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Bacterial Adhesion Phenomenon in Wastewater Treatment Applications
Graeme Williams* and Terence Chan**
*Organic Optoelectronic Materials and Devices Laboratory
**Laboratory of Biopolymers and Nanomedicine
University of Waterloo. Waterloo, ON. N2L 3G1.
Correspondence should be addressed to:
g3willia@engmail.uwaterloo.ca or terencechan@live.ca
1
1. Introduction to Anaerobic Digestion and Upflow Anaerobic Sludge Bed Reactors
Some of the most pertinent and critical applications of adhesion phenomena relate to our needs to treat
and purify wastewater. The development, production and purification of chemicals on large scales have
allowed us numerous technical innovations; however, the waste products from these same processes
also serve to contaminate our water supplies. In some cases, such contamination can lead to
bioaccumulation of hazardous materials and subsequent developmental defects in small organisms,
which become more problematic as the materials are passed up the food chain [1-4]. Beyond these
industrial applications, wastewater treatment also has obvious and critical pertinence in municipalities
and agriculture. In these latter applications, it is feasible to develop a system where wastewater is not
only treated, but the treatment generates fuel as a by-product to power the treatment system itself and
for further uses [4, 5].
This paper focuses on wastewater treatment systems that make use of sludge bed reactors, which treat
wastewater by anaerobic digestion. In such systems, the adhesion of bacteria is the principal interfacial
phenomenon that results in the formation of sludge granules to form the sludge bed. Sludge granules
are “dense, multispecies microbial
communities,” where each individual
species of microbes is incapable of
digesting complex organic material [8];
however, the interplay of the many
species allows for digestion of most
waste products. Optical and scanning
electron microscopy images of example
granules are shown in Figure 2.
In anaerobic wastewater treatment systems, wastewater passes through the sludge granules, and the
bacteria present on the granules promote a four-step digestion process [9]:
I. Carbohydrates, fats and proteins undergo hydrolysis to produce sugars, fatty acids and
amino acids – this is typically the rate-limiting step in digestion of wastewater, especially at
lower operation temperatures [10]
II. The above by-products and remaining waste undergo acidogenesis to produce carbonic
acids and alcohols, as well as H2, CO2 and NH3 gases
III. The above by-products and remaining waste undergo acetogenesis to produce acetic acid,
H2 and CO2 gases
IV. The above by-products and remaining waste undergo methanogenesis to produce methane
and CO2 gas
The resultant effluent has a much lower biochemical and chemical oxygen demand (BOD and COD) than
the influent wastewater – although it must still undergo further processing, typically by reverse osmosis
1 SEM image is a representative micrograph and does not actually correspond to the granules shown in the left.
Figure 1 – Optical (left) and Scanning Electron Microscope (right)
Images of Sludge Granules. Adapted from refs [6] and [7] respectively
1.
2
treatment. Since the digestion process is aided by biomatter and the output product may be used as an
additional fuel source, such biogas plants have been identified as important energy supplies by many
researchers, including those comprising the United Nations Development Programme [5, 11, 12].
Perhaps the most prominent, and therefore most frequently studied, anaerobic sludge reactor is the
Upflow Anaerobic Sludge Bed (UASB) digestion reactor, illustrated in Figure 2. The granules that make
up the sludge bed remain suspended at the base of the reactor. This occurs due to gravity acting on the
granules to counter the upflow of the influent. The result is a reactor with high sludge retention while
still allowing intimate contact between the
influent and the sludge granules. This high
sludge retention grants relatively stable
performance [4]. Some of the most critical
limiting factors to the widespread
implementation of UASB reactors into
wastewater treatment facilities are:
the temperature requirements to drive
anaerobic digestion, and the feasible
organic loading (while still achieving
high treatment efficiency) of the
wastewater when the reactor is kept at
low temperatures [10]
the variation in output efficiency with
changes in influent composition
(including ion content and difficult-to-
decompose molecules such as lignin) and influent pH [4]
the significant start-up time (2-8 months) and seed sludge required to form a healthy, thick
sludge bed for efficient digestion [4, 8].
The latter point is the most critical economic factor, as significant upfront costs and delays tend to limit
investment. Fortunately, this factor can be studied at great depth using the wealth of knowledge and
the many techniques offered by interfacial and surface science.
