Biofilm detachment
by Rune Bakke
A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in
Civil Engineering
Montana State University
© Copyright by Rune Bakke (1986)
Abstract:
Monoculture Pseudomonas aeruginosa biofilms were modeled by mass balances. Measurable
expressions for. substrate removal, cellular reproduction, product formation (extracellular polymeric
substances), and detachment were extracted from the model to determine kinetics and stoichiometry for
the individual processes. This thesis presents a detailed investigations of detachment, the transport of
particulate mass across the biofilm/liquid interface.
Methods were developed to monitor biofilm optical thickness and density in situ at various locations in
the reactor. The optical thickness was converted into actual (mechanical) biofilm thickness by a
geometric analysis of the light path through the sample. Time progressions of biofilm thickness and its
spatial variation within the reactor were obtained by this method. Optical film thickness data from the
literature were also translated into actual biofilm thickness and compared to the data obtained here.
Biofilm optical density was correlated with biofilm cell mass, yielding information regarding biofilm
cell mass distribution, time progression, and density. Transmission and scanning electron microscopy
were used to relate biofilm morphology to biofilm processes. Liquid phase cell, product, and substrate
data, obtained with methods previously published, were analyzed with mass balance equations for the
system together with the biofilm data obtained by the new methods. The fundamental processes of
accumulation, transport, and transformation were separated and factors of significance to detachment
were identified.
P. aeruginosa biofilm thickness reached approximately 35 μm within 24 hours of reactor start-up and
remained more or less constant throughout the experiments even though changes in fluid dynamic
conditions were imposed on the system during this period. Changes in biofilm composition, interface
morphology, and activity were observed throughout the experiments. It was concluded that constant
biofilm thickness can serve as the most appropriate boundary condition linking the liquid phase and the
biofilm mass balances required to model the detachment process. Alternative boundary conditions,
such as constant biofilm density and specific detachment rate proportional to fluid shear force, were not
supported by the data.
BIOPILM
detachment
by
Rune Bakke
A thesis submitted in partial fulfillment
of the requirements for the degree
Of
Doctor of Philosophy
in
Civil Engineering
MONTANA STATE UNIVERSITY
Bozeman, Montana
June 1986
main lie.
D3?g
^/7?
Cop»
© COPYRIGHT
by
Rune Bakke
1986
All Rights Reserved
Ii
APPROVAL
of a thesis submitted by
Rune Bakke
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committee and found to be satisfactory regarding content.,
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Requests
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extensive
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or
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Microfilms International, 300
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and from microfilm and the right to reproduce and distribute
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Signature
Date
I
iv
ACKNOWLEDGMENTS
I wish to express my appreciation to the following:
Bill.Characklis for
providing
advice, support, and the
research environment where I could reach my goals.
Keith Cooksey, Al
Cunningham,
Gordon McFeters, and Dan
Goodman for their large contributions to my thesis program.
Shari McCaughey,
Zbigniew
Lewandowski, Robert Kultgen,
and Warren Jones for help with my thesis.
Andy, Maarten, Pam, Wendy,
Whon Chee, Mukesh, and Ewout
for their cooperation.
Gordon
Willamson
and
Stuart
Aasgaard
for
technical
assistance.
The
staff
and
students
of
the
Civil
Engineering,
Chemical Engineering, and Microbiology Departments whom have
contributed to my education.
-"AU
my
friends
Whom
have
made
my
student
life so
enjoyable.
Montana State University Engineering Experiment Station,
National Science Foundation,
Office
of Naval Research, and
Statens Laanekasse for financial support.
V
TABLE OP CONTENTS
Page
LIST OP TABLES .......... .............................vii
LIST OP FIGURES........ ............. ...........
..viii
ABSTRACT................................ ...............xi
INTRODUCTION............... . ............... .............I
Goal and Objectives................
LITERATURE REVIEW. ..................
4
.5
in Vo o-IO'!
Detachment...*.......
Biofilm Models.......
Erosion......... ...
Fluid Shear Stress
Biofilm Mass.............................. 10
Sloughing...........................
10
Reactor Performance..........
12
Biofilm Properties.............
..14
Biofilm Composition.......
..14
Organism..............
.17
Biofilm-Liquid Interface..................... 17
THEORY.................
20
Model........................
...20
Mass Balance.....................
20
Phase Separation.......
21
Sensitivity Analysis......................... 23
Process Rates...........
....27
Biofilm composition..............
30
EXPERIMENTAL METHODS. ..........
35
Experimental Apparatus...........
35
Operating Conditions............
36
Experimental. Procedures.................
.46
Analytical Methods............
49
Biofilm Thickness......... .................*[49
Optical Density..............................*53
Statistical Methods.......... .........!!!!!!!!! [53
vi
TABLE OP CONTENTS (Continued)
Page
RESULTS. ..........
54
Data Correlation..........................
54
Biofilm Refractive Index...... ..........
54
Light Scattering vs. Biomass..................55
Progression of Biofilm^Variables.................56
Bipfilm Thickness.... ......
...56
Light Scattering by Biofilms.... ............. 62
Fluid Shear Stress.......
63
Progression of Liquid Phase Variables............ 63
Substrate Concentration...... ................ 63
■ Cell Concentration .......................... .66
Extracellular Polymeric Substances (EPS)......66
DISCUSSION.......
69
Biofilm Detachment........
69
Specific Cellular Detachment Rate............ 69
Biofilm Thickness.................
70
Biofilm Roughess...................
78
Biofilm Composition.......... .............. .85
Transitions........
87
Biofilm Aging.........
...89
Specific Cellular Growth Rate.............. ......91
Biofilm Accumulation.............................94
Specific Accumulation Rate....................94
Steady State.. ..................
.96
Modeling........... ....... .................. IOO
CONCLUSIONS. ..............................
NOMENCLATURE. ..................... i..........
BIBLIOGRAPHY.............................
APPENDIX...................
.103
106
.108
114
vii
LIST OF TABLES
Table
Page
1
Relevant kinetic and stoichiometric coefficients
for P. aeruginosa.
18
2
25
3
Mass balances for biofilm reactor with general
terms for process rates.
'
'
Mass balances for biofilm reactor including
kinetic expressions for specific rates (Bakke
et al., 1984) .
26
4
Dimensions of the MRTR.
40
5
Reactor components and dimensions.
41
6
Fraction of substrate removed (reported as mean
* standard deviation of four samples) at various
recycle rates in liquid phase mass transfer
study of MRTR.
45
7
Composition of Nutrient Solution.
47
8
Analytical methods applied.
50
’
.
9-25
Raw data.
114
yiii
LIST OF FIGURES
Figure
Page
1
Schematic representation of two phase biofilm
system described by Equations 10-15. Bulk
transport, substrate diffusion, cell and EPS
production, and detachment are processes
included 6 The coordinate system defined for
this study is illustrated.
24
2
Specific cellular growth rate, m, modeled as a
saturation function of substrate concentration
and product formation rate modeled as the sum
of a growth and a non-growth associated term,
plotted vs. substrate concentration.
34
3
Schematic diagram of experimental system
including flow and temperature control for
gas and liquids.
37
4
Mixed rectangular tube reactor (MRTR). Biofilm
measurements and samples were obtained at
locations labeled 1-8.
38
5
Gross section view of rectangular tube. A-B-C-D
are light paths for biofilm thickness and
optical density measurements. Scale indicated
along the y-axis. Biofilm thickness profile
measurements were taken along the y-axis at
marked positions.250 mm apart.
39
6
Fluid shear stress progression in Experiment I.
Continuous flow operation started at time zero.
42
7
Fluid shear stress progression in Experiment II.
Continuous flow operation started at time zero.
43
8
Fluid shear stress progression in Experiment III.
Continuous flow operation started at time zero.
44
9
Calibration curve for biofilm cell carbon areal
density vs. light scattering by the biofilm
measured as absorbance at wave length = 450 nm.
Line represents best linear fit (R =0.88) .
57
ix
LIST OF FIGURES (Continued)
Figure
Page
10
Calibration curve for liquid phase cell carbon
concentration vs. light scattering in the
liquid phase measured as absorbance at wave
length-= 450 nm. Line represents best linear
fit (Rz=0.94).
58
11
Biofilm thickness progression in Experiment I.
59
12
Biofilm thickness progression in Experiment II..
60
13
Biofilm thickness,progression in Experiment III.
61
14
Progression of biofilm optical density, measured
as absorbance at locations 1-8 (Figure 4), in
Experiment III.
64
15
Progression of liquid phase substrate
concentration, Cg ( x - influent, □. - effluent )
in Experiment IIi. Data points represents the
average of two samples.
65
16
Liquid phase cell mass , Cm, , calculated from
light scattering data (effluent optical density)
in Experiment Tl.
67
17
Liquid phase cell ( □ ) and EPS ( x ) fraction
of particulate organic carbon (POC) in the
effluent in Experiment III.
68
18
Progression of specific cellular detachment rate,
determined according to Equation 17 from optical
density data.,
71
19
Scanning electron micrograph (SEM) of biofilm at
the completion of Experiment III.
73
20
Scanning electron micrograph (SEM) of biofilm at
the completion of Experiment. III.
74
21
Scanning electron micrograph (SEM) of biofilm at
the completion of Experiment III.
75
22
Transmission electron micrograph (TEM) of
biofilm at the completion of Experiment III.
76
X
LIST OF FIGURES (Continued)
Fiaure
Paae
23
Transmission electron micrograph (TEM) of
biofilm at the completion of Experiment III.
77
24
Optical photo micrograph of biofilm-liquid
interface at 50 hours. Corresponding to
Figure 25.
79
25
Bibfilm thickness profile along the y-axis
(i.e. perpendicular to bulk liquid flow
direction) at 50 hours. Corresponding to
Figure 24.
80
26
Optical photo micrograph of biofilm-liquid
interface at 272 hours. Corresponding to
Figure 27.
82
27
Biofilm thickness profile along the y-axis
(i.e. perpendicular to bulk liquid flow
direction) at 272 hours. Corresponding to
Figure 26.
83
28
Progression of standard deviation of nine
biofilm thickness samples in
Experiments II and III.
84
29
Measured cell and EPS fractions of biofilm
carbon (POC) in a) this study, and
b) Trulear1s (1983) experiments.
86
30
Progression of liquid phase specific cellular
growth rate, m, , calculated from Equation 20
and substrate data (Figure 15).
93
31
Specific biofilm cell accumulation rate
progression in Experiments II and III,
calculated as the l.h.s. of Equation 16 from
biofilm optical density data.
95
32
Specific biofilm cell accumulation rate
progression in Experiments II and III on a
natural logarithmic scale. Data from
Figure 31, excluding data obtained during
recycle rate transitions.
97
xi
ABSTRACT
Monoculture Pseudomonas aeruginosa biofilms were modeled
by mass balances. Measurable expressions for. substrate
removal,
cellular
reproduction f
product
formation
(extracellular polymeric substances), and detachment were
extracted from the
model
to
determine kinetics - and
stoichiometry for the individual processes. This thesis
presents a detailed
investigations of detachment, the
transport of particulate mass across the biofilm/liquid
interface.
. Methods were developed
to monitor biofilm optical
thickness and density .in ' situ at various locations in the
reactor. The optical thickness was converted into actual
(mechanical) biofilm thickness by a geometric.analysis of
the light path through the sample. Time progressions of
biofilm thickness and its spatial variation within the
reactor were obtained by this method. Optical film thickness
data from the literature were also translated into actual
biofilm thickness and compared to the data obtained here.
Biofilm optical density was correlated with biofilm cell
mass, yieiding^ information regarding biofilm cell mass
distribution, time progressionf and density. Transmission
and.scanning electron microscopy were used to relate biofilm
morphology to biofilm processes. Liquid phase cell, product,
aiui .s!^k®'t:ra'ke data,
obtained
with methods previously
published, were analyzed with mass balance equations for the
system together with the biofilm data obtained by the new
methods.
The
fundamental
processes
of accumulation,
transport, and transformation were separated and factors of
significance to detachment were identified.
■2-«— ^eruginosa biofilm thickness reached approximately
35 urn within 24 hours of reactor start-up and remained more
or less constant throughout the experiments even though
changes in_ fluid dynamic conditions were imposed on the
system during this period. Changes in biofilm composition,
interface morphology, and activity were observed throughout
the experiments. It was concluded that constant biofilm
2hea“ i o S t
s
au
eppocs
v
t
i;2 ^ t
or
“ °
s"a?ttc
I
INTRODUCTION
Biofilms are microbial populations and their matrices of
noncellular material accumulated on
liquid
phase.
bacterial
The
cells
biofilms
and
surface
this
extracellular
(EPS). The part of the
termed the
in
surfaces submerged in a
study
consisted of
polymeric
substances
biofilm exposed to the liquid phase,
film,
is
of
primary
significance to
bipfilm modeling since all mass transfer between biofilm and
liquid phase occurs here.
main
distinction
These . transfer processes are the
between
biofilm
microbial systems. The base
film
systems
and
dispersed
is the continuous biofilm
matrix between the surface film and the substratum.
Detachment is defined as the transfer of particulate mass
(e.g. cells and EPS)
from
phase. Detachment due to
removal of
individual
physiochemical
cells
and
and
larger
conditions
to the bulk.liquid
particles
referred
portions
of
at the
to as erosion.
biofilm due to
the
biofilm
sloughing
are
treated as separate
and
processes
evidence
erosion and sloughing can
is
small
within
sloughing. Erosion
because
biofilm
shear forces results in continuous
biofilm liquid interphase
Sporadic detachment of
the
suggests
that
the
is
termed
causes of
be distinguished. The distinction
2
is somewhat arbitrary
both kinds of
treated
and
most systems probably experience
detachment.
Erosion
differently • because
they
and
Sloughing are also
may
have significantly
different effects on biofilms.
Understanding
biofilm
of
reactor,
understanding
biofilm
detachment
operation,
of
biofilm
has
biofouling
processes
in
relevance to
treatment,
general.
and
Biofilm
detachment can be the rate limiting process which determines
the metabolic state (average specific cellular growth rate)
.
'•
of steady state biofilms (Bakke et al., 1984). Understanding
of biofilm detachment is,
therefore,
not only necessary to
predict biofilm behavior, but
this knowledge may also serve
as a tool to Control
activity. The central role of
detachment
biofilm
processes
multi-species biofilm
in
composition
reactors,
and
and
performance
the
of
need for further
investigation of detachment processes have been demonstrated
through theoretical analysis of biofilm reactors (Howell and
Atkinson, 1976; Wanner and Gujer, 1985) .
. The
importance
general
is
of
understanding
emphasized
applications
of
by
the
biofilm
transformation processes.
biofilm
increasing
reactors
Biofilm
for
modeling
processes in
number
of
biological
can aid design
and operation of such bio^reactors.
Biofilms
excessive
can
heat
losses in process
also
lead
transfer
to
costly
resistance
equipment.
Improved
problems,
and
such as
fluid frictional
methods for biofilm
3
monitoring, analysis, and modeling can aid in development of
treatment
programs
to
maintain
performance
of
such
equipment.
Very
little
qualitative
and
quantitative
regarding detachment is available,
significance of detachment
in
was, therefore, designed to
biofilms.
Biofilm
to biofilm
biofilm
detachment.
thickness,
appearance.
The
biofilm^liquid
structure, particle
Substrate
Biofilm
properties
surface
and
primary
interest
and
extensively
monitored as measures of
and
its
interface
is
appearance
the
was
roughness. Electron and light
distribution,
consumption
and
monitored were
structure,
of
interface,
used
conditions,
identify factors of significance
interface
were
metabolic
Pseudomonas aeruginosa biofilm
density,
characterized by its
microscopy
study detachment in monoculture
in
reactors were monitored to
in spite of the apparent
biofilm reactors. This study
properties,
fluid dynamic conditions
information
to
investigate biofilm
and interface roughness.
cellular
growth
rate
were
metabolic conditions. Recycle flow
rate was controlled and friction imposed on.this flow by the
biofilm was monitored as
pressure
drop. Fluid shear stress
acting on the biofilm due to liquid phase flow was, thereby,
measured. Since most
of
these
measurements were conducted
throughout the experiments they serve as an investigation of
biofilm aging, and
monoculture biofilm
its
effect
experiment
on
detachment. No previous
reported
in the literature
4
lasted longer
than
two
weeks.
Long-term biofilm behavior
was, therefore, unknown.
Goals and Objectives
The
goal
of
this
significance to biofilm
monitored
in
regarding
a)
pursuit
biofilm
stability (steady state
d) new methods
for
study
was
to
detachment.
of
the
factors of
Several variables were
goal,
boundary
identify
yielding information
conditions
b)
biofilm
conditions), c) biofilm properties,
biofilm
characterization,
term biofilm behavior or aging.
and e) long
5
LITERATURE REVIEW
Detachment
Detachment is the transfer
cells and EPS, from the
et
ale. (1984)
process
which
biofilm
demonstrated
controls
the
specific cellular growth
also
demonstrated
coefficients
that
processes
conceptually
bioreactors.
distinguish
across the
metabolic
in
can
be the
state (e.g.
a biofilm reactor. They
kinetic
and
stoichiometric
transformations
in
a
b10films. This implies that mass
the
only
biofilm
significant
transport of dissolved
Davies, 1974), this
cells
biological
are
Given
detachment
the
describing
to the liquid phase. Bakke
that
rate)
chemostat apply as well in
transport.
of particulate mass, such as
materials
systems
processes
from
information
which
dispersed
regarding
in biofilms (Atkinson and
study
focused On particulate transport
biofilm-liquid
interface. Information available
in the literature
was
analyzed
to determine which factors
may.be of primary significance to detachment of particulates
(i.e. cells and EPS)
and
to
design experiments to further
quantify detachment processes.
Erosion and sloughing were distinguished in the analysis
of detachment because
they,
by
definition, have different
causes. Erosion is the continuous removal of small particles
6
from the
biofilm
at
the
biofilm-liquid
fluid shear stress. Sloughing
large .pieces
of
biofilm.
two
different
The
biofilm
experience both
on
on
film at any depth
by
events,i This
alter
within
the
have significantly
biofilms.
