model for globular
protein, the composition, structure, chemistry and functions of the
proteins contained in EOS is, as yet, little known (Xiao and Zheng,
2016). Furthermore, the type and profile of the proteins secreted by
cultured microalgae may be dependent upon the abiotic conditions and
species. Consequently, to gain further insight, studies on the
adsorption of other model globular proteins may be needed, as is
recommended for animal cells (Attwood et al., 2019). In addition, a
conditioning film may also comprise other macromolecules, such as humic
substances, polysaccharides, or free amino acids present in the water
(Flemming, 2002). All of these can be excreted by the microalgae at high
concentrations in PBR cultures. Generally, the adsorption process of
these macromolecules onto surfaces (within seconds or minutes) is faster
than the cell adhesion (Flemming, 2002). Hence, the degree of initial
macromolecule attachment might also be a good indicator of biofouling
and its study should therefore be addressed.
Microalgae respond to changing environmental conditions by modulating
their extracellular and intracellular metabolites. Within the same
microalgal species, the optimal culture conditions vary depending on the
target metabolite. Therefore, besides the N/P ratio in the culture
medium, the possibilities of combining a wide variety of abiotic factors
are vast. Each of these combinations most likely modifies the
interaction between the PBR surface and the culture due to changes in
the culture and cell properties. From a practical point of view, the
biofouling propensity of different PBR surfaces should be studied
specifically for each cultivation environment and species in order to
the design and/or select the most appropriate surfaces or coatings.
Likewise, transparent H-X3-like coatings should be developed and tested
for PBR use in microalgae cultures where biofouling is likely dominated
by EOS.