Chemically induced biofilm detachment
by Xiao Chen
A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in
Chemical Engineering
Montana State University
© Copyright by Xiao Chen (1998)
Abstract:
Biofilm detachment induced by various chemicals, including metal salts, surfactants and
depolymerization agents, was investigated using an experimental system consisting of a two-species
bacterial biofilm grown in continuous flow annular reactors. Two types of experiments were
performed: 1) in situ environmental step change experiments conducted in annular biofilm reactors to
examine detachment and 2) determination of viscometry of biofilm collected from these reactors.
Experimental results showed that biofilm detachment could be induced by addition of various
chemicals. Monovalent and divalent salts (including NaCl, CaCl2, MgCl2) were the most effective
chemicals in changing biofilm structure, reflected by an average of 73% viscosity reduction. Directly
performing step changes in biofilm reactors with these salts detached 40% of the biofilm in 75 minutes.
Chelants showed similar results, e.g. 19.6% viscosity reduction and 26.3% bilfilm detachment for
EDTA. Surfactants (including sodium dodecyl sulfate, Triton X-100, Tween 20) also altered the
structure of biofilm (e.g. viscosity reduction was 8.7% for Tween 20, 41.9% for Triton X-100, -12.6%
for SDS) and caused a much larger amount of biofilm to detach (average of 61.7%). Addition of
chlorine, monochloramine and some enzymatic lyases (including lysozyme and protease) caused
viscosity reduction and biofilm detachment also. We found that 1) electrostatic (e.g., cation bridging)
and hydrophobic interactions were two major forces that maintain the integrity of biofilm structure; 2)
cells and EPS were the structural components of biofilms. Disruption of biofilm crosslinking forces and
destruction of structural biofilm components could cause biofilm detachment. CHEMICALLY INDUCED BIOFILM DETACHMENT
by
Xiao Chen
A thesis submitted in partial fulfillment
of the requirements for the degree
of
Doctor of Philosophy
in
Chemical Engineering
MONTANA STATE UNIVERSITY
Bozeman, Montana
April 1998
ii
J)312
APPROVAL
of a thesis submitted by
Xiao Chen
This thesis has been read by each member of the committee and has been found to
be satisfactory regarding content, English usage, format, citations, bibliographic style,
and consistency, and is ready for submission to the College of Graduate Studies.
Dr. Phil Stewart
Chairperson, Graduate Committee
Date
Approved for the Department of Chemical Engineering
Dr. John Sears
Head, Department of Chemical Engineering
Date
Approved for the College of Graduate Studies
Dr. Joseph J. Fedock
Graduate Dean
iii
I
STATEMENT OF PERMISSION TO USE
In presenting this thesis in partial fulfillment of the requirements for a doctoral
degree at Montana State University-Bozeman, I agree that the Library shall make it
available to borrowers under rules of the Library, I further agree that copying of this
thesis is allowable only for scholarly purposes,' consistent with “fair use” as prescribed in
the HS. Copyright Law. Requests for extensive copying or reproduction of this thesis
should be referred to University Microfilms International, 300 North Zeeb Road, Ann
Arbor, Michigan 48106, to whom I have granted “the exclusive right to reproduce and
distribute my dissertation in and from microform along with the non-exclusive right to
reproduce and distribute my abstract in any format in whole or in part.”
Signature
Date
vv ?
cn r
iv
ACKNOWLEDGMENTS
First of all I would like to express my gratitude to my academic and research
advisor Dr. Phil Stewart for his guidance, encouragement and support that enabled me to
achieve my goal. Also, I would like to thank all my committee members. Dr. Gordon
McFeters and Dr. Anne Camper counseled and assisted in experimental design. Dr. Max
Deibert and Dr. Jay Rotella also gave me much help. I want to particularly thank Dr. John
Sears for his guidance and admittance to the Ph.D. program in the Chemical Engineering
Department as well as to the Center for Biofilm Engineering. I have learned so much in
such a short time from the unique interdisciplinary nature of the Center that I would like
to thank everybody in the Center for his/her friendship and sharing of his/her expertise.
I especially acknowledge my wife, Xiaobei Wang and my parents, TianChun
Chen and FenMin He, with my love and gratitude for their endless support and care for
all time.