Most
biofilms
and
may be difficult. Erosion
continuous function with only
deeper
hand,
can
removal
the
probably
sloughing, but distinguishing
a
the
other
conditions
also
is
Sloughing, on the
may
to
experiments
at the biofilm surface
effects
due
may
erosion
the two processes in
indirect
is intermittent detachment of
processes
effects
interface due to
layers
of
the
film.
directly influence the
of multiple layers in single
local environment drastically
(e.g. expose a previously anaerobic layer to oxygen), and it
may change biofilm morphology, causing significant roughness
which can alter mass and momentum transfer rates. Separation
of erosion and sloughing
imposing conditions which
due to sloughing
in
this study was accomplished by
favor
insignificant,
erosion, making detachment
as
described in detail in
the experimental methods section.
Biofilm Models
Biofilms are
inherently
are the driving force
for
heterogeneous
since gradients
transport of dissolved substrate
and products from the liquid
phase into and out of biofilms
(i.e. diffusion
Measurements
gradients).
of
temporal and
7
spatial
gradients
are
experimentally
separate
transformation
processes
difficult
the
mass balance analysis.
biofilms,
level
biofilm can be assumed to
present.
detachment . process
in
necessary to reduce the
at
it
To
from the
is, therefore,
of inhomogeneity so that the
be homogeneous in the particulate
Such
homogeneity
EPS, and substrate distribution
in terms of cell,
in biofilms was obtained in
monoculture biofilms (Trulear, 1983)
Wanner and Gujer (1985)
developed a biofilm model which
account for gradients in the biofilm by dividing the biofilm
into
several
layers
perpendicular
gradients. Each layer is
treated
to . the
diffusion
as individual phases (see
Theory chapter) and
appropriate boundary conditions account
for the interaction
between
which couples the
liquid
cell mass balance
for
liquid interface
is
phases. The boundary condition
phase
the
net
cell
biofilm
mass balance and the
layer
cellular
at the biofilm-
detachment.
Wartner and
Gujer stated that a large variety of boundary conditions can
be applied to model detachment in their numerical simulation
of
biofilm
processes.
expression for
detachment
evidence. Trulear and
erosion may be
Selecting
must
be
Characklis
influenced
by
the
based
most
appropriate
on experimental
(1982) found that biofilm
fluid
shear stress, biofilm
density, and biofilm thickness. These observations served as
a
basis
for
evaluate the
the
present
influence
of
study,
these
which
was
designed to
variables on detachment.
8
Identifying
an
appropriate
boundary
condition
for
monoculture biofilm detachment modeling based on fluid shear
stress, biofilm
density,
and
biofilm
thickness data was,
therefore, a major objective in this study.
Monoculture biofilms studied by Trulear (1983) and Bakke
et al. (1984) were
developed
assumed
insignificant
to
be homogeneous because they
gradients
in
terms
of
the mass
balance analysisThis simplified the biofilm reactor system
significantly,
phases
as
(liquid
it
consisted
and
measurements of
biofilm
the
cellular
to
obtain
growth,
described in
biofilms have
in
the
applied
and
quantitative
processes of significance
expressions
for
and
accumulation
Theory
chapter.
coefficients
been
two homogeneous
the mass balance equations
measurable
detail
stoichiometric
of
detachment,
only
phase),
fundamental
were obtained. Manipulations
require
of
obtained
to
Wanner
in
specific
rates are
Kinetic and
"homogeneous"
and Gujer*s (1985)
heterogeneous model to simulate monoculture biofilm behavior
(Wanner,
personal
communication).
described in more detail
in
the
This
simulation
is
Theory and the Discussion
chapters.
Monoculture biofilms in the present study were developed
under conditions similar to
to minimize the
of
level
transformation
and
those applied by Trulear (1983)
of biofilm inhomogeneity. Separation
detachment
processes
was
thereby
possible, and
factors
of
significance
to detachment were
identified.
Erosion
Fluid Shear Stress. Erosion
removal of small particles
liquid interface due
to
from
fluid
liquid. The friction imposed
phase imposes as a shear
energy
of
the
fluid
is defined as the continuous
shear
by
force
is
the biofilm at the biofilm
the
stress from the bulk
biofilm on the liquid
on the biofilm. The kinetic
dissipated
through
breakage of
physical bonds in the biofilm resulting in detachment. Bakke
et
al. . (1986)
detachment data
shear stress
at
correlated
(TruIear
the
mixed
and
culture
Characklis,
biofilm/liquid
biofilm
1982)
interface,
mass
to fluid
r
,in a
turbulent system by the following equation:
ra =.kdr
where r^ is the
1
—I
biofilm detachment rate [t "j and
specific
ky is a detachment coefficient [t ^ P ^].
Previous monoculture
in turbulent flow
Tgrakhia, 1986).
biofilm
biofilm
reactors
Trulear's
experiments
were
at
experiments were conducted
constant
monoculture
modeled
t ('Trulear, 1983;
(
P. aeruginosa )
assuming
specific
detachment rate to be constant (Bakke et al., 1986). To make
10
the model applicable
conditions
to
different
experiments, it
is
fluid dynamics on
therefore, the
systems experiencing fluid dynamic
from
those
necessary
biofilm
control
to
used
in
determine
erosion.
variable
previous
the effect of
Fluid shear force was,
in
this study, regulated
through step functions in fluid recycle rate.
Biofilm
which
Mass.
Rittmann
detachment
rate
(1982)
can
be
presented
equations by
calculated
for
various
experimental systems and conditions assuming that Equation I
is valid. He also
found
that a linear relationship between
total biomass detachment rate, X r^, and biofilm mass, X, is
a reasonable approximation for the data presented by Trulear
and
Characklis
(1982).
Data
obtained
in
monoculture
(Pseudomonas aeruginosa) biofilms by Trulear (1983) can also
be
approximated
by
a
linear
cellular detachment rate,
'(Bakke et
^dM'
al.,
appeared
1984).
-to
relationship
between total
rdM' an<^ biofilm cell mass,
Specific
be
cellular detachment rate,
independent
concentration, CM2, in Trulear*s
of
biofilm
cell
experiments (Bakke et al.,
19.86) .
Slouahina
Sloughing
is
frequently of large
defined
pieces
within the biofilm. These
as
of
intermittent
biofilm,
conditions
detachment,
due to conditions
may evolve slowly and
'^
11
cause sloughing
at .random,
or
they
may
be triggered by
transitions in the environment.
Howell
and
Atkinson
intermittent sloughing
(1976)
however, not available.
data
Even
limitations may play a role
actually
a
model
for
triggered by substrate limitation in
the biofilm. Experimental
that they
developed
supporting their theory is,
though substrate and nutrient
in sloughing, it is not evident
trigger
the
detachment. Several other
factors, such as polymer gel (EPS) strength and density, may
play a significant and varying role in sloughing. Gas bubble
formation has, for example,
of
sloughing
1983).
A
in
been
denitrifying
quantitative
observed as a major cause
biofilms
correlation
(la
Cour Jansen,
between
nitrogen
production and sloughing was
not reported, probably because
other
internal
factors
influencing
transfer were not controlled
(1985) concluded
spatial
that
gradients
in
and
external
mass
or monitored. Wanner and Gujer
direct
observation
biofilms
are
of temporal and
required
to
explain
sloughing but these measurements are difficult at present.
Some
sloughing
quantitative
caused
altered.the free
by
information
is
transients.
Turakhia
calcium,
calcium chelation, and
Ca++,
available
et
regarding
al. (1983)
available for biofilms by
observed immediate biofilm sloughing
of both cells and EPS. Quantitative data relating detachment
rates for both cells
and
obtained. The sloughing was
EPS
to calcium concentration was
presumably caused by removal of
12
calcium important in the biofilm structure.
Substrate
transitions
occur
frequently
in
wastewater
treatment plants. Such transitions can have negative effects
on
biOreaetor
performance,
decreasing
effluent
quality
(Storer and Gaudy, 1969; Der Yang and Humphrey, 1975; Bakke,
1983; Rozich and Gaudy,
1985).
Bakke (1983) found that the
initial response by mixed culture biofilms to step increases
in substrate (glucose,
(imposed by doubling
EPS sloughing.
lactose,
influent
Increased
and lactate) loading rates
substrate concentration) was
biofilm
detected. Therefore, substrate
cell
detachment was not
transitions .caused selective
sloughing of a specific fraction of the biofilm which lasted
for
a
few
minutes.
substrate loading
The
rate
biofilms
Conditions
adapted
to
increased
by increased metabolism
and cell reproduction and re-established steady state within
hours.
Reactor Performance
Effects of detachment on
starting
with
the
reactor performance is reviewed
well-studied
biofilms developed by Trulear
1984). The average
in the biofilm
specific
monoculture
(Trulear, 1983; Bakke et al.,
cellular reproduction rate, m,
was
found
to
cellular detachment
rate.
The
exposed to high fluid
homogeneous
be
proportional to specific
biofilms
investigated were
shear stress (c. = 3.5 Pa), but caused
•
13
no detectable increase in
a
relatively
smooth
fluid frictional resistance (i.e.
biofilm-liquid
interface)
(Trulear,
1983), suggesting that erosion was the dominating detachment
process.
These
biofilms
[effectiveness factors
were
for
quite
homogeneous
diffusion, calculated according
to Atkinson and Davies (1974) ,
experiments ]. So
also
sloughing
were greater than 0.9 in all
due
limitations was not expected
to
since
substrate or nutrient
lack of homogeneity may
be a prerequisite for sloughing (Howell and Atkinson, 1976).
It was demonstrated in
these
experiments that steady state
specific cellular growth rate,
detachment
rate
but
m,
was a direct function of
independent
of
influent
substrate
concentration.
Regulating detachment may serve as a tool to control and
optimize bioreactor processes,
growth rate is so strongly
since
the. specific cellular
influenced by detachment rate in
biofilm reactors (Bakke et al., 1984). Substrate removal is,
for example,
influenced
by
detachment
closely related to the growth
and can, as
suggested
for
since it is
rate of the organisms (Monod,
1942). Product formation is also
growth rate (Luedeking and
rate,
a function of the cellular
Piret,
1959; Mian et al., 1978)
substrate removal, be regulated
through detachment control. Given kinetic and stoichiometric
coefficients for growth and
biofilm
model,
an
optimal
process may be calculated.
product
formation, and a valid
detachment
rate
for
a given
14
In
the
slightly
limitation
is
more
complex
significant,
biofilm is still equal
but its value is less
to
average
inhomogeneity
growth
diffusion
rate
in
the
than that calculated from bulk liquid
longer
optimum detachment
where
detachment rate at steady state,
phase substrate concentration
It is also no
case
(Atkinson
and Davies, 1974) .
possible to analytically determine an
condition
(gradients
in
conditions (biofilm
density,
operation range may
still
for
a
given
the
film).
thickness,
be
process due to
Given
etc.)
diffusion
an optimal
determined by accounting for
diffusion gradients in the film. This may be accomplished by
introducing
an
effectiveness
limitations, f^, according
Alternatively,
diffusion
accounted for by
to
Atkinson
gradients
dividing
(layers perpendicular
factor
to
the
for
diffusion
and Davies (1974).
in
biofilms
can
be
bio.fiIm into several phases
the
diffusion
gradients) within
which gradients may be neglected (Wanner and Gujer, 1985).
The effects of detachment
on
more complex systems such
as multi-species and multi-layer biofilms have been analyzed
theoretically by Wanner
the
lack
specific
quantitative
predictions.
sloughing
detachment
species
of
and
and
erosion
mechanisms
biofilm
Gujer
information
By
comparing
their
play
progression,
Theoretical as well
as
(1984). They emphasized
an
model
necessary
extreme
cases
demonstrated
important
composition,
experimental
to make
of
that
role
in multi­
and
behavior.
work on detachment in
15
multi-species biofilms is required to quantify the effect of
detachment on biofilm behavior.
Riofilm Properties
Biofilm Composition
The effect of
depends on the
fluid
shear
physical
stress on biofilm detachment
properties
of
the biofilm. It is
reasonable to assume that a smooth. dense film with a strong;
structure will experience less erosion than a weak arid rough
film. The quality
substances (EPS)
due
to
and
quantity
may
strongly
its
structural
Quantitative
information
of extracellular polymeric
influence detachment rates,
role
in
regarding
microbial
aggregates.
the
of
role
EPS
on
detachment rates is unavailable, but significant qualitative
information
regarding
EPS
production
and
biofilm
accumulation is available.
A variety of chemical
produced by bacteria
structures
(Sutherland,
considered to be carbohydrate
al.,
is represented in EPS
1982).
with
They are usually
acidic groups (Corpe et
1976;
Fletcher and Floodgate, 1973) , amino groups
‘
■
r
.
(Baier, 1975), and sometimes associated with proteins (Corps
et al.,
1976)o
P.
aeruginosa
produce
EPS
consisting of
mannuronic, giucoronic, and nucleic acids, and small amounts
of proteins (Eagon, 1956;
1962;
Brown
et al., 1969; Evans
16
and Linker, 1973; Mian et al., 1978).
Bacterial
EPS
have
been
shown
to
be
involved
in
selective accumulation of ions (Galanos et al., 1977; Leive,
1974). Turakhia et al.
(1983) stimulated biofilm detachment
by chelation of calcium
decreasing
cellular
ions,
and Turakhia (1986) reported
detachment
with
(1986)
concluded
availability. Turakhia
increasing
calcium
that calcium ions
contribute to biofilm cohesiveness through the cross-linking
of EPS.
EPS has been categorized, based on its spatial association
with the bacterial
cell;
layer attached
the
to
dispersed layer
to
slime
capsule,
and
the
somewhat
a
studies
1983;
EPS
detachment,
suggesting
cells
been
is
influenced
Christensen et
slime,
with
is a
the cell (Brock,
separation
Turakhia,
1986).
without
the
together
which
would
be
attempted in previous biofilm
detachment
portion of the EPS has
b)
is a compact
arbitrary distinction between
presence
categories of EPS with different
ties the
which
quantitative
not
stimulated
and
associated
difficult, and has
(Trulear,
capsule,
cell,
loosely
1979). Due
a)
in
Bakke
(1983)
influencing
of
at
least
cell
two
functions. One kind of EPS
the
biofilm,
while an other
a different, unknown, function which
by
substrate
al.
(1986)
loading
isolated
rate
transitions.
and characterized two
soluble EPS produced by a marine psepdomonas, and found that
the relative production rates
I
1
of
the
two EPS changed from
17
the exponential
growth
phase
to
the
stationary state in
batch
cultures. Sutherland (1977) and Costerton et al.
'
(1978) claim that EPS play at least two significant roles in
I)
structure
of
,microbial
aggregate
processes between cells and
presence
of
at
least
and
2)
transfer
the environment, supporting the
two
functionally
Electron micrographs were applied
in
different
EPS.
this study to seek ah
EPS categorization based on it functional role in biofilms.
Organism
The strain of Pseudomonas
was obtained from the
of Microbiology at
aeruginosa
culture
Montana
used in this study
collection of the Department
State University (Bozeman, MT).
This organism has been studied extensively in both dispersed
and biofilm cultures (Trulear,
1983; Robinson et al., 1984;
Bakkeetal.,
et
1984;
Turakhia
stoichiometric coefficients for
in Table I.
Other
this
strict aerobe (Buchanan
et
1974),
1974) , e) can cause severe
(Woods et al., 1980;
of
growth
infections)
is
in
1986). Kinetic and
organism is presented
characteristics describing Pj. aeruginosa
include: a) negative gram stain
(Buchanan et al.,
al.,
(Buchanan et al., 1974), b)
al.> 1974), c) chemoorganotroph
d)
rod
shape
(Buchanan et al.,
infections in a compromised host
Costerton,
colonies
(Costerton, 1979) .
1979), f) its primary mode
attached
to
surfaces
(e.g.
18
Biofilm-Liquid Interface
Erosion and sloughing are
different effects on
small particles
force.
The
biofilm
from
"peaks"
the
of
therefore, more exposed
smooth the
distinguished partly by their
morphology. Erosion separates
biofilm
a
rough
to
biofilm-liquid
surface
erosion,
interface.
by fluid shear
biofilm
and
surface
are,
erosion tends to
A smoother interface
leads to decreased mass and fluid shear stress. If, as
Table I. Relevant kinetic and stoichiometric coefficients
for P. aeruginosa.
Coef.
Value
V
0.4
I T 1
KgS
2.0
gs m
kgp
0.27
knP
0.035
YPS
y M S
Units
—3
n
n
n
9P Sh"1 h'1
n
gP gS 1
0.34
n
gM gS-1
Howell and Atkinson
in the film cause
(1976) suggested, substrate limitations
sloughing,
then decreased substrate flux
due
to
smoothing
Removal
of
biofilm
sloughing will lead
to
increased biofilm roughness, which.
sloughing.
film
Robinson et al.
(1984)
9P 9M*1
0.56
into the
Source
by
in
erosion
large
may lead to
pieces
during
19
in turn, increases
mass
transport
and fluid shear stress.
Increased fluid shear stress may, in turn, lead to increased
erosion. Therefore, erosion
may
enhance sloughing and vice
verse, so that most biofilms will reach some balance between
the two processes. A biofilm will display a rougher biofilmliquid interface when sloughing,
as
opposed to erosion, is
the dominating process. A rough interface does not, however,
imply that sloughing
is
the
dominating detachment process
because several other factors,
such as biofilm composition,
can also influence biofilm interface morphology.
Increased frictional
resistance
to
fluid
flow
due to
biofilm,roughness has been observed (Trulear and Characklis,
1982).
This
implies
greater
fluid
shear
stress
and,
according to Equation I, higher detachment rate. Filamentous
organisms impose, in general,
liquid
(Trulear
flow
and
than
do
morphology is important for
focus of this study.
and
greater friction on the
non-filamentous
Characklis,
between the liquid,
much
the
1982).
So,
biofilm
biofilm
cultures
interface
both momentum and mass transfer
biofilm
phase
and was a main
20
THEORY
Model
Mass balance
A system can be
compounds
within
divided
each
into
phase
m phases. Accumulation of
is
described
by
a balance
equation of the general form:
net rate of
accumulation
net rate of
=
within the
net rate of
transport
+
transformation
into the
phase
within the
phase
Equation 2 can
be
Bakke et al. 1984;
phase
expressed
wanner
in
and
vector form (Reels, 1980;
Gujerr
1985).