2. Thermodynamics of Sludge Bed Formation
As is evident from the above discussion, the growth and retention of sludge granules, which are few-
millimetre aggregates of anaerobic microbes [12], is critical for efficient operation of UASB reactors. A
greater understanding of their formation is necessary for widespread adoption of this technology.
Unfortunately, there is still much debate regarding the true driving force behind the formation of sludge
granules. In this review, physicochemical models with a focus on interfacial and surface phenomena will
be examined, following the mathematical techniques used by Thaveesri et al. in reference [13].
It has been proposed that inert particles present within the reaction tank may act as seeds to enhance
particle growth [7, 8, 14-16]. Thermodynamic considerations, such as surface tension and free energy,
Figure 2 – Illustration of the Upflow Anaerobic Sludge Bed
Reactor. Adapted from ref [10].
3
have also been identified as likely driving factors for initial granule formation [8]. In later stages of
granule formation, more complex models are required, as it is insufficient to treat the microbes as
lifeless colloids – they move, grow and communicate with each other. These additional complications
and points of understanding will be discussed briefly in Section 4. The reader is further encouraged to
examine reference [8], where Liu and coworkers propose a summative model to fully explain granule
formation growth by encompassing all relevant literature.
If one considers a bacterium contacting a solid substrate, as is the case for bacteria adhering to inert
particles in the reaction tank, one may write the Gibb’s free energy (per area) for the system as:
(1)
, where BS is the surface energy between the bacterium and the substrate
BL is the surface energy between the bacterium and the surrounding liquid
,and SL is the surface energy between the substrate and the liquid.
This follows logically from the fact that the formation of a new substrate-bacterium interface (BS)
requires a loss in area of both the substrate-liquid interface (SL) and the bacterium-liquid interface (BL).
A highly negative Gadhesion implies that the bacteria adhere to the substrate favourably and
spontaneously. As such, by identifying the conditions that allow for a highly negative Gadhesion, one may
promote granule formation. Unfortunately, this is not an immediately useful relationship, as most of
the above variables cannot be easily determined through experimental work.
By Young’s equation:
(2)
, where SV is the solid-vapour surface tension (for example, for the solid substrate or bacteria)
, and LV is the liquid-vapour surface tension (for example, for wastewater or experimental solvent)
One may also apply the empirical relation developed by Neumann and coworkers [17]2, shown below:
(3)
As a critical point of note, this relation is invalid for regimes where 1 - 15(SVLV)1/2 approaches 0, as
equation (3) rapidly diverges. Neumann and coworkers identify this regime for instances where LV is
very high, which would be the case for substances such as pure water, dilute NaCl in water or pure
glycerol. Fortunately, most wastewater contains a number of surface-passivating species (including
synthetic or natural surfactants), which adequately lower the surface tension to allow for this relation to
2 Note: Neumann and coworkers make use of a CGS system of units while we presently make use of the MKS
system of units, accounting for the difference in the denominator.
4
hold. Furthermore, the surface tension of solutions containing bacterial species tends to be on the
order of 40-50 mN/m, which is sufficiently low to maintain validity of this relation [18].
One may also substitute equation (2) into equation (3) to find:
(4)
(5)
Equation (5) provides a route to determine the BL and SL parameters in equation (3) above. For
example, one may find the SV of a solid substrate material by measuring the contact angle it forms with
a liquid of known LV. In order to determine BL, which is the surface tension of bacterial species, one
may perform a similar experiment on a dry bacterial layer formed on a Micropore filter [18]. The
methodologies for making these contact angle measurements are explained further in Section 3.
If one now considers a scenario where the inert particle used for granule growth has been completely
covered with a monolayer of bacteria, which would occur soon after the initial growth stage, the above
analysis can be further simplified. The surface energy of the substrate is the same as the surface energy
of the bacteria, so BS=0 and BL=SL. As a consequence, the free energy of adhesion is:
(6)
Using this relationship, one may plot the free energy
of adhesion versus the liquid-vapour surface tension
for various discrete values of the bacterial surface
tension, as shown in Figure 3. The MATLAB code for
this exercise is available in Appendix A of this paper.
From the data in Figure 3, it is clear that regions
where LV is very low and regions where LV is very
high result in the greatest level of bacterial adhesion.