My research activities have been supported by National Science Foundation and
the Center for Biofilm Engineering, a National Science Foundation-sponsored
Engineering Research Center.
TABLE OF CONTENTS
Page
LIST OF TABLES............................................................................................................vii
LIST OF FIGURES...........................................................................................................ix
ABSTRACT............ .........................................................................................................xi
INTRODUCTION....,........................................................................................................I
LITERATURE REVIEW.............................................................. ,...................................3
HYPOTHESES...............
8
MATERIAL AND METHODS......................................................................................... 9
Microorganism and Culture Conditions....................................................................... 9
Reactor System and Operation.................................................................................... 11
Reactor Treatment and Sampling................................................................................ 13
Analytical Methods for Step Change Experiment.............................
15
Viscometry.................................................................................................................. 18
Source of Reagents.....................................................................................................20
RESULTS.........................................................................................................................21
Data Analysis and Repeatibility..................................................................................21
21
Viscometry..........................
Annular reactor step change experiments............................................................. 26
Electrostatic Interaction.......................................................................................
30
Influence of different salt additions...................................................................... 30
Effect of ionic strength......................................................................................... 31
vi
Cation bridging..................................................................................................... 42
Hydrophobic Interaction.......................................
45
Depolymerization................................................................:......................................48
Relationship between Biofilm Detachment and Viscosity Change............................ 52
Time Scale of Biofilm Detachment.........................................
55
DISCUSSION.............................................................................................
58
Interpretation of Viscometry Study.....................................................................
59
Electrostatic Interation.......................... ....... ....................... ................... *............... 61
Hydrophobic Interaction......................................................................... ............. <..... 63
Depolymerization........................................................................................................ 64
CONCLUSIONS........................................................................................
66
RECOMMENDATIONS FOR FUTURE WORK........ :...... ,........................................67
REFERENCES................
, APPENDICES..........................................................
68
73
Appendix A—Raw Data from Viscometry Experiments...........................................74
Appendix B—Raw Data from Environmental Step Change Experiments................ 91
Raw data from optical density measurements............. ...................... ........... :.... 92
Raw data from total protein and cell enumeration measurements...................... 101
vii
LIST OF TABLES
Table
Page
1. Media composition................. ..............................................................................: 11
2. Annular reactor characteristics................................................................................... 13
3. Statistical analysis of the changes of biofilm viscosity at ionic strength
of 0.3 when compared to the control.........................................................................30
4. Statistical analysis of the changes of biofilm viscosity at different
ionic strength when compared to the control.............................................................37
5. Summary of biofilm detachment caused by environmental step changes
of various salts and related compounds...............................
41
6. Statistical analysis of the changes of biofilm viscosity treated with
EDTA and Bequest 2006 when compared to the control...........................................43
7. Summary of biofilm detachment caused by environmental step changes
of chelants...........................................................................
:....43
8. Metal elements in biofilm samples.............................................................................45
9. Statistical analysis of the changes of biofilm viscosity treated with
surfactants when compared to the control................................................................. 46
10. Summary of biofilm detachment caused by environmental step changes
of surfactants............................ ............. :....................................... ..........................48
11. Statistical analysis of the changes of biofilm viscosity treated with
depolymerization agents when compared to the control..................... ...................... 51
viii
12. Summary of biofilm detachment caused by environmental step changes
of chlorine, monochloramine and lysozyme.................................................... ..........52
13. Summary of linear regression results from correlation of the percentage
of biofilm detachment and the percentage of biofilm viscosity reduction................. 53
ix
LIST OF FIGURES
Figure
Page
1. Viscosity of biofilm sample before and after addition of 0.22M MgCL................... 22
2. Correlation of viscosities of biofilm samples after addition of MgCL to the
initial viscosities. The line was fit using least squares regression......... .................... 24
3. Viscosity changes of biofilm samples with time after addition of
lysozyme. Different symbols represent different trials. The line was
fit using least squares regression...................
25
4. Viscosity of untreated control biofilm samples. The line was fit using
least squares regression...............................................................................................26
5. Correlation of percentage detachment determined by scraping biofilm
from sample slides and by integrating biomass appearing in the
effluent. The line was fit using least squares regression............................................28
6. Correlation of the total amount of biomass detached in effluent by
integrating total protein and by integrating optical density changes.