In the k th
phase:
Ia(CijIZat)k -
Nijk + Rijk
-where: C = chemical state vector
N = flux vector for net transport into phase k
R = intraphase production rate vector
i = component lr2 r3...h
j = process lr2 r3...1
. = phase lr2 r3...m
2
21
The time
progression
of
the
components
is determined by
simultaneous integration of Equation 3 over all i, j, and k,
which can be done by numerical analysis.
Multiplying
Equation
3
matrix of the system yields
be very
useful
This matrix
in
by
the
the
composition
an elemental balance, which may
stoichiometric
contain
elemental
number
analysis (Roels, 1980) .
of
atoms
of
the atomic
species considered per mole of component i.
Phase separation
A system can be divided
into a number of distinct phases
depending on physical characteristics, requirements of model
resolution,
and
available
computing
capacity.
The
most
simple case, m = I, is appropriate for ideal continuous flow
stirred tank reactors (CPSTR),
activity resides in
a
or
chemostats, in which all
homogeneous
liquid phase. Plug flow
reactors can be modeled as CFSTRs
in series and then m > I,
since each
a
CFSTR
is
considered
separate
phase.
If a
biofilm exists in a CFSTR, then it is appropriate for m >. 2,
since a liquid phase and
one or more biofilm phases exists.
Layers in a biofilm with distinct biological activity may be
modeled as different phases within
for
example,
be
perpendicular to
layer of thickness
regarded
the
as
a system. A biofilm can,
several
interacting layers
main
diffusion
gradient, where each
is
described
by mass balances for
■
phase k . By increasing the
biofilm thickness .L^,
22
:
number
the
of
layers m for .a given
thickness
of
each layer,
f
decreases. If m -> go then Lfk -> 0 and the mass balances for
the biofilm becomes a partial differential equation in z and
t, which can
be
activity, and
applied
to predict cellular distribution,
diffusion
gradients
within biofilms (Wanner
and Gujer, 1984) .
The mass balance
model
(Equation .3),
applied to a two
phase system (m = 2)* consisting of one biofilm phase (phase
2, k = 2) and
one
bulk
liquid
schematically described in
defined
for
this
phase
Figure
analysis,
(phase 1> k = I) is
I. The coordinate system
where
x
is
the
bulk
flow
direction, and z is perpendicular to the substratum, is also
described in this figure,.
analysis of this system
1984; 1986) yielding mass
Both theoretical and experimental
has
because
the
performed (Bakke et al.,
balances for substrate, cell, and
EPS (Table 2). These equations
study,
been
are used extensively in this
experimental
system
used
here
is
identical to that analyzed by
Bakke
et al. The same system
has
terms
of
also
been
situation m ->oo
analyzed
(Wanner
communication), yielding
film..
in
the
more, complex
and Gujer, 1985; Wanner, personal
information
on . gradients
in the
23
Sensitivity Analysis
Monoculture
biofilms
were
simulated
integration of the mass balances
4-th order Runge-Kutta routine
determine which parameters
in
on
are
by
simultaneous
Table 2 by a numerical
a VAX 11/750 computer to
more significant to biofilm
progression (Bakke et al., 1986). Kinetic and stoichiometric
coefficients for biofilm processes
al. (1984), Nelson et
al.
published by Robinson et
(1985),
and Bakke et al, (1984)
were applied in this biofilm simulation. It was demonstrated
in this
sensitivity
analysis
adsorption to the substratum
that,
is
of the process
(Bakke
quite
et
though cellular
a prerequisite for biofilm
formation, biofilm progression beyond
of biofilm formation is
even
the very early stages
insensitive to the magnitude
al., 1986). Adsorption processes
were, therefore, not accounted
for
in this study, reducing
the number of terms in the mass balances. The resulting mass
balances, including kinetic
and stoichiometric coefficients
for the processes, are presented in Table 3. Coefficient
values
for
determined
transformation
in
chemostats,
processes
applied
simulations are listed in Table I.
by
in
P.
aeruginosa,
the
computer
24
Figure I. Schematic representation of two phase biofilm
system
described
by
Equations
10-15. Bulk
transport, substrate diffusion,
cell and EPS
production, and detachment are processes included.
The coordinate system defined for this study is
illustrated.
A
OUT
BULK
'TRANSPORT
SUBSTRATE FLUX
DIFFUSION
TRANFORMATIO
LIQUID FLOW
SHEAR STRESS
/</
M DETACHMENT
BASE FILM
BIOFILM
. SURFACE
FILM
MIXING
BULK LIQUID .PHASE
25
Table 2. Mass balances for biofilm reactor with general
terms for process rates.
Liquid Phase Substrate (Eq. 4);
dC,
'SI
(CS0"CS1)D " CM2rS ™ CM1[-dt
YMS
+
J
YPS
Liquid Phase Cell (.5) ;
dC,
"Ml
(CM0 cMl)D + CM2rdM + CMlm “ ^MlraM
"dt'
Liquid Phase Products (6)?
dCT
'Pl
^cPO CP1>D + ^P2rdP + cMlrP
dt
Biofilm Substrate (7);
<3CS2
CM2rS " CM2fD (—
YMS
+ —
YPS
Biofilm Cell (8);
M2
CM2rdM + CM2fDm + CMlraM
dt
Biofilm Products (9) ;
dCT
'P2
"CP2rdP + CM2fDrP
dt
)
26
Table 3. Mass balances for biofilm reactor including kinetic
expressions for specific rates (Bakke et al.,
1984) .
Liquid Phase Substrate (10);
mHtcSl
d c ctd
kgP
knP
+ — — ) + -- ]
1
“ CM2rS “ cMl^------ (---KgS+CSl
^MS
YPS
YPS
Liquid Phase Ceill (11) ;
dC,
'Ml
mIncSl
-cMl0 + .CM2rdM
+ CM1
dt
KgS+CSl
Liquid Phase Product (12);
Pl
Vsi
CP1D + CP2rdP + cMl^ kgp(
dt
) + knP ^
KgS+CS1
Biofilm Substrate (13) ;
dCS,
'S2
mmCSl
1
0 = CM2rS “ CM2fD [ ------ • (—
^gS+cSl
Biofilm Cell (14);
dC,
'M2
YMS
kgP
knP
+ -- ) + ---]
YPS
mmCSl
CM2rdM + CM2fD
dt
KgS+CSl
Biofilm Product (15) ;
dC,
'P2
aM0Sl
CP2rai> + CM2fD^ kgP
dt
+ knP >
Kgs+Csi
27
Process Rates
This section explains
the
manipulations and assumptions
applied to the mass balances
measurable
expressions
for
Kinetics are expressed
(units
=
t
detachment
).
on
in
in
the
terms
Separating
biofilm
Table 3 in order to derive
individual
processes.
of specific process rates
the
effects
accumulation
of
is
growth
of
and
particular
interest.
A balance of specific rates
biofilm cell
mass
balance
is obtained by dividing the
(Equation
14)
by biofilm cell
mass, Cm2
I
dC M2
rdM + m2
CM2
16
dt
where the left
hand
side
(l.h.s.)
specific cellular accumulation rate
the specific cellular
detachment
of
in
Equation 16 is the
the biofilm. r^M is
rate,
m
is the average
■
specific cellular growth rate
in
the bibfilm. The specific
cellular accumulation rate can be obtained by measuring cell
mass with time.
Specific
/'
cellular
detachment
determined from the liquid
given liquid phase
rate,
r^M ,
can
be
phase cell balance (Equation 11)
substrate
mass
and
cell
mass in both
28
phases. The need for liquid phase substrate mass data can be
eliminated by supplying high . reactor, surface area to volume
ratio and liquid dilution
rate,
the liquid phase
neglected
can
be
D,
so that growth rate in
(Bakke
et al., 1984).
Solving Equation 11 for specific cellular detachment yields:
rdM
( D CM1
17
dcJoZdt )/CM2
At steady state. Equation 17 simplifies to:
rdM = D CM1/,CM2
Average specific biofilm cellular growth rate,
estimated from liquid
phase
can be
specific cellular growth rate,
mI 1
.19
fD mI
where f^ is an
effectiveness factor for substrate diffusion
(0 < fD <
which
substrate,
I),
substrate
depends
on
the
concentration,
diffusivity
and
biofilm
of the
density
(Atkinson and Davies, 1974). Values for m1 can.be calculated
after Monod (1940) ,
mI = mmCSl//(Kg S+cSl}
20
Y
29
given maximum
saturation
cellular
growth
coefficient,
concentration, Cg^. mm
rate,
g,
and
m ,
cellular growth
liquid
phase substrate
and Kgg for P. aeruginosa, are listed
in Table I.
An expression
biofilm
for
substrate
m2 . can
balance,
also
which,
be
obtained
due
to
from the
the
short
characteristic time for substrate diffusion in biofilms, can
be assumed
at
steady
state
(Hermanowicz and Ganczarczyk,
1985):
rS " knP//YPS
m2 =
---- —
*------'
.:
21
1^yMS “ kgP^yPS
where rg is specific
and
knp
are
growth
substrate
and
formation rate Coefficients,
stoichiometric yields
for
flux
into the biofilm, k
9"^
non-growth associated polymer
respectively.
products
substrate, respectively (Table I).
the liquid
phase
substrate
(EPS)
rg
balance
assuming negligible activity in
Ypg and
and
are
cells from
can be obtained from
(Equation 10), which,
the liquid phase and steady
state, yields:
'S = D acSI
where Acgj =
- Cgl
/ CM2
22
30
Biofilm Composition
To avoid contamination
system, the biofilm in
end
of
the
and
this
experiments.
physical
study
disturbance of the
was sampled only at the
Indirect
measures
of
biofilm
composition were, therefore, sought.
Rearranging Equation 18 yields:
.
23
CM2 ' CM1 D / rdM
Applying
the
same
assumptions
(steady
state
and
insignificant activity by the suspended cells) to the liquid
phase product balance:
CP2
CP1 D / rdP
According to Wanner and
Gujer
homogeneous biofilm at steady
show r^M = r^p (Bakke
24
(1984) ,
r ^ equals rdp in a
state. Experimental data also
et aI., 1984). Combining Equations 23
and 24, therefore, yields
CM2
CM1
25
CP2
CP1
31
The ratio of cells to polymers
in the.biofilm can, in other
words, be estimated from their
ratio in the liquid phase at
steady state. Equation 25 can
also serve as a good estimate
for non-steady state conditions
as long as the accumulation
terms are negligible compared to detachment rates.
An important
condition
for
coexistence
of particulate
species (e.g. cells and EPS) in biofilms at steady state (0.
Wanner,
personal
communication)
is
that
the
specific
production rates must be the same for all coexisting species
at the substratum. Therefore:
m = rp
where
m
as
a
at
function
z = 0
of
substrate
26
concentration
is
described by Equation 20 and specific product formation rate
can be described by (Bakke et al., 1984):
m +
m and (rp
tnp)
/ Ct
27
) are plotted vs. substrate concentration
in Figure 2 based on the
coefficients in Table I. So, given
Equation 26, the following inequalities emerge:
32
when
m >
^p2 / ^M2
CM2 > CP2
when
m < rp Cp2 / CM2
CM2 < CP2
28
and
where, from Figure 2,
and
29
for
0
inequalities
conditions
<
Inequality
Cg
relate
(i.e.
<
to
concentration). Note that
knp is rather
1984; Turakhia, 1986).
the
EPS
the
mass
on
These
metabolic
vs. substrate
value
for Cg (=
k^p , and the magnitude of
1983;
also
substratum then inequalities 28
to
ratio
transition
(Trulear,
Note
substratum.
composition
strongly
uncertain
28 is valid for Cg > 0.25
at
biofilm
cell
0.25 g m-^) depends
0.25
29
that
and
29
Robinson et al.,
if
Cg
= 0 at the
are not valid, and
from Equations 26 and 27:
30
at the substratum. Equation 30
cells at the substratum,
or
is satisfied if there are no
if
k
nP
0, as suggested by
Turakhia's (1986) data.
Simulation
of
experiments
published
by
Bakke
et al.
(1984) did not
_
content in
This
correlate well with data in terms of EPS
'
the biofilm (Wanner, personal communication).
suggested
that
the
EPS
production
coefficients
33
determined
by
inaccurate.
Robinson
Increasing
formation coefficient,
of
simulation
to
Turakhia, however,
different from zero,
results.
This
et
the
al.
(1984)
non-growth
significantly
data
found
an
apparent
(Wanner,
that
knp
(Table
I)
were
associated product
improved the fit
personal communication).
was
not significantly
apparent contradiction to Manner's
contradiction
is
analyzed
the
Discussion chapter in light of data obtained in this study.
34
Figure 2. Specific cellular growth rate, m, modeled as a
saturation function of substrate concentration and
product formation rate modeled as the sum of a
growth and a non-growth associated term, plotted
vs.- substrate concentration.
o 0.3
O 0.2
S u b s tra te
C o n c e n tr a tio n
(g m ~ 3 )
35
EXPERIMENTAL METHODS
Methods were developed
density
data
obtain biofilm thickness and
non-intrusively . and
biofilm reactor was
which are
to
constructed
described
in
non-destructively.
A
to .accomodate the methods
detail
in
this
chapter. Methods
previously published are referenced.
Experimental Apparatus
The experiments were
conducted
tube reactor (MRTR) described
Tables 4 and
5.
Figure
3
in
is
in
a mixed rectangular
Figures
a
3, 4, and 5, and
schematic diagram of the
entire experimental apparatus describing liquid and air flow
and temperature control. The reactor (Figure 4) is a recycle
loop
with
air
and
effluent. The air
liquid
travels
inflow to the outflow ports
tube loop to avoid an
inflow
the
a combined
distance from the
additional (gas) phase in the system.
to the water, mixes the liquid
transports
port and the effluent
shortest
and
and not through the rectangular
The air flow supplies oxygen
phase, and rapidly
ports
port.
recycle flow through the
liquid
A
between the influent
peristaltic pump drives the
rectangular
(pyrex) tubes. One of
these tubes is equipped with a manometer to measure pressure
36
drop
due
to
friction
at
the
biofilm-liquid
interface.
Samples were obtained at locations labeled I through 8.
The rectangular tubes were constructed from a square and
a rectangular
capillary
tube
section view (Figure 5).
as
The
fastened to the center of
described
by
the cross
capillary tube was sealed and
the
square tube by silicone glue
at both ends, creating
two parallel rectangular channels as
the liquid phase *
rectangular channels are 1.9x5.0x300
mm. The
sampling
The
coordinate
system
locations
for
is
defined
biofilm
labeled along the y-axis.
in
thickness
Thickness
Figure
5, and
profiles
are
measurements (in the z
direction) were obtained at y = -1.00, -0.75, ..., 1.00 mm.
Characteristic dimensions and
parts
description for the
MRTR are listed in Tables 4 and 5.
Operating Conditions
Both bulk liquid
flow.rates)
nutrient,
and
transport
liquid
phase
temperature,
controlled to
maintain
buffer
imposing the
was
fluid
(i.e. mixing and
composition
etc.)
(i.e. influent
were
carefully
constant
environmental conditions.
during
an experiment was recycle
The only parameter varied
flow rate, which
conditions
varied
shear
as
force
a
step function in time,
progressions described in
Figures 6, 7, and 8 for the three specific experiments.
Figure 3, Schematic diagram of experimental system including
flow and temperature control for gas and liquids.
38
Figure 4. Mixed rectangular tube reactor (MRTR). Biofilm
measurements
and
samples
were
obtained at
locations labeled .1-8.
Nutrient
Solution
Manometer
Rectangular
tubes
Recycle line
39
Figure. 5. Cross section view of rectangular tube. A-B-C-D
are light paths for biofilm thickness and optical
density measurements. Scale 20:1 (Biofilm not to
scale). Biofilm thickness profile measurements
were taken along the y-axis at marked positions
.25 mm apart.
y A (mm)
BULK
BULK
LIQUID.
LIQUID
PYREX
I mm,
•
V
40
Table 4. Dimensions of the MRTR
Wetted Surface
Area [ m m
2
J
Rectangular tubes ..........16500
Recycle tubes and
+ Mixing chamber ...... .......3340
= Total
19840'
(~
0.02 m2)
Liquid Volume
[ mm
3
]
Rectangular tubes ......... .11400
Recycle tubes ...............1600
+ Mixing chamber ............. .5000
= Total ....................
==>
18000
(=
18 ml)
Surface area to volume ratio, A/V = 1100 m~^
41
Table 5. Reactor components and dimensions
Descriotion
Catalog #
Square Glass
Tubes
Wale Apparatus
S-105
. 5x5x300
inside dim.
Rectangular
Glass Tube
Wale Apparatus
RT-2540
1.2x4.8x300
Recycle tubing
(nylon)
Recycle pump,
peristaltic
Recycle pump
tubing, silicone
.
Dimensions fmm)
Outside dim.
I.D. 4
Cole-Parmer
WZIRO57
Masterflex
6411 - 13
I.D. I
42
Figure
6.
Fluid shear
stress
progression
Continuous flow operation started
Experiment
in E x p e r i m e n t
at t i m e zero.
I
700
Time (h)
I.
43
F i g u r e I.
Fluid shear stress
progression
in E x p e r i m e n t II.
C o n t i n u o u s f l o w o p e r a t i o n s t a r t e d a t t i m e zero.
Experiment Ii
o .12
I
OL
I
I
I
I
I
I
*1
E .04
I
I
------------------- — —
-50
I --------------—
I------------------------------ 1 - —
100
150
Time (h)
200
250
300
44
Figure
8.
Fluid shear stress
p r o g r e s s i o n in E x p e r i m e n t III.
C o n t i n u o u s f l o w O p e r a t i o n s t a r t e d at t i m e zero.
Experiment III
100
150
Time (h)
200
350
45
Recycle rate, r, was varied
at the
liquid-biofilm
interface.
mixing characteristics of the
since it creates more
for
a)
rate/recycle
which
establish
rate,
was tested to determine the minimum
liquid
phase - substrate transport
b)
The
steady.
monitor
7.7, 6.4, 4.0 ml/min. and
is
test
state
reducing recycle rate as a step
function of r
r also changes
system, an undesirable effect
limitations were insignificant.
follows:
Changing
variables. Liquid phase mass transfer
rate as a function of r
recycle rate
to change fluid shear force
was conducted as
at
a
substrate
high
removal
mixing
while
function in time ( r = 9.0,
t = I h ), Substrate removal as a
presented
in
Table 6. Reduced substrate
removal implies liquid phase (external to the biofilm) mass
transfer limitations. Reduced mass
detected at r
=
4
ml/min.