This is clearly indicated by a large, negative free
energy term, which implies favourable or
spontaneous adhesion. Furthermore, there exists a
region from LV=30 to 60 mN/m, where, regardless of
the properties of the bacteria, bacterial adhesion and
granule growth are severely restricted.
Unfortunately, as will be detailed below, this region encompasses much of the natural and feasible
growth conditions in most UASB reactors.
Figure 3 - Free Energy of Bacterial Self-Adhesion for
Various BV values
5
In order to further understand the data in Figure 3, one may associate high bacterial surface tension
with hydrophilic cell characteristics. Oppositely, one may relate low bacterial surface tension to
hydrophobic bacterial cells. Using this generalization, bacterial adhesion is clearly enhanced in very low
LV solutions if the bacterial cells are inherently hydrophilic. Furthermore, if the cells are very
hydrophobic, they preferentially form granules in very high LV solutions; however, granule formation in
low LV solutions is still feasible for hydrophobic cells. As a point of note, for BV equal to or greater than
60 mN/m, the calculated free energy shows an erroneous inflection at high LV values. This inflection
artificially lowers the free energy in this regime, and is due to the limitations of the relationship
proposed by Neumann and coworkers in equation (3), as discussed earlier [17].
Sludge granule microbes have varying
roles according to the anaerobic
digestion process: acidogens perform
both hydrolysis and acidogenesis,
acetogens perform acetogenesis and
methanogens perform
methanogenesis. Most acidogens are
hydrophilic, with high bacterial surface
tension values. Most acetogens and
methanogens, as well as most
hydrogenotrophs, are considered
hydrophobic, with low bacterial
surface tension values [13]. The
organization and arrangement of
these species within the granule will
depend on the properties of the
surrounding liquid, or in the case of a
UASB reactor, the properties of the
influent wastewater, as detailed
below (and illustrated in Figure 4):
Wastewater with high LV will favour initial granule growth of hydrophobic acetogens and
methanogens, with acidogens dispersed throughout the outer shell of the granule.
Wastewater with low LV will favour initial agglomeration of hydrophilic acidogens, but will still
allow for spontaneous adhesion of hydrophobic acetogens and methanogens. The hydrophilic
acidogens may act as a scaffold for further adhesion of the hydrophobic species, resulting in a
layered granule structure. The resulting granule has a hydrophobic core and a hydrophilic
acidogen shell.
In order to test the data obtained from this thermodynamic analysis, Thaveesri et al. ran two UASB
reactors in conjunction, with one reactor (reactor S2) maintained with low LV, and the other reactor
(reactor S1) with an untreated (higher) LV [13]. The researchers achieved a low LV in reactor S2 by the
Figure 4 - Illustration of Granule Structure for Different Wastewater
Surface Tension Values
6
regular addition of the surfactant Linear AlkylBenzeneSulfonate (LABS) to the influent wastewater. This
treatment served to decrease the liquid surface tension of the prepared feed influent from 68 mN/m to
49 mN/m. Over the period of the experiment, reactor S2 maintained a relatively low liquid surface
tension of 46 to 48mN/m. In contrast, reactor S1 gradually increased in liquid surface tension from 50
to greater than 55 mN/m.
The effect of the variation in the reactors’ liquid surface tension is twofold:
The granules formed in reactor S2, where the liquid surface tension is lower, were generally
larger than those formed in reactor S1, with mean radii of 2.1mm versus 1.7mm. This may be
due to the fact that the overall free energy of adhesion for the bacteria is larger at this given LV.
It may also be due to the fact that the initial acidogen agglomerations provide scaffolds for
efficient granule growth for all bacterial species, as noted above.
As the liquid surface tension in reactor S1 increased, biogas bubbles adhered more strongly to
the sludge particles in S1, resulting in an eventual loss of active biomass. The loss of biomatter
resulted in complete deterioration of the reactor efficiency. This phenomenon has been
attributed to the hydrophobic surface nature of the granules formed in a high LV environment,
which adhere more strongly to biogas bubbles.