The line was fit using least squares regression...........................................................29
7. Viscosity measurement of biofilm samples with addition of
various salts, all at ionic strength of 0.3.................................................................... 31
8. The influence of ionic strength on biofilm viscosity when treated
with NaCl. The initial viscosity was 150 cP............................................................... 33
9. The influence of ionic strength on biofilm viscosity when treated
with CaCL- The initial viscosity was 150 cP.......................................... ................. 34
10. The influence of ionic strength on biofilm viscosity when treated
with FeCL- The initial viscosity was 150 cP............................................................. 35
11. The influence of ionic strength on biofilm viscosity when treated
with Fe(NOs)S. The initial viscosity was 150 cP........................................................36
12. The measurements of biomass concentration versus time in the
effluent of a biofilm reactor following biofilm treatment with 0.3M
NaCL Treatment was initiated at t=0 and terminated at t=60 minutes.
Biomass was determined by turbidimetry (A), total protein (B), and
by colony formation on agar plates (C). In panel C, (•) denotes
P. aeruginosa and (■) denotes K. pneumoniae.........................................................38
13. The measurements of biomass concentration versus time in the
effluent of a biofilm reactor in an untreated control. Biomass was
determined by turbidimetry (A), total protein (B), and by colony
formation on agar plates (C). In panel C, (•) denotes P. aeruginosa
and (■) denotes K. pneumoniae................................................................................39
14. Comparison of biofilm species composition before and after treatment.
The y-axis is the ratio of the percentage of P. aeruginosa in the sample
after testament to the percentage of P. aeruginosa in the sample before
treatment. Symbols indicate effluent (O) and biofilm ( • ) samples...........................42
15. Viscosity measurement of biofilm samples treated with EDTA or
Dequest 2006..............................................................................................................44
16. Viscosity measurement of biofilm samples with additions of various
surfactants...................................................................................................................47
17. Viscosity measurement of biofilm samples treated with chlorine (pH 7.7)
and monochloramine (pH 7.3). The concentration refers to total chlorine................. 49
18. Viscosity measurement of biofilm samples with additions of enzymatic
lyases........................................................................................................................... 50
19. Correlation of the percentage of biofilm detachment and the percentage
of biofilm viscosity reduction. Symbols indicate results from salt group
( • ) , including pH and sucrose, from surfactant group (A) and
depolymerization group (O)....................................................................................... 54
20. Comparison of relative rapidity of biofilm detachment with various
treatments. Peak time (A) and relative rate (B) are defined in the text.
The dashed line in panel B indicates the relative rate that would be
measured if the detachment rate were uniform over the 75 minutes
measurement period.................................................................................................... 57
xi
ABSTRACT
Biofilm detachment induced by various chemicals, including metal salts,
surfactants and depolymerization agents, was investigated using an experimental system
consisting of a two-species bacterial biofilm grown in continuous flow annular reactors.
Two types of experiments were performed: I) in situ environmental step change
experiments conducted in annular biofilm reactors to examine detachment and 2)
determination of viscometry of biofilm collected from these reactors. Experimental
results showed that biofilm detachment could be induced by addition of various
chemicals. Monovalent and divalent salts (including NaCl, CaClz, MgCl2) were the most
effective chemicals in changing biofilm structure, reflected by an average of 73%
viscosity reduction. Directly performing step changes in biofilm reactors with these salts
detached 40% of the biofilm in 75 minutes. Chelants showed similar results, e.g. 19.6%
viscosity reduction and 26.3% bilfilm detachment for EDTA. Surfactants (including
sodium dodecyl sulfate, Triton X-100, Tween 20) also altered the structure of biofilm
(e.g. viscosity reduction was 8.7% for Tween 20, 41.9% for Triton X-100, -12.6% for
SDS) and caused a much larger amount of biofilm to detach (average of 61.7%). Addition
of chlorine, monochloramine and some enzymatic lyases (including lysozyme and
protease) caused viscosity reduction and biofilm detachment also. We found that I)
electrostatic (e.g., cation bridging) and hydrophobic interactions were two major forces
that maintain the integrity of biofilm structure; 2) cells and EPS were the structural
components of biofilms. Disruption of biofilm crosslinking forces and destruction of
structural biofilm components could cause biofilm detachment.