The
transfer in the MRTR was
minimum
experiments was, therefore, chosen
safety factor
of
5
in
terms
of
r applied in the
at 20 ml/min, allowing a
avoiding
mass transfer
limitations.
Table 6. Fraction of substrate removed (reported as mean ±
standard deviation of four samples) at various
recycle rates in liquid phase mass transfer study
of MRTR.
r
(ml/min)
:
^cSO-cSl ^ cSO :
±
9.0
7.7
6.4
4.0
0.872
0.877
0.011
0.859
0.013
0.841
0.006
0.020
46
Liquid flow rate, F, was set
ml reactor volume,, results in
3 h
. The minimum r
the MRTR
a
to
continuous
at I ml/min which, for a 20
a liquid dilution rate, D , of
F ratio was, therefore, 20, making
flow
stirred
tank reactor (CFSTR)
(Characklis et al., 1986) .
Influent liquid composition
that
in
except
annular
that
reactor
(Table
experiments
substrate
Ca
was identical to
by
Turakhia (1986)
concentration
concentration were maintained at 20
2+
7)
and
calcium
g m-^ , as carbon and as
, respectively.
Experimental procedure
The
experiments
were
initiated
recycle rate according to schedule
a sterile system containing
of a stationary
state
through the effluent
P.
port
operation of the reactor
by
establishing
the
(Figures 6, 7, and 8) in
influent liquid. One milliliter
aeruginosa
of
the
culture was injected
MRTR, followed by batch
till stationary state was reached.
Stationary state was reached when optical density stabilized
at sample locations 1-8 (Figure 4) .
, was then
established
monitored. Recycle
appeared
stable
rate,
and
r,
Dilution rate, D = S h ' 1
progression of the experiment
was
(pseudo-steady
changed
state)
when the system
based
on
biofilm
thickness, biofilm absorbance, and effluent absorbance data.
47
Table 7. Composition of Nutrient Solution.
Constituent
Influent Concentration (g m
Glucose
20.0
NH4Cl
MgSO4 7H2O
Na2HPO4
(Buffer)'
7.2
2.0
213.0
KH2PO4
204.5
(Buffer)'
CaCO3
(HOCOCH2)3N
50.0
0.40
(CK4)6 HO7O24 4H20
0.001
FeSO4 7H20
ZnSO4 7H20
MnSO4 H2O
CuSO4 SH2O
Na2B4O7 IOH2O
0.112
0.10
0.008
' pH = 6.8
0.002
0.001
—3
)
48
The liquid effluent was
cell count, and
sampled for substrate, SOC, TOC,
absorbance
throughout the experiments. The
biofilm was sampled for cell count and TOC at the end of the
experiments only,
while
biofilm
were measured throughout.
thickness
Biofilm
and absorbance
absorbance, biofilm cell
samples, and biofilm thickness measurements were obtained at
positions labeled in Figures 4 and 5. Biofilm cell, TOC, and
electron microscopy samples were obtained from the capillary
tube (Figure 5) which was
in I cm
long
removed from the reactor and cut
2
(I cm ) placed directly into 2 %
samples
formaldehyde for cell count,
2% gluteraldehyde for electron
microscopy, and in TOC vials for TOC measurements.
The various
(Figures
6,
schedules
7,
and
transitions between
chosen
8)
r,
allow
2r,
for
testing
and
Sr,
information regarding the influence
rate (i.e.
recycle
history)
rate.
reproducibility
on
The
the
all
possible
of the previous recycle
also
bio-films.
significance, first recycle rate
of
potentially yielding
behavior
design
between
varying recycle rate
for
during
allows
If
biofilms
the present
testing
history
of
has
2 and 3 are
the only actual replicates.
Analytical methods
Analytical methods
Robinson et al., 1984;
previously
Bakke
published (Trulear, 1983;
et al., 1984; Turakhia, 1986)
49
are listed in Table
8.
New
methods for biofilm thickness,
refractive index, and in situ biomass determination by light
scattering are described below.
Biofilm thickness
Biofilm thickness
measurement
by
light
microscopy has
previously been applied in biofilm studies to determine film
thickness of samples removed ' from biofilm reactors (Trulear
and Gharacklis,
1985). It has,
1982;
Trulearf
however,
1983;
erroneously
Shieh
and Mulcahyf
been assumed that the
vertical displacement of the sample measured by the vertical
stage
micrometer
of
an
optical
vertical distance between
and
the
biofilm-substratum
differences in
etc.,
the
unity,
and
specimens.
indices
proportionality
in
general
Bennett
and
thickness.
Biofilm
for
constant
biofilm
will
vary
focus.
the
for
(1967)
Due
to
air, water, glass,
between
thickness
Bennett
phenomenon as the difference
equals
two Surfaces (biofilm-liquid
interfaces) . in
refractive
stage displacement and
microscope
is
the vertical
not equal to
different biofilm
referred
to this
between optical and mechanical
. thickness
measurements
by
light
microscopy reported in previous studies are, in other words,
apparent or optical thickness
thickness.
and not the actual mechanical
50
Table 8. Analytical methods applied.
Component
Method
References
Cm , Cell Mass
Epifluqrescent
Paul, 1982
Direct Count,
Hoechst Dye 33258
. Bakke et al., 1984
Robinson et al., 1984
cS' Substrate
Mass
Sigma 510 Glucose
Analysis Procedure
Trulear, 1983
TOC & SOC
Oceanography Int.
Bakke et al., 1984
RobinSon et al., 1984
Carbon Analyzer
POC
( TOC - SOC )
Bakke et al., 1984
Robinson et al.,1984
CP' EPS Mass
( POC - Cm )
Bakke et al., 1984
Robinson et al., 1984
Ap ,
pressure
drop
manometer
51
To obtain a
relationship
biofilm thickness, a
between optical and. mechanical
geometric
from the lamp through
lens was performed
•
the
biofilm
(Bakke
and
displacement of the
biofilm
the
interface
biofilm-liquid
analysis
of the light path
sample to the objective
Olsson, 1986) . The vertical
sample
to
required to focus from
the
biofilm-substratum
interface, measured by the vertical stage micrometer, is the
optical thickness,
z^.
The
mechanical
thickness, L^, can
then be calculated as:
where n^ = biofilm refractive index
n
9
= refractive index of the medium interfacing the
film between the film and the objective lens.
(n = 1.474 for the glass used here).
9
Biofilm
thickness
readings
obtained on samples removed
in
previous
studies
were
from the biofilm reactor, which
cause disturbance of the reactor.
The reactor in this study
had transparent (pyrex) walls
that the light microscope
could be focused on any
so
plane
obtain biofilm optical thickness
the biofilm. The
optical
in the reactor and, thereby,
in situ without disturbing
thickness
was
determined as the
52
vertical distance required to move
the focal plane from the
liquid/biofilm interface to the biofilm/substratum interface
(e.g. from B to C
in
along the
(perpendicular
y-axis
Figure 5). Biofilm thickness profiles
to
direction) were obtained at sample
described in Figure
5.
the
bulk liquid flow
position 4 (Figure 4) as
Mechanical thickness was calculated
from the optical
thickness
This calculation
require
data
according to Equation 31.
knowledge
of
biofilm refractive
index, n^, which, due to the high water content of biofilms,
can be assumed equal to that of water (n^ = I.33) (Bakke and
Olsson,
1986).
Small
variations
composition changes are, however,
determination of biofilm
in
n^
due
to
biofilm
possible. Two methods for
refractive
index were, therefore,
also applied.
The
reactor
(MRTR)
was
thickness by the eyepiece
designed
to
measure
biofilm
micrometer from a cross sectional
view of the biofilm in the
x-z
plane (focal plane // AD in
Figure 5). This method yields mechanical thickness directly,
but is not very sensitive. Biofilm refractive index, n^, was
then
determined
from
Equation
mechanical thickness data. Values
directly by placing
biofilm
the end of the experiments.
31
based
on
optical and
for n^ were also obtained
samples
on a refractometer at
53
Optical Density
Light scattering by
biomass
non-destructive,'in
situ
biofilm and in the
liquid
was
measure
of
as an indirect,
biomass
both in the
phase. Optical density, measured
as absorbance, is proportional
to concentration of bacteria
(analogous to Beer-Lambert law)
and shape (Koch, 1970; 1984).
vs. absorbance were
measured
within fixed ranges of size
Standard curves for cell mass
generated
for
both biofilm and liquid
phase data.
Liquid phase absorbance was
spectrophotometer and biofilm
measured
absorbance
colorimeter (Sybon/Brinkman PC801)
probe, both operated at wave
path from the probe,
through
with
length
the
I
on a Varian DMS 90
was obtained by a
a fiberoptic light
= 420 nm. The light
reactor, off the mirror,
and back through the reactor to the probe is shown in Figure
5 (A - D - A) .
Statistical Methods
Linear
regression
concentration to light
was
used
absorbance
function correlated TOC standards
to
correlate
cell
mass
data, while a saturation
to TOC readings according
to the BMDP statistical software package (BMDP, 1983).
54
RESULTS
A comprehensive listing of all
Appendix. Cellular, polymer and
raw data is found in the
substrate data are reported
as carbon equivalents. Transitions in fluid shear stress are
labeled by arrows indicating
magnitude and direction of the
change in figures showing progression of variables.
Data Correlation
Biofilm Refractive Index
Biofilm thickness
was
light microscopy. Biofilm
as the distance.between
liquid
and
the
measured
optical
the
by
focal
planes for the biofilm-
biofilm-substratum
thickness
methods using
thickness was determined
■
actual mechanical
two
interfaces,
while the
L
was
measured
directly by the
eye-piece micrometer. By comparing measurements from the two
methods,
biofilm
refractive
index
was
calculated
from
Equation 31 based on 20 samples from Experiments II and III:
nf = 1.33 ± 0.17
Biofilm refractive index
was
also measured directly on
.55
biofilm samples removed from
of the experiments with
and
2
samples
a
from
the reactors at the completion
refractometer
Experiments
(8 samples; 4, 2,
If
IIf
and
IIIf
respectively):
nf = 1.348
± .013
These biofilms had refractive indices close to that of water
(nw = 1.333) , as expected, due to their high water content.
Light Scattering vs. Biomass
■ ■
Light
scattering
■
data
absorbancer was correlated
at
with
1=450
nmf
reported
as
biomass in both suspension
and in the biofilm. Biofilm mass data were only available at
the end of the
are
based
experiments, while liquid phase correlations
on
data
cell
mass
throughout
three
experimental
progressions.
Biofilm
measured
as
is
absorbance,
plotted
in
vs.
light scattering,
Figure . 9.
Linear regression
yields (r =0.89)
CM2 = 2.072 AbS. . (g nf2)
The
highest
cell
mass
regression because it
was
reading
was
excluded
approximately
.
from
this
twice a s .large as
56
total
biofilm
mass
(TOC)
at
this
location,
which
is
impossible (i.e. an outlier).
Effluent cell mass
is
plotted
vs.
light scattering in
Figure 10. Linear regression yields (r^=0.94)
-
CM1 = «095 AbS
(g m-3)
Progression of Biofilm Variables
■
Progression
presented
in
of variables
this
section
-
X
measured
and
through
analyzed
to
time
are
determine a
boundary condition for detachment.
Biofilm Thickness
Progression
of biofilm
Experiments I, II, and lit
and
13,
thickness,
Lf,
data
for
are presented in Figures 11, 12,
respectively.
Biofilm
approximately 35 micrometers
within
thickness
reached
24 hours of continuous
flow operation in all experiments. Average biofilm thickness
remained more or
Average
less
constant throughout the experiments.
standard deviation
and III were 37 + I (216),
of
Lf
for Experiments I, II,
38.2 ± 0.6 (161), and 32.2 ± 0.5
(161), respectively (number of samples in parenthesis).
57
Calibration curve for biofilm cell carbon areal
density vs. light scattering by the biofilm
measured as absorbance at wave length = 450 nm.
Line represents best linear fit (Rz=0.88).
film Cell Carbon (g m 2)
Figure 9.
.05
,
.1
Biofilm Optical Density
.15
.2
(Absorbance)
58
Figure 10
Calibration curve for liquid phase cell carbon
concentration vs. light scattering in the liquid
phase measured as absorbance at wavelength = 450
nm. Line represents best linear fit ( R = 0 .94).
Effluent Optical Density ( Abs. )
59
Figure
11.
Biofilm thickness
progression
Experiment
Time (h)
I
in E x p e r i m e n t
I.
60
Figure
12.
Biofilm thickness
progression
in E x p e r i m e n t
II
E x p e r i m e n t Il
350
Tim e ( h )
61
Figure
13.
Biofilm thickness
progression
Time (h)
in E x p e r i m e n t
III.
62
Optical .biofilm thickness data
were reported by Trulear
(1983) for monoculture
P.
aeruginosa biofilms in turbulent
reactors at more
an
order
than
shear stress than in the present
of substrate loading
rates.
study and for a wide range
This
mechanical thickness according to
equal 33 — 11 mm
(mean
of magnitude higher fluid
data was translated into
Equation 31, and found to
±_ standard
deviation
for 20 data
points from six experiments reported by Trulear). Therefore,
biofilm thickness is
relatively
biofilms within a wide
range
constant for P. aeruginosa
of physical and physiological
conditions tested. The fluid dynamic range tested by Trulear
and in this research was
3.5 Pa, and
a t
<
0.08
laminar
and turbulent, 0.04 < r <
Pa. Substrate concentrations ranged
from Cso = 2 0 g nf3 (IOKgg)
to Cgl = 0.2 g nf3 (O1IKgg) and
the substrate loading rate range was
m ^ h
Since biofilm
interface and it is
appropriate boundary
0.02 < DCJV/A < 0.34 g
thickness defines the biofilm-liquid
constant
condition
in
time,
it can serve as an
linking
biofilm and liquid
phase mass balances in the simultaneous integration of these
equations (Table 3).
Light Scattering by Biofllms
Light scattering by- the
cell areal density,
biofilms, correlated to biofilm
increased
significantly throughout the
experiments as illustrated for
Experiment III in Figure 14.
63
Since biofilm thickness was constant, this change was due to
changes in the
biofilm
cell
increased light scattering
volumetric density. Thus, the
within
the
biofilm
was due to
changes in biofilm composition.
Fluid shear stress
Fluid shear stress acting
phase flow
was
Figures 6 - 8 .
monitored
No
ort
and
changes
the
biofilm due to liquid
progressed
in
pressure
as described in
drop due to other
factors than recycle rate were detected. The biofilm did not
impose a detectable
drag
force
on
the
recycle flow. The
method was, however, insensitive
to
changes in fluid shear
stress below 0.02 Pa.
Progression of Bulk Liquid Phase Variables
Substrate Concentration
Progression of
substrate
concentration
in the reactor
effluent, Cgl, for Experiment III is presented in Figure 15.
Progression
trend.
A
for
rapid
observed at the
the
other
decrease
same
time
experiments,
in
substrate
showed
a similar
concentration was
as biofilm thickness increased.
Effluent, substrate concentration
did,
however, not reach a
constant level, as biofilm thickness did. Substrate
64
Figure 14.
Progression of biofilm optical density, measured
as absorbance at locations 1-8 (Figure 4), in
Experiment III.
Experiment III
K **
150
Time (h)
o
8
x
7
V
6
*
5
O
4
•
3
A
2
B
I
65
Figure 15. Progression
of
liquid
phase
influent,
substrate
concentration, Cg ( x
-
□ - effluent
), in Experiment III.
Data points represents the
average of two samples.
Experiment III
. ISO
Time ( h )
300
66
concentration is plotted on a natural, logarithmic (In) scale
to better illustrate this point. .
Cell Concentration
Effluent
constant
cell
throughout
concentration,
the
shear stress transitions
cm i '
experiments
as
remained
except
illustrated
in
quite
during fluid
Figure 16 for
Experiment I I I .
Extracellular Polymeric Substances (EPS)
Progression
presented
as
of
EPS
EPS
in
fractions
Experiment III. This figure
the
effluent
of
POC
also
POC.■It appears from this figure
remains
relatively
constant
in
with
time
Figure
17
is
for
includes cell fraction of
that the EPS to cell ratio
at
0.4
±. 0.2,
(average -
standard deviation of 22
samples) throughout the experiment
in the liquid phase. The
cell
to EPS ratio in the biofilm,
therefore, also remains constant according to Equation 25.
67
Figure 16. Liquid
phase
cell
mass,
C^,
calculated from
light scattering data, in Experiment
Experiment Il
O
10 -
<v 5 -
150
Time
( h )
II.
Figure 17. Liquid phase cell ( □.) and EPS ( x ) fraction of
particulate organic carbon
(POC) in the effluent
in Experiment III.
Experiment III
Call
EPS
Lu
X X
Time (h)
69
DISCUSSION
A
major
objective
of
these
experiments
was
to
quantitatively estimate accumulation, growth, and detachment
in the biofilm balance
(Equation 16). The estimated process
rates
identify
were
used
to
and
analyze
factors
of
significance to biofilm detachment.
Biofilm Detachment
Specific Cellular Detachment Rate
Specific cellular
detachment
from the cell concentration
rate,
rdM, was calculated
measurements
experiments according to Equation 17. rd
from
the
same
equation
based
on
mass,
is
detachment
presented
rate
based
in
is
establishment of steady
were also
returned to
observed
its
18.
observed
to
state
of r^^ in which only
A
peak
coincide
in
cellular
with
the
for biofilm thickness. Peaks
the
pre-transition
liquid residence times).
scattering data
on direct measurement of cell
Figure
during
was also estimated
light
(correlated to cell mass). Progression
the last data point is
at the end of the
Specific
flow
transitions, but rdM
magnitude
within hours (5
cellular detachment rate
70
was f in other
word,
influenced
stress, but not by the
by
changes in fluid shear
magnitude of the fluid shear stress.
It was therefore concluded that
Equation I is not valid and
can not serve as
condition required to model
the
boundary
detachment. Constant biofilm thickness, on the contrary, may
serve as an accurate boundary
condition to link the biofilm
and the liquid phase mass balance in.biofilm models.