Thaveesri et al. verified the hydrophilic surface nature of reactor S2 granules and the hydrophobic
surface nature of reactor S1 granules by metabolic activity tests. These results stress the importance of
the acidogen shell and the layered structure in granule formation. The hydrophilic surface
characteristics serve to make the granules more resilient to wash-out of active biomass, which is
critically important for the initial growth as well as the ultimate lifetime of the granules in the UASB
reactor. While it has not been shown experimentally, this stabilizing hydrophilic shell may also serve to
make the granules more resilient to changes in the composition of the influent wastewater.
3. Experimental Techniques: Wilhelmy Balance Tensiometry and Drop Volume Techniques
Granule structure and formation are key areas of research for improving UASB reactors because of the
critical role that the granules serve in anaerobic digestion. By determining the hydrophobicity of
individual bacterial species, researchers are able to predict their arrangement during initial aggregation
stages. This knowledge ultimately leads to an understanding of the finalized granule structure, which
can allow researchers to improve several performance factors, including granule formation time,
granule retention in the sludge bed and granule-biomass interactions. As discussed in Section 2, it is
clear that the calculation of the surface energies of granule bacteria is essential to the efficient
operation of UASB reactors. However, direct measurement of the surface energies of bacteria is
impractical. Instead, one may measure the contact angle of a specific bacterial film and then apply
equation (5) to determine the corresponding surface energy, as noted above in Section 2.
7
A commonly used technique for contact
angle measurements is the Wilhelmy
balance tensiometry method (illustrated
in Figure 5). The reader is encouraged to
examine reference [18], where a more
detailed and thorough explanation on
contact angle measurements for sludge
bed granules is provided. The
methodology is described briefly herein:
A bacterial species of unknown BV is dropcast onto a Micropore filter paper and dried, forming a
bacterial layer on the filter (henceforth denoted as the experimental substrate). Next, the substrate is
attached to a thin metal wire that is connected to a tensiometer/microbalance and dipped into a solvent
of known SV. In practical application, a beaker or container with the solvent of known SV is raised
upward to contact the substrate, instead of the substrate being lowered into the solvent – more
accurate force measurements can be made by the balance if it is held stationary. As the substrate
comes into contact with the solvent, the solvent will wet the substrate, resulting in some force that may
be measured by the tensiometer. Since SV, Ftensiometer and the perimeter of the substrate are all known
variables, the contact angle θ can be calculated using the Wilhelmy equation, equation (7), shown
below:
(7)
Another common method of bacterial contact angle measurement can be accomplished by goniometry.
However, it has been reported that goniometric methods may not produce as accurate measurements
as the Wilhelmy balance tensiometry method [20]. This follows as a consequence of the inherent
averaging effect in Wilhelmy balance tensiometry, as the contact angle measurement is effectively
averaged over the entire perimeter of the substrate. Furthermore, in the case of tensiometry, force
values are provided by accurate microbalances, avoiding the need for the subjective contact angle
measurements associated with goniometry.
As demonstrated by Thaveesri et al., the wastewater surface tension (denoted as LV in much of Section
2) also strongly affects granule formation and granule retention in UASB reactors. As such,
measurement of wastewater surface tension is also frequently necessary in UASB reactor studies. This
is commonly accomplished by the drop volume technique [21]. In this technique, the wastewater of
interest is collected in a syringe and pushed through a vertical capillary at a constant flow rate to
generate a drop at the bottom tip of the capillary. The time to grow the drop is controlled such that the
drop reaches its steady state shape and size, allowing researchers to approximate the instantaneous
surface tension values with the equilibrium state values. The drop is allowed to grow to its maximum
size and fall off the tip of the capillary, and is then detected by an optical sensor. The drop volume is
measured by recording the number of volume increments required to form the maximum drop size. This
value is incorporated into equation (8) – derived from the Young-Laplace equation – to determine the
liquid surface tension, as described by Gunde et al. [21]:
Figure 5 - Illustration of the Wilhelmy Balance Tensiometry Method.
Adapted from ref [19].
8
(8)
, where Vf is the volume at which the drop falls off
, g is the acceleration due to gravity (~9.81 m/s2)
, Δρ is the density difference between the liquid and vapour phases
, r is the radius of the capillary
, γ is the surface tension of the wastewater
, and f is the correction factor developed by Harkins and Brown to account for the remaining liquid at
the capillary tip after the droplet falls [22].