I
INTRODUCTION
BiofIlm detachment refers to the transfer of biomass and other particulate
constituents of a surface-attached microbial film to the fluid phase surrounding the
biofilm. Detachment is one of the fundamental phenomena governing biofilm
accumulation and activity. As the primary process balancing microbial growth in most
biofilm systems, detachment is a key determinant of the extent of biofilm accretion.
Detachment probably plays a role in the development of the heterogeneous structures
observed in some biofilm systems (Murga et ah, 1995) and it may also influence biofilm
ecology (Rittmann, 1989; Stewart et al, 1997). Despite its fundamental importance in
virtually every biofilm system, detachment is one of the least well understood processes
in biofilms.
Detachment is.also interesting as an alternative strategy for controlling unwanted
biofilms such as those that foul cooling water towers, oilfield produced water pipelines,
or food processing plants. Biocides and antibiotics have been the principle weapons used
to combat biofouling. These agents work by killing microorganisms. This strategy is
invariably frustrated by the universally observed reduced susceptibility of biofikn
microorganisms to disinfection (Costerton et al, 1987; Brown and Gilbert, 1993).
Furthermore, in many biofilm fouling problems the desired end result is a clean surface
rather than an inactive, yet physically intact, biofilm. Antimicrobial agents achieve this
2
indirectly by stopping growth and allowing the natural detachment process to slowly
remove the biofilm. Promoting the detachment process directly would appear to be an
attractive and obvious alternative approach. This approach could have the added
advantage Of reducing reliance on inherently toxic control agents whose continued use is
fundamentally limited with the trend towards increasingly restrictive environmental
regulations.
LITERATURE REVIEW
Earlier research on detachment of biofilm has investigated the influence of shear
stress, growth rate, limiting nutrient, oxidizing regents, and enzymes. Trulear and
Characklis (1982) first determined a relationship between biofilm detachment rate and
shear stress. Subsequent analyses have not arrived at a consensus regarding the
significance of shear stress (Rittmann, 1982; Bakke et ah, 1990, Peyton and Characklis,
1993). Detachment in fluidized bed reactors is thought to occur predominantly by an
abrasion mechanism (Chang et ah, 1991; Gjaltema et ah, 1995). A few analyses suggest
that detachment can be growth-associated; that is, specific detachment rates depend on
the net growth rate in the biofilm (Speitel and DiGiano, 1987; Stewart, 1993; Peyton and
Characklis, 1993; Tijihuis et ah, 1995). Howell and Atkinson (1976) proposed that
sloughing occurs when the substrate concentration at the substratum falls below a critical
value. Applegate and Bryers (1991) found that P. putida biofilm grown under oxygen
limitation exhibited detachment rates 20-40% of those measured in a biofilm grown
under carbon limitation. The oxygen-limited biofilm contained more extracellular
polymeric substance (EPS) and more calcium than did the carbon-limited biofilm.
Characklis (1980) found that biofilm detachment resulting from chlorination was much
higher between pH 7.5 and 8.5 than it was between 6 and 7. An enzyme blend of
cellulase, amylase and protease was claimed effective in digesting microbial slime
4
(Wiatr, 1991). Johansen et al. (1997) reported that a complex mixture of polysaccharide­
hydrolyzing enzymes, including protease, cellulase, pectinase, p-glucanase, and xylanase,
was able to remove bacteria from steel and polypropylene substrata.
Biofilm is mainly composed of a variety of bacteria embedded in a matrix of
extracellular polymeric substances (EPS) of bacterial origin with water channels inside its
structure (Costerton and Lewandowski, 1997; Costerton et al., 1995; Christensen and
Charaklis, 1990; Siebel, 1987; Bakke, 1986). The adhesion forces that maintain the
structural integrity of biofilms or floes (i.e. biological aggregates from activated sludge)
are thought to include long range forces, such as van der Waals forces, and short range
forces, such as chemical bonds and hydrophobic interactions (Marshell, 1990; Tadrosi
1980; Porter and Lewin, 1972).