Biofilm Thickness
The observation that P.
biofilm
thickness
importance
to
of
approximately
biofilm
biofilm-liquid
aeruginosa has a characteristic
modeling,
interface
distribution of biomass
biofilm reactor
is
it
thereby
the
simulation
pm
because
and
in
35
of
great
defines the
the
spatial
biofilm reactor system. In
by
simultaneous integration of
the mass balances for the system, constant biofilm thickness
can serve as
the
boundary
transport from the biofilm
condition
to
for particulate mass
the liquid phase required to
account for detachment. Constant biofilm thickness may be an
accurate
boundary
significantly
with
condition
time
because
even
though
physiological conditions changed.
was not
influenced
loading rates,
and
by
biofilm within the conditions
did
both
not
change
physical
and
Average biofilm thickness
substrate
physical
it
concentration, substrate
shear
tested
forces .acting
on the
in this and Trulear's
71
Figure 18. Progression of specific cellular detachment rate,
determined according
to
Equation
17 from light
scattering data.
Experiment Il
I
iso
Time
( h )
A t
72
(1983) study (0.02 < DCgV/A < 0.34 g m 2 h 1 , 0.2 < Cg < 20
-3
g m
, laminar and turbulent liquid phase, and 0.04 < x. <
3.5 Pa). Fluid shear stress
transitions
(Ar = 0.08 Pa) did
not have a detectable influence on biofilm thickness either.
P. aeruginosa biofilms
have
a characteristic thickness
which suggests a very regular and specific biofilm structure
not influenced by metabolic conditions within the biofilm or
by
external
information
forces
acting
regarding
Electron
of
which
contention
is regular,
specific
cells
a
distributed
biofilm.
of
biofilm
Lacking
polymers
factors regulate biofilm
micrographs
however, support the
i.e.
the
composition
precludes determination
thickness.
on
(Figures
19
-
23)
do,
that the biofilm structure
polymeric
throughout
the
strand connecting
biofilm
matrix
is
responsible for the structural integrity of the biofilm. The
electron
micrographs
illustrate
(32 nm) polymer bundles
(maybe
together in the biofilm
(Figures
polymer coils, they may
be
that
a
coils)
20
network
of thin
are tying the cells
and
21). if they are
protein structures, since small
amounts of proteins are found in EPS from P- aeruginosa (see
Literature
Review
chapter).
micrographs (Figures 22 and
The
transmission
electron
23) illustrate that the stringy
EPS constitute only a small fraction of the total EPS. It is
the matrix of cells and
thickness,
and
not
polymers which has a characteristic
the
complicates the search for
polymers
the
per
se.
This
clearly
limiting factor for biofilm
73
Figure
19.
Scanning
electron micrograph
the completion
of Experiment
(SEM)
III.
o f b i o f i l m at
74
F i g u r e 20.
Scanning
the
electron micrograph
completion
of
Experiment
(SEM)
III.
of b i o f i l m at
75
Figure
21.
Scanning
electron micrograph
the completion
of
Experiment
(SEM)
III.
o f b i o f i l m at
76
Figure
22.
Transmission
at
electron micrograph
the completion
of E x p e r i m e n t
(TEM)
III.
of b i o f i l m
77
Figure
23.
Transmission
electron micrograph
at the c o m p l e t i o n
of Experiment
(TEM)
III.
of b i o f i l m
78
thickness. It
is
not
even
limiting factor(s) may
following questions
be
may
clear
at
this
determined,
lead
biofilm structure: a) what is
to
point how the
but
better
answers to the
understanding of
the polymer composition ?
b)
since calcium concentration transitions influence detachment
(Turakhia et al., 1984),
calcium availability ?
characteristic
is biofilm thickness influenced by
c) do other bacterial species have a
biofilm
thickness
?
d)
do
mixed culture
biofilms have characteristic biofilm thickness ?
These questions can
be
addressed
with
the method for
biofilm thickness determination developed in this study. The
method also
permits
a
detailed
analysis
of
the biofilm
liquid interphase characteristics, or biofilm roughness.
Biofilm Roughness
Biofilm-Iiquid
time
even
interface
though
characteristics
average
biofilm
changed with
thickness,
Lf,
was
constant. In the early stages (first week) the interface was
Very smooth (Figures 24 and
25). A rougher interface with a
patchy appearance and
cracks
and 27)
developed
deviation in
channels
are
deep
with
time,
(Figure 28).
frequently
(Figures 20, 26, and
27),
causing
It
quite
so
or channels (Figures 26
increased standard
should be noted that these
deep
that
(O.5L^)
the average
but
narrow
and the
total biofilm volume are not significantly influenced by the
79
Figure 24. Optical
photo
interface at
50
micrograph
hours.
of
biofilm-liquid
Corresponding to Figure
80
Figure 25. Biofilm thickness profile
along the y-axis (i.e.
perpendicular to bulk . liquid
flow direction) at
50 hours. Corresponding to Figure 24.
50-i------------------------------- — ---------------------------------------- -------------- ----------------:
E
Distance from Center of Tube, y ( mm )
81
roughness. This change in interface morphology may be due to
hydrodynamic interaction between
biofilm
and
enhances
interface
advantage to the cells
uptake with time
15)
was
Increasing
surface area of
transport
phase and the
which
is
an
supports the contention that
improved
Interface roughness may create
transport.
liquid
in the biofilm. Increasing substrate
(Figure
interface transport
the
by increasing roughness.
local turbulence and enhance
roughness
biofilm
will
exposed
shortens diffusion distances
to
from
also
the
the
increase
the
liquid phase and
liquid phase to the
deeper biofilm layers. Alternatively, the changing roughness
may be regarded as
a
and less base film
(see
denser and. more
transition
Figure
efficient
at
towards more surface film
I). The biofilm is growing
substrate
removal while its
interface morphology is obtaining a rougher appearance.
Scanning electron micrographs (SEM) of the biofilms were
also obtained to
investigate
on a smaller scale. Roughness
26 is apparent
in
the
included to demonstrate
on a cellular
scale
on
electron
the
optical photo-micrographs and
the system. Higher
the interface characteristics
micrograph
in Figure 19,
correspondence between JLn situ
SEMs
magnification
(Figure
a similar scale as Figure
20
of samples removed from
SEMs illustrate roughness
and
21).
Note that high
magnification SEMs have a short depth of field, exaggerating
the apparent roughness.
82
Figure 26. Optical
photo
interface at 272
27.
micrograph
hours.
of
biofilm-liquid
Corresponding to Figure
83
Figure 27. Biofilm thickness profile
perpendicular to bulk
along the y-axis (i.e.
liquid
flow direction) at
film Thickness (pm)
272 hours. Corresponding to Figure 452.
-1.00
-0.75
-0.50
-0.25
0
0.25
0.50
0.75
Distance from Center of Tube, y ( mm )
1.00
84
EiIgure 28. Progression of standard deviation of nine biofilm
Standard Deviation in Lf ( jjm )
thickness samples in Experiments II and III.
o ..
X
150
Time ( h )
2
P
250
85
Biofilm Composition
t.
The biofilms in this
study
consisted of cells and EPS.
The relative magnitudes and distribution of cells and EPS in
the
biofilms
were
investigated
to
determine
their
significance to biofilm structure and detachment.
The EPS to cell mass
constant at about
17),
suggesting
increased with
0.4
throughout these experiments (Figure
that
time
ratio in the liquid phase remained
EPS
mass
proportional
according to Equation 25.
EPS
of
this
The
are fluid dynamic
major
here than
(1983).
the
Note,
determination
mass density
in other word, a
and EPS mass for the
is
compared
(1983;
the
and
to biofilm
Bakke et al., 1984)
state"
however,
that
been
biofilm
age. The EPS to
end of the experiments reported
"steady
has
bibfilm
differences between these studies
conditions
cell ratio is lower at
the
cell
was,
cell
study
composition observed by Trulear
in Figure 29.
in
throughout these experiments.
Biofilm composition in terms
in
with
mass
significant biofilm component
three experiments
density
ratios
the
improved
reported by Trulear
method
by
for
cell
mass
computerized
image
analysis since Trulear*s experiments.
Transmission and scanning
SEM)
of
the
films
were
experiments (SEMs; Figures
23)
to
further
electron micrographs (TEM and
obtained
19-21
investigate
and
biofilm
at
the
end
of
the
TEMs; Figures 22 and
composition.
These
86
Figure 29. Measured cell and EPS fractions of biofilm carbon
(POC) in a) this
study,
experiments.
Cl
STUDY
and b) Trulear's (1983)
87
electron
micrographs
show
throughout the biofilms.
different
EPS
a
dense
They
components,
also
one
packing
of
cells
show two distinctively
forming
a
capsule around
individual cells and the
other stringy EPS inter-connecting
cells. These EPS strings
are
considerably smaller
than
33
nm
flagella.
in diameter, which is
Their
composition is
unknown. Estimates from several TEMs suggests a volume ratio
of capsular EPS to EPS strings
TEM in Figure 23 illustrates
of at least one hundred. The
that the EPS strings penetrate
the capsular EPS, suggesting that the stingy EPS is the main
contributor to
serve some
biofilm
other
structure,
purpose(s).
while
The
the capsular EPS
capsular
EPS
is found
between the cell and the environment, which suggests that it
serves as a
diffusion
surroundings.
Effects
detachment may yield
regulator
of
between
various
further
the cell and its
transitions
clues
on biofilm
regarding the roles of
these groups of EPS in biofilm structures.
Transitions
Effects : of
transitions
in
substrate
loading
rates,
calcium concentration (Literature Review chapter), and fluid
shear
stress
analyzed to
(this
further
study)
investigate
structure. Turakhia et al.
available in
biofilm
on
(1983)
reactors
by
biofilm
roles
of
detachment
were
EPS in bipfilm
reduced the free calcium
chelation, and observed
88
immediate biofilm cell and EPS sloughing. Fluid shear stress
transitions in this study also caused immediate sloughing of
both cells and EPS.
stress
and
These
calcium
stringy EPS bonds,
observations suggests that shear
transitions
since
these
both
EPS
cause
strands
breakage
of
appear to be
responsible for biofilm structure. Substrate transitions, on
the
other
hand,
influencing
stimulated
cellular
detachment
observation can be explained
two functionally
by
different
flux transitions had
EPS
a
detachment
(Bakke,
without
1983) .
This
the separation of EPS into
groups,
significant
as
follows: substrate
impact on the capsular
EPS, since it serves as a diffusion regulator or buffer zone
between the cell and the . environment, while the stringy EPS
which ties cells
together
is
conditions such as substrate
insensitive to physiological
transitions (also discussed in
terms of biofilm thickness).
Transient biofilm behavior does, in other words, support
the proposed separation of EPS in two functionally different
groups. I)
Capsular
individual
cells
influenced by
Stringy
EPS
and
flux
is
EPS
serve
their
transitions
the
integrity of biofilms.
main
as
a
surroundings
(e.g..
contributor
Calcium
element in these EPS strands.
buffer zone between
appear
and
can
be
substrate flux). 2)
to
to
the structural
be an important
89
Biofilm Aging
Biofilm optical density, measured as absorbance, changed
dramatically (by an order Of
magnitude) during the 15 to 23
days of essentially constant biofilm thickness. This implies
major
changes
density.
in
Other
biofilm
factors
composition
such
distribution, may also have
and their significance
are discussed here.
as
in
EPS
terms
of mass
composition
and
changed. Clues for such changes
to
biofilm detachment and structure
Progression of biofilm-liquid interface
characteristics are discussed in more detail in the "Biofilm
Roughness" section.
Certain
observations
suggest
that
EPS
composition
changed with time in these experiments. Visual inspection by
light
microscopy
supplied
the
transitions. During the first
the biofilm appeared
to
be
cells scattered throughout.
consist of more cells
apparent.
This
distributed,
low
structures. Transmission
(TEM and SEM) support
TEMs of a few
days
a
and
for
such
continuous polymer gel with
the
that
density
clue
few days of reactor operation
Later,
while
suggests
initial
the biofilm appeared to
polymer gel was no longer
the
gel
EPS
to
changed
denser,
from
a
localized
scanning electron micrographs
this interpretation. Trulear's (1983)
old
distributed throughout the
here for fifteen days old
P. aeruginosa biofilms display EPS
biofilm,
while in TEMs obtained
biofilms EPS appear to be limited
90
to a dense capsular layer
close
(33 nm) EPS strands (sample
capsular EPS
is
also
transition in EPS
preparation was identical). The
seen
in
composition
distributed gel (referred to
by Brock
to the cells and long thin
(1979)),
to
Trulear's
is,
as
dense,
in
TEMs.
The slow
other words, from a
the slime component of EPS
stringy
EPS
stands. It is,
therefore, proposed that a polymer gel supply the structural
integrity
of
formation.
the . biofilm
Stronger
taking over the
during
polymer
structural
the
strands
early
develop
responsibility
stages
with
of
time,
in the biofilm,
which allows for a denser packing of cells while maintaining
a significant liquid
phase
for
diffusion of substrate and
other dissolved substances within the biofilm.
Data published by Christensen
contention
according
changed
that
to
its
bacteria
adjust
environmental
EPS
their
conditions.
composition
conditions occured.
et al. (1986) support the
Metabolic
when
EPS
Their pseudomonad
changes
conditions
composition
in
may
metabolic
have been a
regulating factor in the present study also, since substrate
availability per cell decreased with time.
To summarize, biofilm
thickness
measured which does not change
early stages of
roughness
is
the only parameter
with biofilm age, beyond the
biofilm formation. Biofilm-liquid interface
increased
with
increased both in terms of
experiments, while the ratio
time.
Biofilm
mass
density
cell and EPS mass throughout the
of
cell
to EPS mass remained
91
constant. EPS composition appeared
a distributed gel to
made the
biofilm
to change with time from
stronger polymer stands. These changes
less
sensitive
(e.g. fluid shear stress
to physical disturbances
transitions) and more efficient at
substrate removal (Figure 431).
/
'
:
;
Specific Cellular Growth Rate
Specific cellular growth
with time since
rate
in
substrate concentration decreased (Equation
20). Specific cellular growth rate
was calculated based on
30),
the
Monod
Trulear
(Equation
(1983;
(Table I). A steep initial decrease
but
detectable
decrease
Given constant stoichiometric
growth and
product
in the liquid phase, m^,
liquid phase substrate data (Figure
equation
coefficients from
slow
the reactor decreased
rate implies increasing cell
al., 1984). Increasing
cell
and
et
kinetic
al., 1984)
in m^ was followed by a
the experiment.
kinetic coefficients for
decreasing
cellular growth
mass, in the reactor (Bakke et
biofilm
conclusion that biofilm
Robinson
throughout
and
formation,
20)
optical density support the
mass increased throughout the
experiments.
Average specific cellular growth rate in the biofilm, m2 ,
can
be
estimated
effectiveness
from
factor
Values for fD are
not
for
Equation
19,
diffusion,
known,
its
given
f .
range
m^
and
Although
is
I
the
exact
> f^ > 0,
92
implying m^ <
. Since
decreases with increasing biofilm
mass density (Atkinson and
Davies, 1974), fD decreased with
time in the present study. This suggests that m^ = m^ in the
early stages
of
similarly to m^
the
experiments,
(Figure
30),
and
but
that m2 progressed
its magnitude decreased
faster than for m^.
Alternatively, m^ can be found
given substrate concentration
Cm2 was measured
directly
and
at
allowing determination of m2
from Equations 21 and 22,
biofilm, cell mass, CM2.
the
and
end of the experiments,
of fD (Equation 19). This
calculation is, however, very sensitive to the value of knp,
which precludes a
meaningful
result,
since an exact value
for knp is not available, as demonstrated in the Litterature
Review.
Finally, average specific cellular
be estimated from the
state
and
at
Equation 16 is
biofilm
cell
"pseudo-steady
zero
or
terms. Specific biofilm
was small compared
to
mass balance at steady
state"
negligible
cell
growth rate, ny,, can
when
compared
the
l.h.s. of
to the other
accumulation rate (Figure 31)
specific
cell detachment rate, r^M ,
(Figure 30) throughout most of the experiments except during
the first days
of
biofilm
growth, and
during fluid shear
stress transitions. The biofilms were, therefore, at pseudo­
steady state and m2 =
r^M . Average specific cellular growth
rate in the biofilm progressed
rdM
(Figure
30)
except
in other words, similarly to
during
the
first
few
93
Figure 30. Progression
growth rate,
of
liquid
phase
, calculated from Equation 20 and
substrate data (Figure 15).
E x p e rim e n t 111
Liquid Phase Specific Cellular Growth Rate (1 /h )
specific cellular
150
Time ( h )
94
days and during fluid shear stress transitions.
Riofilm Accumulation
Specific Accumulation Rate
Specific biofilm
cell
the l.h.s. of Equation
accumulation
16
from biofilm absorbance data, is
plotted vs. time for Experiments II
steep initial change, coinciding
thickness,
is
apparent.
rate, calculated as
and III in Figure 31. A
with the change in biofilm
Accumulation
did
not,
however,
establish itself at zero following the rapid initial change.
The biofilm did not reach
density at the
same
steady
time
as
state in terms of optical
it
reached steady state for
thickness.
To further investigate
specific accumulation
the
rate
change in accumulation rate,
data
(excluding
during transitions) were plotted
on
a
data obtained
log scale in Figure
32. It is apparent from this figure that biofilm density.did
not
reach
experiment.
decrease
steady
state
Specific
with
time.
within
accumulation
Which
imply
approaching steady state. This
the
completion
rate
that
did,
the
of
the
however,
biofilm
was
figure also illustrates that
specific biofilm cell accumulation rate is much smaller than
specific cellular
detachment
steady state assumption
rate,
applied
to
supporting the pseudo­
obtain an estimate for
95
Figure 31. Specific
biofilm
cell
progression in Experiment
accumulation
rate
III, calculated as the
l.h.s. of Equation 16 from light scattering data.
Time
(h )
96
specific cellular growth rate in the biofilm.
Constant
biofilm
boundary condition
mass
density
mass
for
density
detachment
changed
is
not
modeling
significantly
an
accurate
since biofilm
throughout
the
experiments.