4. Additional Modes of Analysis and Limitations of the Thermodynamic Approach
While the experimental data reviewed in this paper is strong, and the thermodynamic approach is
scientifically sound, the surface tension model is overly simplistic for an inherently complex, biological
system. It is worthwhile to consider other factors in a UASB reactor, including: DLVO theory and its
application to granule formation, the role of extracellular polymers in granule formation, hydrophobic
force-induced organization and cell-to-cell communication effects.
From simple DLVO theory, when two
bacteria approach each other, they will
undergo attractive van der Waals forces as
well as electrostatic repulsion forces, with
the strength and intensity of the forces
dependent on their size, geometry and
surface potential. For two spherical
species in solution, the potential energy
versus separation distance may be similar
to the plot shown in Figure 6. Two
bacteria in close contact with each other
first undergo reversible adhesion, as
shown by the secondary minimum in
Figure 6. In order to irreversibly bind to
each other, the bacteria must overcome
the energy barrier, denoted as Wirreversible in
Figure 6. Several methods detailed below
have been proposed for bacteria to
overcome this energy barrier:
Bacterial cells inherently hold negative surface charges. The addition of positive divalent or
trivalent ions to solution would lower the energy barrier, as per DLVO theory. By introducing the
critical coagulation concentration of positive ions, the double layer repulsion between bacteria
could be strongly reduced, decreasing the Wirreversible energy barrier to zero. This would promote
bacterial aggregation, allowing bacteria to reach the irreversible adhesion minimum [23].
Figure 6 - Representative Interaction Energy of Two Spherical
Particles (ie. Bacteria), with Attractive van der Waals forces and Repulsive Electrostatic Forces Shown Separately.
(Note: Negative = Attraction; Positive = Repulsion)
9
Some bacteria possess extracellular appendages, such as fimbriae and protruding fibrils. By
acting as physical bridges, these appendages overcome the energy barrier and reduce the gap
between bacteria to promote cell aggregation [24].
Beyond the simple physicochemical models
detailed above, in a complete analysis of
this system, one must also take into
account a number of biochemical
considerations. For example, most bacteria
naturally produce exopolysaccharides –
polysaccharides targeted for delivery to the
extracellular environment – that aid in
adhesion and cohesion between bacterial
cells in a similar fashion to extracellular
appendages (illustrated in Figure 7). In addition, exopolysaccharides act as support matrices in aggregate
communities [25].
The role of exopolysaccharides is the key mechanism of granule formation in the Capetown’s model
[26]. This model proposes that under certain conditions, a specific methanogen (Methanobacterium
strain AZ) overproduces amino acids, causing the formation of an exopolysaccharide matrix around the
bacteria. Another model known as the Spaghetti model proposes that the filamentous bacteria known
as Methanosaeta form an initial branched network, promoting cell aggregation in a similar fashion to
the action of exopolysaccharides [27].
Hydrophobic forces are also believed to be key driving factors of granule formation [28]. Wilschut and
Hoekstra developed a local dehydration and hydrophobic interaction model proposing that dehydrating
the local environment of cells would increase the hydrophobicity of cell surfaces, thereby reducing the
excess surface Gibbs free energy required to promote bacterial aggregation [24, 29]. Furthermore,
highly hydrophobic bacterial surfaces are often indicative of the presence of extracellular appendages as
well as the cell wall proteins responsible for the recognition and binding of exopolysaccharides [28].
Such surface characteristics have been discussed above for their ability to enhance bacterial adhesion.
As living organisms, bacteria inherently partake in intercellular communication, allowing for the
effective exchange of nutrients. Through cell-to-cell communication, bacterial communities can
optimize the spatial organization of individual cells to achieve the most effective interactions with their
environment and to maximize their metabolic efficiency. Instead of random aggregation, bacteria
strategically position themselves to maximize substrate accumulation and waste product clearance, thus
maximizing their chances of survival. In systems of varying bacterial species, this concept is referred to
as the syntrophic microcolony model [30]. In this fashion, individual bacteria arrange themselves to
most effectively interact with wastewater during granule formation [31].
Figure 7 - Illustration of the Effects of Exopolysaccharides and
Extracellular Appendages in Bacterial Adhesion. Adapted from ref [8].