The long range adhesion forces can be addressed in part by the theory of
Derjaugin, Landau, Verwey, and Overbeek (DLVO theory) (VerWay and Overbeek, 1948;
Derjaugin and Landau, 1941). Since surfaces of bacterial cells are normally negatively
charged (Marshall, 1976), the net interaction of cells in biofilm would be expected to be
repulsive at very low electrolyte concentrations. In an ionic milieu, the net charge on the
cell surface is counter-balanced by cations. The result is an electrical double layer at the
interface between the cell and the aqueous phase. The double layer is made up of the
charged cell surface and the layer of opposite charged counter-ions that are attracted
electrostatically to the cell surface. In a high ionic strength aqueous medium the double
layer is compressed. At electrolyte concentrations corresponding to ionic strength about
1=0.001, a shallow secondary minimum may be formed at some separation distance
where the long range attractive van der Waals forces balances the short range repulsive
electrostatic forces. The decrease of electrical double layer is insignificant with further
addition of electrolytes. Zita and Hermansson (1994) reported that microbial floes from
wastewater activated sludge behaved in close accord with the predictions from DLVO
theory. It was found that the floe dissociation coefficient (a measure of the tendency of
floes to disperse) decreased gradually to its smallest value when the ionic strength of the
solution increased to about 0.01; then it remained almost unchanged with increasing ionic
strength from 0.01 to 0.1. However, it was found in the same study that ,when ionic
strength increased from 0.1 to I, the dissociation coefficient was drastically increased.
The later could not be explained by DLVO theory.
Cation bridging (e.g. Ca+) and hydrophobic interactions can be considered as short
range adhesion forces that maintain the structural integrity of biofilms, floes, and
biological gels. Turakhia et al. (1983) showed that immediate and substantial detachment
of a P. aeruginosa biofilm could be effected by addition of the calcium-specific chelant
ethylene glycol-bis-(|3-aminoethyl ether)-N,N-tetraacetic acid (EGTA). Gordon et al.
(1991) found that the gel strength of the alginate gel separated from mucoid
Pseudomonas aeruginosa was significantly reduced upon addition of NaCl, EGTA,
EDTA, and sodium dodecyl sulfate (SDS). SDS is a surfactant which may disrupt the
hydrophobic interactions that maintain gel strength. Singh and Vincent (1987) found that
the hydrophobic character of the isolated Pseudomonas sp from sewage sludge was
associated with the capacity to form aggregates. Valin and Sutherland (1992) even found
a correlation between flocculation in activated sludge and hydrophobicity on the basis of
6
contact angle measurement from sludge sample. The hydrophobicity of the cell surface is
also believed to be an important factor in determining the extent of adhesion of bacteria
to solid surfaces (Rosenberg, 1986).
The majority of the extracellular polymers in a biofilm are thought to be
polysaccharides. Research focused on polysaccharides or polymer matrices may well bear
on some phenomena in biofilms, such as biofilm detachment. For example, with regard to
the solution properties of polysaccharides, it was found that the viscosity decrease that
occurred when salt was added to solutions of different polysaccharides varied
considerably with the type of polysaccharide presented (Smidsrod and Hang, 1967; Cox,
1960). It was also found that the viscosity of an exopolymer produced by a Pseudomonas
sp. decreased by 75% of that in fresh water when 2.7M CaCl2was added, and by 85%
when subjected to 4.4M NaCl (Dasinger et ah, 1994). Parker et. al. (1996) determined the
influence of a whole range of metal cations on the viscosity of capsular polysaccharide
from Microcystis flos-aquae. He observed a biphasic effect of metal ion concentration on
viscosity of this polysaccharide. Initially the viscosity increased with increasing metal ion
concentration until a maximal viscosity occurred at a concentration of 1-1 OmM. The
viscosity decreased with further addition of that ion.
In summary, cells and extracellular polymers are considered major components of
biofilm. The structural cohesiveness of various types of biological aggregates including
biofilms, microbial floes, microbial gels, appears to result from a combination of forces
including long range van der Waals forces and short range forces like electrostatic and
hydrophobic interactions. These cohesive forces can be disrupted by numerous ways,
7
such as addition of salts (NaCl, CaCl2), addition of chelants (EGTA, EDTA), and
disturbance of hydrophobic interactions by adding surfactants. Treatment of a biofilm
with oxidizing reagents (e.g., chlorine) or with enzyme lyases (e.g., protease) can also
cause biofilm detachment.
