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