Steady state
All
parameters
changed
throughout
therefore, not at
however,
measured,
the
biofilm
experiments.
steady
approach
except
state.
steady
The
thickness,
system
was,
The biofilm reactors did,
state,
since
specific
biofilm
accumulation rate was decreasing
with
should also be noted
that
accumulation rate was very
low compared to . the
other
this
terms
biofilm cell mass except during
time (Figure 32). It
in
the mass balance for
the first days. In terms of
mass balance calculations to separate growth and detachment,
the accumulation
term
may,
therefore,
be neglected. This
situation is referred to as "pseudo-steady state".
Specific
biofilm
logarithmic scale
linear function
accumulation
(Figure
in
442),
time.
The
exponential decay with time,
rate,
appear
rate,
plotted
to
on
a
decrease as a
therefore, follow an
suggesting that the biofilm is
asymptotically approaching a steady state. Investigating the
progression of other
system
the ultimate steady state
parameters
may point out what
conditions might be like. Biofilm
97
Figure 32. Specific
progression
biofilm
in
cell
Experiment
accumulation
III
on
a
rate
natural
logarithmic scale. Data from Figure 31, excluding
data obtained during recycle rate transitions.
U
- 4
.2
-
6
-
-
Time
(h )
98
density data
and
liquid
phase
substrate data demonstrate
that this evolution towards steady state involves increasing
biofilm cell mass
concentration.
density
Since
consumer density is
and decreasing reactor substrate
less
substrate
higher,
is
available
and
substrate concentration at the
substratum must become very low, maybe nil.
A
boundary
condition
species, such as
cells
(Wanner and Gujer,
for
and
1985)
EPS,
was
steady state approached by
coexistence
of particulate
in steady state biofilms
analyzed to characterize the
the biofilms. Their mass balance
analysis of steady state biofilms revealed that the specific
production rate
of
all
coexisting
Steady state biofilms must
This condition
is
terms in the theory
be
the
described
and
(Relations 26-30). Literature
(personal
in
boundary
this
analyzed in mathematical
composition at steady state
data and computer simulations
performed by Wanner
of
same at the substratum.
chapter, yielding relationships between
measured parameters and biofilm
terms
particulate species in
communication) were analyzed
condition,
and
an
apparent
contradiction was discovered. If the data presented by Bakke
et al. (1984) were steady state
value than determined in
the other
hand
measured
biofilm reactors. This
data errors or that
the
not at steady state,
or
data then knp has a greater
chemostats
a
(Table I). Turakhia on
smaller
contradiction
situation
both.
knp,
can
equal
be
zero, in
due to large
simulated by Wanner was
Data obtained in this study
99
demonstrate that it can
suggesting that the
take
weeks
biofilms
simulated
state since they were only a
approached here
high
biofilm
has
very
mass
concentration
at
low
29 are
hot
valid,
substrate concentration and
suggesting
biofilm
approaching zero (Cg -> 0 at z
C e is indeed zero at
were not at steady
few days old. The steady state
density
the
to reach steady state,
that
substratum
substrate
interface
was
= 0), as discussed above. If
steady State, then Inequalities 28 and
and,
therefore,
yield
no information
regarding biofilm composition. Some information may still be
salvaged
from
this
analysis
satisfied (i.e. knp
= 0
at
concentration, Cjyf2, did not
there were no
apparent
Equation
30
must be
steady state). Biofilm cell
appear
cell
depth at the end of the
since
to approach zero, since
density gradient with biofilm
experiments (Figure 22). If so, knp
must equal zero, as measured by Turakhia (1986) .
The EPS.to cell mass
be the same as in
Equation 25
is
ratio did not
maintained
at
the
valid
change
the
ratio
liquid
at
with
real
in the biofilms appeared to
phase (0.4; Figure 17) since
pseudo-steady
time
steady
it
state. Since this
will probably also be
state
approached
by the
system.
In summary, it appears
approached a stable Steady
cell ratio of 0.4
that the biofilms asymptotically
state
conditions with an EPS to
(Figure 17) where substrate concentration
at the wall is zero.
100
Modeling
To put the results
from
this study in perspective they
are discussed in context of
related to results
biofilm modeling in general and
previously
obtained
in our laboratory.
Biofilm modeling has progressed from.a "black box" to a well
understood structured system
through continuous interaction
between experimentation and theoretical analysis.
A mono^culture biofilm
ago
to
investigate
system
the
metabolic
biofilms (Trulear, 1983). It
that the same
kinetic
was
was
and
chosen several years
activity
of
cells in
concluded from this study
stoichiometric coefficients for
biological transformation processes are valid in biofilms as
in suspension (Bakke et
al.,
1984). This implies that mass
transfer processes between the
liquid phase and the biofilm
and within the biofilm are the only processes complicating a
biofilm mass balance model over a dispersed culture model.
Biofilm-liquid
interface
categorized based on
being
transported
transport.
understood
the
as
Transport
and
transport . processes
characteristics
either.
of
modeled
based
Particulate transport,,
on
understood. Wanner and
Gujer
physical boundary condition
the
mass
on
other
be
of the component
dissolved
dissolved
can
or
particulate
is
quite
diffusion
hand,. was
well
theory.
not well
(1985) demonstrated that some
at the biofilm-liquid interface
101
is required to model detachment of particulate mass from the
biofilm to the liquid phase.
Nelson et al.
(1986)
investigated
cell transport from
the liquid phase and attachment to submerged surfaces, which
are important
initiation.
processes
A
in
the
sensitivity
analysis
balance model described earlier
initial attachment
events
insignificant effect on
early
stages
of
the
of biofilm
biofilm mass
revealed, however, that the
modeled
by
Nelson
et al. have
active pseudo-steady state biofilms
unless substrate loading rate
is
very
low (DCgo << HimKgg)
(Bakke et al., 1986) .
Detachment is, therefore, the main particulate transport
process
in
detachment
active
have
biofilms.
been
Several
proposed,
literature review, but conclusive
was lacking. All these models
will serve as a
boundary
EPS, etc.) transport from
It was determined in
serve as
since
the
it
appears
to
the characteristics
culture
biofilm
balance model.
of
evidence
in
the
for any of them
biofilm to the liquid phase.
study that biofilm thickness can
condition
be
Given
discussed
have in common that that they
the
a
independent of physiological
the limits'tested.
for biofilm
condition for particulate (cells,
this
boundary
as
models
in mono-culture biofilms
constant
biofilm
property,
and physical conditions within
the interface boundary condition,
the
behavior
cells
can
be
and
the reactor, mono­
predicted
by
the mass
102
Alternative boundary conditions
proposed in the literature,
were
for biofilm detachment,
not supported by the data
analyzed. Comparing biofilm detachment
stress progression (Figures 7 and
the
idea
that
specific
rate and fluid shear
18) yields no support for
cellular
detachment
rate
is
proportional to fluid shear stress. Constant biofilm density
is clearly not a valid
assumption for the biofilms analyzed
in this study, precluding its
use as boundary condition for
biofilm detachment
It
modeling.
that constant biofilm
Condition for P.
was, therefore, concluded
thickness
aeruginosa
is
the preferred boundary
biofilm
detachment in biofilm
mass balance simulations.
Through
determination
and
parameters, monoculture biofilm
Understood.
The
analysis
behavior
theoretical . and
developed to obtain this knowledge
more complex biofilms. These
the development
species biofilms.
of
biofilm
is presently well
experimental
methods
should now be applied to
methods
mathematical
of
may, for example, aid
models
describing multi­
103
CONCLUSIONS
Both theoretical
analysis
were
investigation
and
experimental
improved
of
or
biofilm
particulate and soluble
developed
detachment.
components
methods for biofilm
to
facilitate
the
balances
for
Mass
were analyzed to derive
measurable expressions for the fundamental process rates. An
experimental system was
fluid dynamic
methods for
and
biofilm
developed
conditions
biofilm
density
thickness
thickness based on a
geometric
through the
Biofilm
biofilm
sample.
optical
permitting
monitoring.
optical
mechanical biofilm
while
to maintain well-defined
density.
The
drawn from
these
experiments
within the
range
of
use of optical
Biofilm optical thickness
were
was
measured
in-situ. The
determined
from optical
analysis
cell
mass
following
with
experimental
of the light path
was correlated to
conclusions were
P. aeruginosa biofilms
conditions
tested. The
useful range of some of the conclusions has been extended by
considering data from Trulear
organism:
(1983) obtained with the same
104
1.
Biofilm thickness
reached
within
of
24
hours
approximately 35 micrometers
reactor
relatively constant
start-up
throughput
the
and
remained
experiments (15-23
days).
2.
Biofilm thickness was
conditions
within
not
a
influenced
wide
range
conditions tested: laminar
flow (Trulear, 1983) ,
3.5 Pa and step
(this
fluid
changes
of
fluid
dynamic
thesis) and turbulent
shear
in
by fluid dynamic
stress from 0.04 to
fluid shear stress of 0.04
and 0.08 Pa.
3.
Biofilm
thickness
was
conditions within the
not
influenced
by
metabolic
range of substrate concentrations
tested: IOK
'> Cc, > 0.1K _ where K 0 is the cellular
gs
S
gs
gs
growth saturation coefficient.
4.
Constant
condition
biofilm
required
thickness
to
can
account
transfer processes between
the
serve
for
as
a boundary
particulate
biofilm
mass
and the liquid
phase (detachment) in mass balance biofilm models.
105
5.
Although
biofilm
quickly, other
and biofilm
thickness
variables
optical
levels, i.e. the
reached
did
Process
rates,
not
reach constant
reactor, system did not reach
steady state.
6.
constant level
(e.g. substrate concentration
density)
biofilm
a
.
particularly
detachment
significantly during transitions
but quickly returned
(within five liquid
fluid shear force,
to their pre-transition magnitudes
residence
term biofilm reactor
in
rate. Changed
times). Therefore, long­
performance
was not significantly
influenced by fluid shear force transitions.
7.
Biofilm thickness standard deviation increased with time
while average biofilm
constant.
Thus,
thickness
biofilm-liquid
remained
more or less
interface
increased, resulting in a thicker surface film.
roughness
106
NOMENCLATURE
substratum area [L ]
Components:
P
product (EPS)
substrate (glucose)
S
cells fP. aeruainosaV
M
D
0
O
1
O
CO
H
A C S1
CO
11
Component concentrations, C:
product mass concentration in phase, k [M. L"3]
cPk
cell mass concentration in phase k [Mh l '
CMk
L"3]
substrate mass concentration in phase k
CSk
dilution rate [t 1]
effective substrate mass diffusivity in I
DSf
d
hydraulic diameter [L]
F
f
bulk liquid flow rate [L3 t 1I
friction factor [ - ]
Ji
diffusive mass flux vector for component
.
cellular growth saturation coef. IMs L"3
I
cellular detachment coef. [t ]
product detachment coef.
[t 1J
growth assisted product formation coef.
non-growth assisted product formation
coef.[Mp Mm"1 t 1J
maximum cellular growth rate, [t 1]
■
L"2 t'1]
Coefficients:
KgS
kdM
kdP
2,-1
-I.
107
Ii£
yf
total biofilm thickness [L]
. thickness of biofilm layer [L]
optical biofilm thickness [L]
N
transport (flux) vector [M E-^ t~^]
n^
biofilm refractive index ['-J
n
glass tube refractive index [ — ]
9
Specific process rates, r:
r^M
net cellular detachment rate [t-^]
r
product formation rate [t-^]
*
rdP
rs
m
-I
product detachment rate [t
substrate uptake rate [t-1]
cellular growth rate [t~^J
time [t]
3
reactor volume [L ]
flow velocity vector [L t-"*"]
yield of product from substrate [Mp Mg-"1']
yield of cells from substrate [Mm M 0-^]
effectiveness factor for substrate diffusion [ - ]
fluid density [M L
biofilm density coef. [M L ^J
momentum flux (fluid shear force)
[Pa]
Subscripts:
i
j
k
f
component (M-cell; P-products; S-substrate)
process (a-adsorption; D-diffusion; d-detachment;
g-growth; p-product formation)
phase (0-inflow; 1-bulk liquid; 2-biofilm)
biofilm
108
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114
APPENDIX
RAW DATA
Table 9. Fluid dynamic conditions in Experiment I.
T ime
( h )
-65
-48
-12
0
30
49
54
96
127
151
196.5
222
270
296
318
341.5
365.5
388
410
411
411.3
411.74
419.5
460
485
529
530. B
531.2
531.38
531.65
652
676.5
*
Sample Dilution Recycle
( # ) Rate
Rate
(1/h)
(ml/min)
I
2
5
6
13
16
18
23
25
26
29
30
33
34
35
36
39
41
45
46
47
51
54
55
59
60
61
67
69
0
0
0
3
3
3
3
3
3
3
3
3 ..
3
3
3
3
3
3
3
3
3
3
3
3
3 .
3
3
3
3
3
3
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
40
40
4Q.
40
40
40
40
20
20
20
20
20
h - measured head loss
- air" in rectangular tubes;
h
(mm)
Fluid Shear Stress Technical
Ideal
Measured. Problems
(Pa) -
2.0
2.5
,
1.0
.5
2.0
1.0
.8
.6
2.0
3.5
2.9
1.1
1.4
2.2
1.3
3.0
4. 0
4.5
6.5
2.5
3.0
2.5
1.5
1.6
physical
.04
.04
.04
.04
.04
.04
.04
.04
.04
.04
.04
.04
.04
.04
.04
.04
.04
.04
.04
.04
.08
.08
.08
.08
.OS
.08
.08
.04
.04
.04
.04
.04
.0540
.0675
*
.0270
.0135
.0540
.0270
.0216
.0162
.0540
.0945
.0783
.0297
.0378
.0594
.0351
.0810
.1080
. 1215
. 1755
.0675
.0810
.0675
*
.0405
.0432
disturbance of biofilm
115
Table
Time
<h>
10.
Fluid dynamic
conditions
Sample Dilution Recycle
<#>
Rate
Rate
(ml/miri)
(1/h)
-47.33 I
-.33 . 17
O
IS
.42
19
1.67
21
11.67 22
22.17
23
34.67
25
48.17
26
101.67 31
120.67 32
123.67 33
124.57 35
124.67 36
125.67 38
133.17 40
143.17 41
147.67 42
153.17 43
196.67 46
216.67 48
218.67 50
218.75 51
219.67 53
220.67 54
228.17 56
246.67 58
291.17 62
291.59 63
291.67 64
292
67
299.17 71
311.67 72
322.17 75
0
0
3
3
3
3
3
3
3
3
3
.3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
.3
3
3
.
40
40
40
40
40
40
40
40
40
40
40
40
40
20
20
20
20
20
20
20
20
20
60
60
60
60
60
60
60
40
40
40
40
40
'h — measured head loss
*
air in rectangular tubes;
in E x p e r i m e n t
II.
h ' Fluid Shear Stress Technical
(mm> Ideal
Measured
Problems
(Pa) -
2.8
3.8
4. I
3.0
2,5
2.0
2. I
2.8
2.6
1.8
.9
.9
1.4
1.9
2. I
8.1
4.9
9. I
4. I
7.3
.3*5
3.3
3.9
.08
.09
.08
.08
.08
.08
.08
.08
.08
.08
.08
.08
.08
.04
.04
.04
.04
.04
.04
.04
.04
.04
. 12
.12
.12
, 12
.12
.12
.12
.08
.08
,OS
.OS
.08
.0756
.1026
.1107
.0810
.0675
.0540
*
.0567
.0756
.0702
.0486
.0243
.0243
.0378
*
.0513
.0567
.2187
. 1323
.2457
.1107
.1971
.0945
.0891
. 1053
physical disturbance of biofilm
116
Table
T ime
( h >
11.
Fluid dynamic conditions
Sample DilutionRecycle
Rate
Rate
(#)
(1/h)
(ml/min)
-47.33
I
-.33
17
0
18
19
.42
20
I
21
1.67
22
11.67
23
22. 17
25
34.67
.1Q1.67. .31
120.67
32
123.67
33
124.57
35
124.67
36
125.67
38
40
133.17
41
143.17
147.67
42
196.67
46
216.67
48
218.67
50
218.75
51
219.67
53
220.67
54
228.17
36
246.67
58
291.17
62
291.59
63
291.67
64
67
292
299.17
71
311.67
72
322.17
75
0
0
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
.
. 40
40
40
40
40
40
40
40
40
40
40
40
40
60
60
60
60
60
60
60
60
20
20
20
20
20
20
20
40
40
40
40
40
' h - measured head loss
h'
(mm)
2.8
2.9
4. I
3.1 .
2.4
2.4
2.2
3.2
2,6
6.5
4.6
4.9
4
4.6
5.7
2
I. I
I. I
/1.6
1.9
2.5
3
3.7
in E x p e r i m e n t
III.
Fluid Shear Stress Technical
Ideal
Measured
Problems
- (Pa) .08
.08
.08
.08
.08
.08
.08
.08
.08
.08 .
.08
.08
.08
.12
.12
.12
.12
.12
. 12
. 12
.12
.04
.04
.04
.04
.04
.04
.04
.08 .
.08
.08
.08
.08
.0756
.0783
.1107
.0837
.0648
.0648
_0594
.0864
.0702
. 1755
. 1242
.1323
.1080
.1242
. 1539
.0540
.0297
.0297
.0432
.0513
.0675
.0810
.0999
117
Table
12.
Biofilm thickness
Experiment I;
Sample
< # )
I
II
12
13
15
18
21
24
27
29
30
nZSsZS
34
35
36
39
41
42
43
45
50
53
54
55
56
57
58
59
62
65
66
67
68
readings
si
43
42
41
39
35
38
30
32
46
45
48
65
44
I).
Biofilm Thickness Readings
Biofilm Optical Thickness, z
(urn)
at locations ( y ):
*
-1.00 -0.75 -0.50-0.25
O
0.25
0.50
I
40
36
36
52
33
41
51
53
55
37
46
46
46
39
35
34
40
(Experiment
I
20
31
27
38
50
44
51
44
52
30
33
42
43
40
41
42
30
44
44
45
51
35
39
44
31
40
37
35
57
.51
54
0
35
28
11
35
44
49
51
52
50
36
40
36
38
38
34
37
31
52
44
43
54
40
35
32
42.