HYPOTHESES
I hypothesize that there are two broad types of crosslinking interactions in the
biofilm matrix: electrostatic and hydrophobic. Electrostatic crosslinks could involve
direct interactions between the two charged elements or they could be mediated by
bridging substances, for example divalent cations or charged proteins. Similarly,
hydrophobic interactions could be direct or they might be mediated by a hydrophobic
protein.
With this structural model of the biofilm in mind, biofilm detachment could be
induced by a number of different treatments, including, for example, I) degradation of an
EPS polymer chain, 2) loss of cellular structural integrity (i.e., cell lysis), 3) disruption of
a cell-polymer or polymer-polymer electrostatic crosslinking interactions, and 4)
disruption of cell-polymer or polymer-polymer hydrophobic crosslinking interactions.
The overall goal of the work reported in this dissertation was to perform
preliminary experimental investigations of these hypotheses in order to understand the
mechanisms that cause biofilm detachment.
9
MATERIAL AND METHODS
Two types of experiments were conducted to investigate the influence of various
chemicals on biofilm detachment. The first type of experiment involved directly
measuring the amount of biofilm detachment caused by a step change addition of a
treatment chemical added directly to a biofilm reactor. Second type of experiment used
viscometry to. measure changes in the viscous properties of resuspended biofilm after
chemical treatment. Biofilms were grown on the inner surface of a continuous flow
annular reactor for about 7 days to reach a pseudo-steady state. Then they were
challenged with an instantaneous concentration change of certain chemicals which for the
most part were not biocidal. Any changes of biomass on the surface and in the effluent of
reactor were recorded, analyzed, and quantified using in-line optical density
measurement, total protein measurements, and a cell enumeration method. Biofilms
scraped from the surface of an untreated reactor were used for viscometry experiments.
Biofilm viscosity changes before and after addition of various chemicals were recorded
and quantified using a viscometer.
Microorganism and Culture Conditions
Pseudomonas aeruginosa (ERCl) and Klebsiella pneumoniae (KPl) were co­
cultured on a minimal salts medium to grow binary species biofilms. These two species
10
were stored in an autoclaved medium of peptone (2%) and glycerol (20%) at -70 0C after
enrichment. Medium was prepared in two 20-liter carboys containing 62-fold
concentrated stock solution. One was filled with phosphate buffer alone, i.e. Na2HPO4
and KH2PO4; the other held the remaining components of medium. The desired final
medium concentrations in the annular reactors were achieved by mixing flows of these
two carboys with an additional one from a continuously aerated water tank. The medium
components were sterilized by autoclaving and dilution water was sterilized by filtration
using two filters (0.2 pm capsule filter, Gelman Sciences) in series. The culture
preparation protocol and media compositions are listed below.
Culture preparation protocol
1)
2)
Pure species isolation
•
Transfer the stock culture onto R2A agar.
•
Streak the liquid in T-shape on the R2A agar using a flame sterilized loop.
•
Incubate the plates at 3S0C for 1-2 days.
•
Find a single colony of the target species.
Enrichment
•
Pick that single colony and restreak onto TSB agar thoroughly (Tryptic
Soy Broth, Difco).
•
3)
Incubate the plates at 35°C for 1-2 days.
Transfer 5ml autoclaved solution of 2% peptone and 20% glycerol to each of
TSB plates.
11
4)
Scrape off colonies and mix them with the liquid solution using a loop.
5)
Transfer Iml of culture solution to each cryo vial and save them under -70°C.
Table I. Medium composition for annular reactor.