10
5. Conclusions
Upflow anaerobic sludge bed reactors have received increasing interest over the past few decades as a
wastewater treatment strategy due to their relatively simple designs and their beneficial production of
biogas. However, one of the main drawbacks to their widespread implementation has been the
relatively large start-up time required for the formation of the anaerobic bacteria granules. These
bacteria granules have been identified as the key component responsible for anaerobic digestion of
wastewater. As such, much research has focused on understanding the concepts behind granule
formation.
Of particular importance are the thermodynamics involved in granule formation. From the analysis of a
simple identical species system, it is apparent that the surface energy of the bacterial cell and the
surface tension of the surrounding wastewater are critical to spontaneous bacterial aggregation. From
the analysis completed in this article, it was found that the granule structure, especially the outer core,
can vary in bacterial composition considerably with very minor variations in influent wastewater surface
tension. Such alterations to granule structure were found to have significant impact on granule
retention and, as a consequence, on UASB reactor life.
The simple thermodynamic analysis of bacterial adhesion makes many assumptions and is thus limited
in its practical application. Granules are formed of multiple species, each with different surface energies,
and do not resemble lifeless, spherical particles. Thus, additional models must be considered, and can
include variations due to hydrophobic interactions, exopolysaccharides, and cell-to-cell communications.
By understanding the various models of bacterial adhesion, researchers should be able to improve
granule formation times, and thus make UASB reactors more attractive for widespread use. Ultimately,
the various models indicate that improving formation times cannot simply be accomplished by adjusting
a single factor, but will involve the manipulation and optimization of many interconnected parameters.
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Appendix A – MATLAB Code for Free Energy Calculations and Plots
analysis.m
%CHE612 - Wastewater Treatment Project
%Plot of free energy of adhesion for varying liquid and bacterial
%surface tension values
%Constants and initial input values
eps0 = 8.854187817*10^-12; %F/m
epsr = 78.5; %H2O
kB = 1.3806503*10^-23; %J/K
T = 298; %K
q = 1.602176462*10^-19; %C
NA = 6.02214199*10^23; %/mol
g_BV = [30:10:60].*(10^-3);
g_LV = [0:0.01:70].*(10^-3);
%Passing the surface tension values to the free energy calculation function
dG_gbv30 = dGadh(g_BV(1),g_LV);
dG_gbv40 = dGadh(g_BV(2),g_LV);
dG_gbv50 = dGadh(g_BV(3),g_LV);
dG_gbv60 = dGadh(g_BV(4),g_LV);
%Generating & saving plots
mkdir('DataFigures');
figsavepath = ['DataFigures\'];
figsave = figure;
plot_gbv30 = plot(g_LV.*(10^3),dG_gbv30.*(10^3));
hold on
plot_gbv40 = plot(g_LV.*(10^3),dG_gbv40.*(10^3));
plot_gbv50 = plot(g_LV.*(10^3),dG_gbv50.*(10^3));
plot_gbv60 = plot(g_LV.*(10^3),dG_gbv60.*(10^3));
set(plot_gbv30,'Color','green','LineWidth',2);
set(plot_gbv40,'Color','red','LineWidth',2);
set(plot_gbv50,'Color','magenta','LineWidth',2);
set(plot_gbv60,'Color','blue','LineWidth',2);
plotpx = xlabel('\gamma_L_V (mN/m)');
set(plotpx,'FontSize',16)
plotpy = ylabel('Free Energy of Adhesion (mJ/m^2)');
set(plotpy,'FontSize',16)
xlim([0 70])
ylim([-150 0])
plotleg = legend([plot_gbv30, plot_gbv40, plot_gbv50, plot_gbv60],
'\gamma_B_V=30 mN/m','\gamma_B_V=40 mN/m', '\gamma_B_V=50 mN/m',
'\gamma_B_V=60 mN/m', 'Location', 'SouthEast');
set(plotleg,'FontSize',16)
grid on
%Creating a filename
figname = [figsavepath 'FreeEnergyPlot' '.png'];
print(figsave, figname, '-dpng');
close
dGadh.m
function [ dG ] = dGadh( g_BV, g_LV )
%This function outputs the free energy of adhesion of a bacterial granule
%for a given gamma(Bacteria-Vapour) & gamma(Liquid-Vapour) - where gamma =
%surface tension
dG = -2.*((g_BV.^(1./2) - g_LV.^(1./2)).^2)./(1-15.*((g_BV.*g_LV).^(1./2)));
end
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