37
48
36
33
84
57
O
33
24
33
33
45
42
55
53
49
33
30
29
32
31
27
30
37
42
47
47
39
34
31
37
45
44
58
40
44
43
51
O
30
26
34
41
46
.44
41
53
49
34
28
30
36
32
26
32
37
46
36
45
48
37
32
34
. 36
42
43
39
38
52
64
Bi ofiTm Refractive Index, n f ,
measured at the end of experiment
by refractometer.
I
31
29
26
38
48
57
53
48
47
28
29
36
35
31
27.
34.
51
41
40
40
44
32
36
42
42
39
53
46
36
48
52
1.356
1.362
I.35S
1.362
I
31
28
32
37
44
47
60
51
35
29
33
38
35
36
28
23
26
38
39
28
38
32
23
35
33
34
35
49
34
21
51
Average
0.75
I
29
34
30
46
52
49
51
36
38
11
33
32
39
34
31
' 28
27
34
35
32
30
29
40
25
44
40
41
37
42
30
59
1.00
32
36
35
39
34
34
35
23
38
27
39
30
32
33
36
29
41
30
37
. 40
42
z
(urn)
1.0
31.1
29.5
28.6
40. O
45.3
46.6
51.6
48.8
. 46.9
29.8
33.8
36. I
37.7
35. 6
31.4
32.7
34.9
41.2
40.7
38. B
42.7
34.2
33.7
35. 6
37.7
37.4
44.7
39.7
41.0
48.2
52.7
118
13.
Experiment
Biofilm thickness readings (Experiment
II;
§
Sample
( # )
Biofilm Optical Thickness; z
at locations ( y ):
-O.. 75 -0.50 -0.25
O
0.25
0.50
O
I
22
-23
24
25
28
29
31
32
38
39
40
41
46
48
53
56
57
60
61
69
71
72
.75
0.0
.3
.5
25.0 ' 29.0
39.0
40.0
32.0
38.0
32.0
32.0
37.0
40.0
38.0
41.0
39.0
42.0
43.0
50.0
47.0
46.0
50.0
52.0
45.0
37.0
48.0
61.0
56.0
50.0
46.0
47.0
38.0
45.0
39.0
45.0
48.0
40.0
42.0
39.0
42.0
45.0
47.0
36.0
42.0
32.0
0.0
.7
26.0
42.0
35.0
35.0
39.0
40.0
43.0
49.0
51.0
43.0
40.0
46.0
44.0
42.0
44.0
34.0
43.0
34.0
39.0
40.0
0.0
.3
27.0
37.0
35.0
35.0
43.0
42.0
39.0
0.0
.5
29.0
33.0
38.0
38.0
37.0
41.0
48.0
0.0
.5
27.0
43.0
41.0
41.0
44.0
42.0
42.0
37.0
49.0
49.0
46.0
50.0
49.0
35.0
38.0
45.0
42.0
40.0
48. 0
■42.0
46.0
50.0
43.0
42.0
28.0
32.0
47.0
55.0
37.0
49.0
48.0
41.0
45.0
31.0
42.0
43.0
36.0
32.0
47.0
38.0
42.0
38.0
33.0
39.0
Biofilm Refractive Index, n f , 1
B*
measured at the end of experiment,
by refractometer.
I
II).
Bio-film Thickness Readings
O
Table
I.3350
(urn)
0.75
1.00
0.0
.7
30.0
40.0
41.0
41.0
39.0
42.0
47.0
0.0
.3
28.0
39.0
39.0
39.0
39.0
40.0
46.0
0.0
.4 .
27.0
39.0
44.0
41.0
42.0
44.0
50. 0
38.0
46.0
49.0
45.0
47.0
47.0
49.0
46.0
38.0
40.0
37.0
47.0
35.0
38.0
48.0
51.0
43.0
50.0
40.0
39.0
43.0
40.0
42.0
49.0
39.0
36.0
41.0
48.0
50.0
44.0
47.0
50.0
37.0
41.0
45.0
39.0
37.0
36.0
44.0
40.0
and I.3352
ave.
0.0 .
.47
27.56
39.11
38. 11
37. 11.
40.00
41.11
44.00
43.00
41.67
46.67
50.56
42.44
48.67
48.44
42.33
42.22
39.44
39.89
41.17
41.22
38.22
38.44
Mech.
Thick.
Lf <um>
0
0
20
40
30
40
40
40
30
40
40
40
T a b l e 14.
Experiment
Biofilm thickness
III;
readings
Bi ofiIm Optical thickness; z
at locations ( y .) :
( # > — I -OG —0 ■75 -0.50 --0.25
O ' 0.25
0.50
0
.8
28
33
34
32
34
46
45
36
44
47
40
31
29
33
41 '
32
26
42
39
31
26
0
.5
24
41
33
43
39
47
39
36
40
33
35
36
27
30
33
45
35
43
28
43
III).
Bidfilm Thickness Readings
Sample
I
. 22
23
25.
28
29
31
32
38 .
39
40
41
46
48
53
56
57
60
61
69
71
72
75
(Experiment
O
.667
. 24
41
36
43
40
43
43
. 34
40
34
35
35
37
30
36
25
20
38
27
27
0.75
1.00
0 /
.3
25
41
36
42
33
44
0
I
25
46
34
40
35
44
0
.9
27
35
36
40
42
43
37
38
40
34
35
40
37
18 .
31
43
42
26
39
33
30
25
22
28
25
36
34
26
27
22
O
.9
25
36
37
40
41 .
43
0
.8
28
35
32
44
39
45
0
.4
26
36
33
35
39
39 .
38
42
42
32
29
34
32
39
37
40
40
36
23
32
41
41
34
40
38
39
35
36
35
27
32
37
31
42
41
38
39
38
28
32
37
28
41
38
40
36
24
32
34
36
33
31
■ 34
26
37
.30
30
42
Bipfil m Refractive Index, n f , =
at the end of the experiment.
measured by refractometer
:
1.3341
(urn)
and
1.3401
.
ave.
0
.70
25.78
38,22
34.56
39.89
38.00
43.78
45.00
39.40
39,67
41.22
34.78
34.00
34.44
33.33
33.89
31.56
32.11
32.80
33.67
29.44
32.67
Mech.
Thick.
Lf <um)
0
0
20
30
30
40
40
30
30
30
30
30
.
120
T a b l e 15.
C e l l m a s s data.
Liquid Phase Cell Mass
(Epifluorescent Direct Count)
Ex pt. Time
(.# ) ( h )
Number/Volume
Diameter
+/- .< u m3 ) (mg/1)
( um ) +/— ( #/ul )
Length
< urn )
+/-
.0009
.0006
.0164
.1752
.2112
... 0894
.1416
.0005
.0003
.0028
.0540
.0750
.0127
.0260
.43
.34
.89
.40
.39
.11
.25
.045
,027
1.759
8.475
9.987
1.232
4.285
1.20
I.12
.77
.91
.46
.64
.37
.37
1.67
11.67
22. 17
29. 17
34.67
124.67
■124.83
127.67
322.17
1.06
1.09
1.08
1.01
1.05
.95
■88
1.01
.88
.55
.58
.41
.35
.27
.40
«38
.39
.52
.63
.57
.62
.60
.73
.61
.55
.63
.54
.25
.24
.17
.15
.14
.20
.21
.17
.22
.0010
.0648
' .1104
.1200
.0347
.0768
.0744
.0763
.1140
.0005
.0095
.0338
.0343
.0174
.0377
.0206
.0145
.0390
.33
.28
.33
.29
.44
.28
.21
.32
.20
.039
2. 139
4.384
4. 107
1.825
2.553
1.886
2.894
2.761
1.67
11.67
22. 17
29. 17
34.67
124.83
127.67
322.17
.98
1.07
I.15
1.04
.98
.88
.86
.86
.31
.34
.25
.26
.22
.23
.25
.25
.60
.57
.72
.64
.60
.60
.57
.52
.16
.15
.13
.15
.11
.13
.13
.12
.0003
.0442
.1260
.1440
.1140
.3120
.1680
.2568
.0001
.0176
.0295
.0390
.0384
.0770
.0312
.0333
.28
.28
.47
.33
.28
.25
.22
. 18
.009
1.464
7.058
5.709
3.836
9.219
4.359
5.667
0
4
9
21
28
671
674.5
II
II
II
II
II
II
II
.11
II
III
III
III
III
III
III
III
III
1.19 .50
1.24 .79
(Epifluorescent Direct Count)
Biofilm CelI Density
Length
( urn ) +/—
Expt .SampIe
( # )( # )
I
I
I
II
II
III
III
.23
.25
.30
.24
.32
.20
.20
.67
.60
.84
.65
.67
.44
.60
I
I
I
I
I
I
I
I
4
7
3
6
3
7
.94
.88
.85
.77
.86
.80
.94
.35
.29
.29
.27
.25
.34
.30
Number/Area
Diameter
+/—
( um ) +/— (#/um2)
.62
.58
.58
.48
.62
.52
.59
.19
.20
.21
.21
.18
.22
.21
14.4900
8.5680
7.0140
11.5500
30.2400
14.2800
15.3300
2.0100
1.9900
1.3500
3.1500
7.1000
6.2200
5.5700
* Vc
- average cell volume (micrometers cubed)
:
■ Cml - liquid phase cell carbon concentration
Cm2 - biofilm cell areal carbon density
Cm2^
Vc
( um3 ) <g/m2)
.29
.23
.23
. 14
.26
. 17
.26
.490
.236
.187
.191
.937
.284
.461
121
T a b l e 16.
B i ofilm total
organic
Biofilm Total Organic Carbon
Experiment Sample
( # )
Location
carbon
(TOC)
(TOC)
TOC
( g/m2 )
I
I
I
I
I
4
5
7
.526
.376
.291
.327
+ /—
+ /”
+ /—
+/-
.005
.021
.019
.003
II
II
II
II
I
3
5
7
.489
.538
.442
.610
+/+/“
+ /—
+/-
.004
.018
.006
.041
III
III
III
III
I
3
5
7
.474
.474
.634
.536
+/+/+/+/-
.006
.006
.015
.008
data.
122
Table
17.
Liquid phase TOC,
S O C , and POC
( E x p t . I).
Experiment
I;
Liquid Phase Total, Soluble, and Particulate Organic Carbon
TOC
SOC
POC ( = TOC - SOC )
Time
< h )
-65.0
2.0
. 6.0
23.0
30.0
54.0
78.0
101.5
127.0
151.0
173.0
179.0
196.5
222.0
251.0
270.0
296.0
318.0
341.5
365.5
388.0
407.0
410.0
411.5
412.5
436.5
460.0
503.0
509.0
526.0
529.0
531.2
533.7
558.0
576.0
652.0
673. 0
Sample
( # )
I
9
.10
12
13
18
21
24
25
26
27
28
29
30
32
33
34
35
36
39
41
43
45
48
50
53
54
56
57
58
59
61
62
65
66
67
68
TOC
( mg/1 >
22. 17
38.65
31.21
15. 16
11.02
14.53
10.99
14.05
17.70
13.19
22.59
11.81
6.91
4.66
10.19
7.60
8-48
7. 15
10.57
15.68
14.22
13.24
14.57
45. 12
14.94
13.96
14.36
18. 14
43.47
17.79
16. 19
18.24
14.86
9.38
7.73
7.75
6.32
+/+/+/+ /—
+/+ /—
+/+/+/+/+/+/+/+/+/+ /+/+ /^+ /+/+ /—
+/+ /—
+/+/+/+ /—
+/+/+/+ /+/+/+/+/+/+/-
.26
.85
-60
.09
1.25
.69
.27
.46
.59
.22
I. 19
.22
.26
.35
.30
.28
.33
.05
. 10
.07
.05
.08
.24
. 11
. IO
.17
. 14
.17
. 15
.33
. 13
.32
.24
. 12
.21
. 14
.19
SOC
( mg/1 )
POC
< mg/I
)
38.98 +/18. 13 +/8.28 ■+/ —
5.11 +/6. 15 +/7.22 +/8.00 +/4.43 +/5.51 +/3.76 +/-
.21
4.43
I ■68
. 14
.30
I. 13
.19
.29
.29
.24
7.77
4.22
4.56
3.60
2.87
5.07
3.83
3.39
2.78
4. 14
3.96
3. 86
3.38
3.52
2.95
9.55
24.96
+/-.
+/+/+/+/+ /—
+/+/+/+ /—
+/+ /—
+/+/+/+/+/-
.12
.40
.35
.08
.19
. 11
»06
. 15
.07
.04
.05
.18
.24
.27
.10
.29
.15
-.85
.44
5. 63
4.00
5.62
2.09
6.74
12.29
11.44
9. 10
10.61
41.26
11.55
10.44
11.41
8.59
18.51
15. 12
10.91
11.74
2.83
2.99
1.92
7. 87
+/+/+/+/+/+/+ /—
.25
.05
.07
.28
.11
.03
.11
1.07
7.33
3« 12
6.55
4.74
5.83
-1.56
values are average +/-■ standard deviation of 4 samples
• -.32
13.08
6- 88
5.92
8.38
3. 77
6.05
13.27
7.68
18.83
123
Table
18.
Liquid phase TOC,
Experiment
Time
< h )
1.67
11.67
22. 17
34.67
54. 17
101.67
120.67
123.67
125.67
133.17
143.17
196.67
218.17
219.67
220.67
222.67
228.17
240.67
274.67
288.17
291.17
292.00
293.17
299.17
311.67
322.17
( E x p t . II).
IX; Liquid Phase
Total, Soluble, and Particulate .Organic Carbon
TOC
SOC
POC
Sample
( # )
21
22
23
25
28
31
32
33
38
40
41
46
49
.53
54
55
56
57
60
61
62
67
69
71
72
75
S O C f and POC
TOC
( mg/1
29.49
14.47
10.58
9.52
36. 66
10.91
10.21
9.90
10.81
9.08
7.91
9.51
9.95
21.88
8.68
7.58
7.20
7.53
11.72
14.33
13.64
. 11.31
13.09
11.00
9.83
8.98
SOC
( mg/1
)
+/- .326
+ /— .246
4*/— . 180
+/- .068
+/- .335
+/- . 106
+ /— .074
+/- . 191
+/- .397
+/- .262
+/- .713
+/- .345
+/- .034
+/- . 183
+/- .212
+ /- .034
+/- .592
+/- .283
+/- 1.84
+/- .601
+/- .745
+/- .455
+ /“ .441
+/- 1.43
+/- .460
+ /— .448
.
24.87
1.1.70
4.48
3.92
32.84
14.55
7.87
5.31
6.33
5 .43
6. OS
4.67
4.65
4.00
3.62
4.09
3.59
3.24
7.29
8. 03
10. 12
10. 10
9.65
6.16
9.21
6.25
+/+/+/+/+/+/+ /“
+ /—
+ /+/+/+/+/+/+/+ /—
+/+ /+/+ /—
+/+/+/+/+/+/-
)
.27
.19
.03
.59
I. 19
. 13
.19
. 16
.73
.05
.16
. 12
.03
.23
.02
.12
.07
.31
2. 14
.30
1.04
1.40
1.66
.71
.52
.56
values are a v e r a g e +/— standard d e v i ation of 4 sa m p l e s
POC
( mg/1
)
4.62
2.77
6.09
5. 60
3.83
-3.64
2.34
4.59
4.48
3.65
1.83
4.84
5.31
17.88
5.06
3.49
3.61
4.29
4.43
6.30
3.51
1.20
3.43
4.84
.62
2.72
124
Table 19. Liquid phase TOG, SQCr and POG (Expt. III).
Experiment 111;
Liquid Phase Total, Soluble,
TOC .
SOC
Time
< h >
Sample
<: # )
1.67
11.67
22. 17
. 34.67
54. 17
IQl,67
120.67
123.67
125.67
133*17
143.17
196.67
218.17
219.67
:220.67
222.67
228.17
240.67
274.67
288.17
291.17
292.00
293.17
299.17
311.67
322,17
21
22
23
25
28
31
32
33
38
40
41
46
49
53
54
55
56
57
60
61
62
67
69
71
72
75
IOC
( mg/1
26.63
14.86
11.16
9.95
28.48
9.67
11.17
10.76
19.40
11.28
9.94
7.73
13.51
14.07
9.59
13.45
12.09
12.32
17.90
15.93
18.44
34.90
18.95
14.42
17 .09
13.67
+/+/+/+/+/+/+ /T+ /+ /+ /+ /—
+ /—
+/+/+ /”
+/+/+ /—
+ /+ /+/+ /+ /—
+ /—
+/-
and Particulate Organic Carbon
POC
(= TOC - SOC)
)
.03
. 17
.09
.15
.15
.30
.04
. IO
.39
.08
.02
.07
.15
.95
.07
.12
.18
.32
.20
.03
—
.24
.91
.29
1.25
.60
SOC
< mg/1
22.44
12.29
4.84
3.77
20.31
23.92
8.08
5. 16
4.91
5.73
5. 10
5.43
3.87
3.72
3. 19
3.38
3.66
2.88
7.63
6.97
10.57
9.11
9.40
6. 65
7.73
5.30
+/+ /+ /—
+/+/+ /+ /“
+ /—
+ /—
+ /—
+/+/+/+ /+/+/+/+ /+/+/.+/ —
+ /—
+/+/+/+/-
>
.06
.69
.09
•03
.43
.21
.07
. 19
.15
.09
.14
.79
.IO
.14
.04
.04
.03
.01
.15
.14
.93
.84
1.57
2.02
. 14
.09
POC
( mg/1
)
4.19
2.56
6.31
6. 18
8. 17
-14.26
3.09
5.60
14.49
5.54
4.83
2.30
9. 64
10.35
6.41
10.07
8. 43
9.44
10 .27
8.96
7.88
25.79
9.55
7.77
9.36
8.37
, 125
Table 20. Substrate Concentration Data (Expt. I)
Experiment
Time
< h )
I; Liquid Phase Substrate Concentration,
Csl - Effluent
and
CsO - Influent
( reported as mg carbon per liter )
Sample
( # )
2. 0
6.0
23.0
30.0
46.5
54.0
.78.0
101.5
151.0
196.5
222.0
251.0
270.0
296.0
318.0
341.5
365.5
388.0
407.0
410.0
411.5
412.5
436.5
. 460.0
485.0
503.0
526.0
529.0
531.2
533.7
538.0
558 .0
. 576.0
652.0
9
10
12
13
15
18
21
24
26
29
30
32
33
34
35
36
39
41
43
45
48
50
53
54
55
56
58
59
61
62
63
65
66
67
.