Chemicals
Final concentration (mg/L)
Glucose
40
NH4Cl
14.4
MgSO4 7H20
4.0
Na2HPO4
382
KH2PO4
408
Trace
(NH4)6Mo7O24 4H20
0.002
ZnSO4 7H20
0.2
MnSO4 H2O
0.016
CuSO4 5H2O
0.004
Na2B4O7 IOH2O
0.002
FeSO4 7H20
0.224
(HOCOCH2)3N
0.8
CaCl2 ZH2O
0.08
Reactor System and Operation
Biofilms were grown on wetted inner surfaces of an annular reactor with
continuous flow. There were three areas covered with biofilms: I) the surface of the
12
rotating polycarbonate drum, 2) surfaces of twelve removable 316L stainless steel slides
attached to the inner surface of reactor, and 3) the rest of the polycarbonate inner surface
of the reactor. Table 2 summarizes.features of this reactor. Since there was no significant
statistical difference of biofilm coverage between stainless steel slides and the
polycarbonate surfaces based on the measurement of total mass and total protein, the
wetted surface area was simply calculated by summation of these three individual areas.
Biofilm was sampled without interruption of nutrient flow or inner cylinder rotation by
removing sample slides through stoppered holes in the top of the reactor. Because of the
relatively fast growth rate of Klebsiella pneumoniae, this organism could dominate in
biofilms if the two species were inoculated simultaneously (>99% based on cell
enumeration). To avoid this situation, the reactor was inoculated first with 1.0 mL of
thawed stock culture (I O8 cell mL"1) of Pseudomonas aeruginosa for 24 hours in batch
mode, then with K. pneumoniae for an additional 18 hours. After a total of 42 hours of
incubation in batch mode with the drum rotating, the influent flow was introduced at a
rate of 31 milliliters per minute. The influent flow consisted of three inlet streams: I) a
phosphate buffer flow (0.5 mL/min), 2) a nutrient (lacking phosphate buffer) flow (0.5
ml ./min), and 3) a dilution water flow (30 mL/min). The biofilm was allowed to grow for
7 to 9 days before a treatment was initiated. The inoculation procedure is listed below.
Biofilm annular reactor inoculation protocol
1) Fill up reactor with medium.
2) Adjust the rotation rate of inner drum to lOOrpm.
13
3) Inject Iml of thawed frozen culture of Pseudomonas aeruginosa into reactor.
4) Inject Iml of thawed frozen culture of Klebsiella pneumoniae 24 hours later.
5) Switch the reactor to continuous mode 18 hours later.
Table 2. Annular reactor characteristics.
Characteristic
Value
fluid volume
.570 mL
flow rate
31 mL min"1
dilution rate
3.26 h"1
Wetted surface area
1600 cm2
gap width
0.8 cm
inner cylinder diameter
10 cm
rotation rate
100 min'1
Reynolds number
4800
Reactor Treatment and Sampling
After seven days in continuous flow mode, the reactor was subjected to a step
change experiment. Step changes of treatment chemical concentration in the reactor were
achieved by adding several milliliters of concentrated treatment chemical directly into the
reactor and simultaneously switching to a continuous supply of this reagent at the desired
concentration for 60 minutes. The pulse dose was the calculated amount to
14
instantaneously raise the bulk concentration in the reactor to the desired final
concentration.
Biofilm was sampled approximately 30 minutes prior to the initiation of treatment
and every 15 minutes after the step change. The total sampling period was 75-90 minutes
or about 4 residence times. Effluent samples of biofilm were continuously collected
through a stopcock in the effluent tubing. Samples were homogenized for I minute using
a tissue homogenizer operated at 20,000 min"1. A sub-sample (100-200 mL) was
centrifuged for 10 minutes at 4 0C and 12000 xg for total protein analysis. Biofilm
covered slides were removed using aseptic sampling techniques at various times during
the experiments. They were scraped into 100 mL phosphate buffer (same concentrations
as in medium). This biofilm suspension was homogenized for 0.5-1 minute. Part of the
solution (40 mL) was then centrifuged for 10 minutes at 4 0C and 12000 xg for total
protein analysis. The step change protocol, slide coupon sampling protocol and effluent
sampling protocol are detailed below.
Step change protocol
1) Prepare 3 liters of treatment chemical solution (solution A) with concentration of
1.033-fold of final concentration.
2) Prepare solution B by dissolving X mg of target chemical into 5 - 1 5 mL water.
X = target chemical concentration (mg/L) x reactor volume (L)
3) Autoclave solution A and B.