Csl
Csl
(mg/1> (mg/1) mean
+/-
,04
26. 38 26.32 26.35
.55
20. 17 19.39 19 .78
.16
1.87
1.99
1.76
.04
1.01
1.03
.98
.00
1.20
1.20
1.20
.94
.41
1.23
■65
.00
,70
.70
.70
.36
1.38
.59
.87
.00
.BI
.BI
.81
. 15
.13
.24
.07
.00
.07
.07
' .07
.06
.15
.20
.11
.03
.15
.13
.11
.06
.29
.24
,33
. 13
1.32
1.41
1.23
. .51
.56 . .,153 ■ ,03
.03
.24
-27
.29
.03
.27
.29
.24
.09
1.03
1.09
.96
.06
.96
1.00
.91
.16
.71
.82
.60
.09
.85
.91
.78
.OO
.51
.5:1
.51
.06
.47
.42
,51
5 .20
6.72
5.29
8.91
6.85
5.25
6.72
5,25
9.36
6.81
5.22
6.72
5.27
9. 13
6.83
.03
.00
.03
.32
.03
.43
.43
.34
.25
.16
.38
.34
.08
.06
. 13
.11
CsO
CsO
(mg/1> (mg/1>
mean
+ /—
' 27.04 26.16 26.60
.63
■
■" -
25.34 25.79 25.57
.32
11.10 11.36 11.23
.19
10.54 10.76 10.65
9.02
8.97
9.06
.16
.06
126 .
Table
21.
Substrate Concentration Data
Experiment
Time
< h )
II)
II; Liquid Phase Substrate Concentration
Csl - Effluent
and
CsO - Influent
( reported as mg carbon per liter )
Sample
Csl
Csl
( # ) (mg/I) (mg/1) mean
-47.33
1.67
11.67
22. 17
34.67
48. 17
54. 17
101.67
120.67
123.67
125.67
127.67
133.17
143.17
196.67
218.17
219.67
220.67
222.67
228.17
240.67
274.67
288.17
291.17
292
. 293.17
299.17
311.67
322.17
(Expt.
I
21
22
23
25
26
28
31
32
33
38
39
40
41
46
49
53
54
55
56
57
60
61
62
67
69
71
72
75
20.53 21.49 21.01
4.91
4.99
4 .95
1.06
I. 14
I. IO
.71
.71
.71
1.32
.84
.84
.91
1.4.1
1.84
1.38
1.93
1.15
1.15
.46
.69
.83
.42
.60
.37
.37
.28
.46
.51
.65
.55
.37
.92
.79
.78
.75
1.41
1.23
1.32
1.50
1.18
I. 18
.33
.78
. .55
.46
.51
.64
.33
.39
.55
.55
.57
.49
.46
I. 12
.82
.BI
.83
1.41
1.53
1.35
1.71
1.17
1.17
.39
.73
.69
.44
.55
.51
;35
.33
.51
.53
.61
.52
.42
CsO
CsO
(mg/I I (mg/1)
mean
+/-
19.60
20.00
19.80
.29
20.86
21.04
20.95
. 13
21.36
21.45
21.41
.07
22.65
20.95
21.40
19.64
22.02
20.30
.88
.93
21.41
20.86
21.13
.39
13 >80
18.87
18.83
.05
19.07
19.48
19.28
.29
.68
.06
.06
.00
.28
.03
.04
. 11
.OO
,43
.04
.30
.02
.02
. 10
.06
.20
.03
.07
.19
.03
.OS
.06
.03
.05
.05
.06
127
22.
Substrate Concentration Data
Experiment
Time
( h )
Hi)
III; Liquid Phase Substrate Concentration,
Csl - Effluent
and
CsO - Influent
( reported as mg carbon per liter ) .
Sample
Csl
Csl
( # > (mg/1> (mg/1) mean
-47.33
1.67
11.67
22. 17
34.67
48. 17
54,. 17
101.67
120.67
.123.67
125.67
127.67
133.17
143.17
196.67
218.17
219.67
220.67
222.67
228.17
240.67
274.67
. 288.17
291.17
292.00
293.17
. 299.17
311.67
322.17
(Expt.
I
21
22
23
25
26
28
31
32
33
38
39
40
41
46
49
53
54
55
56
57
60
61
62
67
69
71
72
75
+/—
CsO
CsO
(mg/1)(mg/1)
20.00
20.71 20.92 20.82
6.59
6.53
6.66
1.67
1.84
1.49
.97
.92
1.01
.15
.09
.25
.06
20.81 20.27
.97
1.41
I. 19
.88
■66
1.20
.87
.97
.97
.78
.87
.97
.87
.69
.74
.46
.46
.46
.19
.37
.37
.87
.32
.92
1.36
I. 18
.87
.69
.73
.96
.82
.60
.46
.82
1.00
.78
.73
.73
.51
.51
.64
.33
.37
.31
.44
.33
.95
1.38
1.19.
.87
.67
.96
.92
.89
.78
.62
.85
.98
.83
.71
.73
.48
.48
.55
.26
.37
.34
.66
.33
.03 .
.03
.00
.01
.02
.33
.06
.10
.26
.23
.04
.03
.07
.03
.00
.03
.03
. 13
. IO
.00
.04
.31
•OO
21.82 21.41
21.87 22.53
20.32 21.55
22.28 21.08
21.18 19.09
19.39
mean
8
1
"I
81I
Table
19.48
+/—
128
Table 23. Biofilm and liquid phase optical density (Ex. I ) .
T i me
Sample
< .h )
( #
-65
-48
-41
-24
-12
O
2
6
11
23
30
36
46.5
51.5
54
60
77
78
85
96
101.5
127
151
173
179
196.5
222
244
251
270
296
318
341.5
347
360
365.5
383
388
396.5
407
410
411.3
411.34
411.74
412
412.5
419.5
432.5
436. 5
460
485
503
509
526
529
530.8
531.2
531.28
531.38
5 3 1 ^65
532.12
533.7
538
558
576
652
6/3 .
676.5
I
2
3
4
5
6
9
10
11
12
13
14
15
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38,
39
40
41
42
43
45
47
49
50
51
52
53
*54
55
56
57
58
59
60
61
62
63
65
66
67
68
69
>
Biofilm Optical
at locations:
1 . 2
3
.031
.031
.024
.021
■ .033
.034
.058
■058
.069
.065
. .051
■058
.029
.039
.061
.064
.Ii
. 102
.246
.213
.225
■ 196
.282
.235
.347
.28
.371
.304
■401
.325
■408
.346
.435
■337
.429
.418
.467
.359
.448
.361
.44
.322
.495 . .373
■612
.43
.714
.512
.634
.424
.487 ,.338
.437
.316
.436
.305
.506
.368
.516
.387
.547
.399
■529
.407
.651
.528
.719
.562
.698
.545
. .749
.575
.788
.601
.767
.612
.76
.601
.94
. 74
.965
.742
.971
■989
1.086
1.086
I. 16
1.341
1.625
1.644
1.64
1.64
.735
.77
.785
.87
.887
I .058
I .395
1.44
I .476
I .607
I .648
I .608
I.608
I .607
I.609
1.663
1.51
1.637 I.514
I.182 I.051
1.179
.961
1.304 I,.122
1.303 I..137
1.296 I,.163
Density
■4
(abs.),
7
B
.031
.047
.032
.056
.051
-. 009
-.013
-.013
.02
.015
-033
.088
.231
.28
.225
.255
.3
.283
.314
.341
.011
.024
.071
.206
. 138
. 179
.234
.223
.267
.247
.334
.38
.366
.355
.387
.457
.549
.448
.355
.297
.333
.399
.376
.411
.452
.57
.584
.589
.616
.652
.651
.644
.766
.815
.219
.29
.245
.244
.263
.314
.384
.291
.234
.201
.238
.28
.288
.309
.334
.424
■458
.441
.456
.508
.5
.489
.592
.638
.321
- .348
.437
.496
.404
.478
.537
.578
.514
.622
.657
.808
.829
.898
.901
.983
1.175
1.494
1.494
1.455
I .474
.639
.596
.656
.605
.713
.671
.717
.66
.777
.711
.973
.907
1.337 1.244
1.37 1.314
1.41
1.34
1.445 1.374
I
1.477
1.463
.582
.992
.56
.98
.985 1.075
.843 1.019
.85 I. 023
1.457
I .444
.472
.528
.737
.688
.696
1.392
1.383
.659
■666
.821
.768
.771
Liquid
Phase
Cabs.)
.08
.088
.075
.068
.053
.078
.071
.085
..034
.071
.062
.067
.028
.054
.03
.04
■024
.063
.068
.071
. 101
.088
.097
.027
.074
.072
.062
.358
. 184
.097
.076
.077
.079
.078
■097
■081
. 107
.096
.062
.06
.063
.063
.055
.044
■042
.035
.04
.049
.06
.038
.04
.054
129
T a b l e 24.
B i o f i l m and
Time
( h )
-47.33
-46.33
-45.33
-44.33
-43.33
-42.33
-41.33
-40.33
-38.33
-37:33
-25.33
-23.33
-20.33
-17.33
-11.83
-1.33
0.00
0.42
1.00
1.67
11.67
22.17
29.17
34.67
5 4 . 17
78.67
101.67
120.67
123.67
• 124.47
124.57
124.67
124.83
125.67
127.67
133.17
143.17
153.17
191.17
196.67
215.67
216.67
218.17
218.67
. 218.75
218.92
219.67
220.67
222.67
228.17
240.67
246.67
274.67
288.17
-291.17
291.59
292.67
291.75
291.89
292.67
293.17
293.67
298.17
299.17
311.67
316.67
318.17
3 2 2 . I7
Sample
( # )
I
2
3
4
5
6
7
8
9
10.
11
12
13
14
15
16
18
19
20
21
22
23
24
25
28
29
31
32
33
34
35
36
37
38
.39
40
41
43.
45
46
47
48 .
49
50
51
52
53
54
55
56
57
58
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
liquid phase
optical
Biofilm Optical Density
at locations:
6
5
4
3
density
(Absorbance),
2
.036
.054
.035
.042
.032
.036
.035
.036
.034
.041
.049
.042
.054
. .067
.075
.063
.034
.039
.037
.043
.032
.034
.039
.037
.032
.041
.039
.043
.055
.067
.082
-066
.086
.054
.077
.069
.068
■074
.059
.059
.054
.062
.075
.082
. 123
. 137
. 131
.115
.079
.045
.073
.055
.052
.056
.051
.057
.047
.059
.071
.079
.093
.078
. 137
. 145
.041
.042
.035
.076
. 124
. 153
. 132
.051
.038
.037
.086
. 137
. 179
.211
. 107
.097
.066
. 189
. 192
.214
.262
. 129
. 121
. 125
.241
.250
.301
.336
.032
.025
.042
■ 111
. 146
. 194
.228
.217
.307
.502
.521
.247
.451
.563
.583
.306
.413
.485 . .616
.619
.774
.629 . .803
.511
.628
.749
.752
-533
.544
.585
.660
.688
.865
.896
.594
.604
.644
.718
.763
.963
■981
.646
.661
.708
-780
.822
1.018
1.043
.804
.822
.863
.943
.994
1.190
1.212
.762
.767
.822
.876
■930
.964
I. 053
1.115
1.282
1.173
.976
.975
.981
.999
1.042
1.066
I. 164
1.198
1.205
1.065 I. 124
1.066 1.122
1.075 1.131
1.091 1.144
1.147 1.191
1.176 1.214
1.276 1.312
1.309 1.344
1.318 1.348
1.288
1.290
1.299
1.305
1.357
1.374
1.456
1.488
1.412
1.178
1.183
I. 185
I. 193
1.228
1.261
1.322
1 .354
1.208
1.320
1.347
i.495
•1.212
1.325
1 .352
1.496
1.365
I .224
1.252
1-337
1.363
1.365
1 .394
1.509
1.536
1.373
1.402
1.275
1.384
1.410
1.547
1.411
I
.028
.020
.001
.016
.007
■008
.006
.007
.010
■009
.042
.035
.047
.058
.083
.060
.041
.029
.056
1.117
.091
.059
Ave.
(Ex.
Liquid
Phase
(Abs.)
.047
.048
.038
.039
.034
.036
.046
.047
■042
.051
.053
.052
.071 '
.081
. 100
.085
.055
.023
.008
.005
.03
,052
.046
.043
i036
.471 . .361
.024
.533 . .052
.629
.758
.661
.037
.779
.678
.046
.031
.031
.028
■.026
,792
.689
.025
.802
.700
.027
.843
.744
.025
.919
.816
.03
■968
.861
■016
1.009
.029
I. 173 1.070
.029
.03
1.246 1.139
.026
.031
.031
■401
.411
1.247 1.146
.206
1.246 I. 147
.038
1.245 1.153
.029
1.254 1.164
1.292 1.210
.024
1.312 1.234
.021
1.396 1.321
.041
1.425 1.353
.028
1.321
.047
.033
.03
.027
.02
1.343
.018
.012
1.431 1.364
.012
.015
1.444 1.375
.017
1.470 1.403
.016
.018
■017
1.482 1.418
.023
.034
.030
.037
.067
. 140
.204
.243
.066
.059
.057
. 128
. 165
.208
.235
130
T a b l e 25.
B i o f i l m and
Time
(h)
-47.33
-46.33
-45.33
-44.33
-43.33
-42.33
-41.33
-40.33
-38.33
-37.33
-25.33
-23.33
-20.33
-17.33
-11.83
-1.33
0.00
0. 42
1.00.
1.67
11.67
22. 17
29. 17
34.67
54. 17
78.67
8
;0ia
.010
.020
.021
.022
.027
.027
.038
.023
.031
.022
.029
.034
.060.
.063
.061
liquid
phase optical
Bio-film Optical Density
at locations:
7
6
5
4
.014
.005
.020
.017
.016
.022
.036
.032
.023
.027
.022
.026
.041
.055
.061
.064
.040
.032
.026
.030
.025
.031
.053
.058
.100
.105
.116
.136
.129
.159
.271 . .396
.366
.354
.409
.455
.579
.648
.597
.600
.029
density
(absorbance)
3
2
I
Ave.
.021
.021
.030
.027
.023
.032
.023
.025
.032
.032
.041
.062
.069
. 100
i024
.059
.009
.052
.024 . .047
.025,
.052
.038
.053
.025
.072
.028
.033
.023
-031
-077
.079
.072
.073
.078
.078
.098
.091
.149
.100
.146
.097
.060
.063
.066
.072
.063
.086
.058
.051
.045
.050
.065
.056
.070
.061
.057
.063
.076
.080
. 126
. 142
. 147
. 155
. 139
. 187
.072
.063
.071
.080'
.094
. 107
.117
.082
.090
. 100
, 102
.127
.042
.007
.038
.040
.045
.049
.029
.034
.023
.029
.068
.065
.073
.088
.097
-111
.067
.056
.055
. 150
. 156
. 180
.211
.373
.424
.524
.718
.745
.128
.133
.135
.232
.271
.294
.322
.516
.569
.686
.893
.912
.055
.071
.072
.121
.163
.180
.224
.376
.392
.498
.688
.705
. 140
. 105
. 103
. 139
. 161
.212
.246
.390
.445
.571
.784
.809
.080
. 133
.060
.110
.057
.111
.118
. 179
.176
.227
.211
.288
.250 - .341
.381
. 474
.454
.580
.564
.672
.766
.926
.802
.907
.084
.074
.074
. 131
. 170
.202
.235
.397
.411
.547
.751
.760
.941
.947
.980
1.035
1.024
1.200
1.230
.713
.708
.743
.822
.832
.817
.808
.850
.919
.930
.808
.782
.819
.897
.895
.916
.892
.938
1.031
1.024
1.017
1.121
1.091
1.243
.784
.778
.817
.886
.889
1.016
1.089
1.357
1.131
1.231
1.201
1.367
1.203
101.67
120.67
123.67
124.47
124.57
124.67
124.83
125.67
.612
.703
.761
127.67
.612
.696
.777
133.17
.645
.745
.814
143.17
.694
.788
.898
153.17
.704
.808
■894
191.17
.849
.955
1.058
196.67
.892
I.009 1.106
215.67
216.67
1.003 1.110 1.221
218.17
218.67
218.75
218.92
219.67
1.033 I. 158 1.224
220.67
I.022 1.140 1.238
222.67
1.047 1.159 1.256
228.17
1.082 1.177 1.282
240.67 .1.140 1.248 1.328
246.67
1.152 1.253 1.337
274.67
1.263 1.371 1.444
288.17
1.283 1.400 1.463
291.17
1.305 1.429 1.467
291.59
291.67
291.75
291.89
292.00
1.311 1.444 1.473
292.62
293.17
1.316 1.436 1.471
297.67
299.17
1.323 1.409 1.486
311.67
1.352 1.462 I .502
316.67
318.17
322.17
1.388 1.458 1.527
1.380
1.377
1.588
1.414
1.462
1.473
1.560
1.568
1.585
1.154
1.163
1.175
1.197
1:258
1.290
1.402
1.401
1.252
1.262
1.272
1.292
1.356
1.360
1.461
1.476
1.222
1.236
1.245
1.268
1.317
1.344
1.451
1.475
1.576
1.385
1.384
1.397
1.454
1.480
1.553
1.574
1.593
1.250
1.228
1.266
1.264
1.320
1.336
1.438
1.455
1.447
1.455
1.585
1.408
1.484
1.476
1.572
1.469
1.601
1.627
1.421
1.448
1.492
1.519
1.486
1.515
1.585
1.475
1 .504
1.641
1.455
1.524
1.520
1.616
1.516
(Ex.Ill).
Liquid
Phase
(abs.>
.050
.018
.005
.004
.021
.052
.046
.043
.047
.021
.018
.031
.047
042
.032
.067
. 107
- 106
.061
.041
.035
.026
■018
.027
.060
■079
.070
.085
.077
.069
.044
.067
.060
.069
.089
.060
.073
.079
.280
.479
.409
.284
. 115
.093
.064 '
.064
.058
.060
.059
.063
MONTANA STATE UNIVERSITY LIBRARIES
CO
III ill III Il III
762 100 15!34 3 1
D3T8
B179
cop. 2