4) Inject solution B directly into reactor at the beginning of step change experiment.
15
5) Switch feeding of the water source to solution A and continuously maintain it for 60
minutes.
6) Switch back to water after 60 minutes and disconnect from solution A.
Slide coupon sampling protocol
1) Gently pull out one of the stainless steel slides.
2) Scrape biofilms from the surface into 100 mL autoclaved phosphate buffer solution
with scraper.
3) Homogenize biofilm solution using Homogenizer at 20000 min"1for 45 sec.
4) Take Iml out of the 100ml sample for viable cell count.
5) Centrifuge biofilm sample (40ml out of 100ml) for total protein measurement.
Effluent sampling protocol
1) Collect whole effluent sample for 15 minutes.
2) Homogenize biofihn solution using Homogenizer (High-Speed Homogenizer, Cole
Parmer E-04719-00; Generators (probes), Cole Parmer E-04720-11 (100mm x 8mm),
and E-04720-12 (200mm x 18mm)) at 20000 min'1for 45 sec.
3) Take Iml out of the 100ml sample for viable cell count.
4) Centrifuge 100 or 200ml of biofilm sample for total protein measurement.
' Analytical Methods for Step Change Experiment
Biofilm samples were subjected to various analyses, including viable cell
enumeration, total protein measurement and optical density measurement. Viable cells
16
were enumerated by performing serial dilution and plating on R2A and Pseodomonas
isolation agar plates using the drop plate method (Hoben and Somasegaran, 1982; Miles
and Misra, 1938). The number o f P. aeruginosa colonies was counted from Pseudomonas
isolation agar. The number of K. pneumoniae colonies was calculated by subtracting the
number of P. aeruginosa colonies from the total number of colonies counted from R2A
agar. Cell pellets recovered after centrifugation were further treated with TEP solution
(IOmM Trizma Hydrochloride, ImM EDTA and ImM phenylmethylsulfonyfhioride) at
pH 8 to extract soluble proteins from biofilms. The supernatants after an additional
centrifugation were used to analyze for total protein by a modified Lowry method using
Sigma kit No. 690-A. Effluent samples were also analyzed for viable cell counts and for
total protein. The optical density at 650 nm of the reactor effluent was continuously
monitored by a spectrophotometer (Spectrbnic 20D, Milton Roy Co.) installed in-line.
This spectrophotometer was connected to a computer that had an A/D data acquisition
board (CIO-DAS08PGA, Computer Boards, Inc.) installed. A LABTECH NOTEBOOK
(Laboratory Technology Co.) application running on that computer logged data from the
spectrophotometer every 60 seconds. Data recording was started at 15 minutes before the
step change and maintained for 90 to 105 minutes. Viable cell enumeration and total
protein measurement protocols are detailed below.
9
/
17
Viable cell enumeration protocol
1) Sequentially dilute sample into dilution tubes containing 9ml of phosphate buffer
(consisting of Na2HPO4i 2.691mM and KH2PO4, 2.998mM) by transferring Iml
aliquots.
2) Transfer Iml aliquots using Drop Plate method from ones with the most likely
concentration for colony formation, to R2A agar and PIA (Pseudomonas isolation
agar).
3) Incubate all plates in 37°C incubator for 24 hours, then count the colonies.
4) The number from PIA is just Pseudomonas aeruginosa concentration and the big,
white, opaque colonies on R2A are Klebsiella pneumoniae.
Total protein measurement protocol
1) Discard the supernatant of the centrifuged biofilm samples. Mix the pellet with 2 or
4mI of TEP solution (IOmM Trizma Hydrochloride, ImM EDTA and ImM
phenylmethylsulfonyfluoride) at pH 8.
2) Disrupt cells using sonication (TORBEO 36810, Cole-Parmer) by three pulses at 10
seconds for each pulse.
3) Centrifuge again and use the supernatant for total protein assay.
4) Set the wavelength of spectrophotograph at 725nm.
5) Follow the Sigma 690-A diagnostic kit instruction for the rest of assay.
18
Viscometry
A set of experiments was conducted using viscometry to detect changes in biofilm
mechanical properties when challenged with different chemicals. Viscosity is the measure
of the internal friction of a fluid. It depends on the concentration of the polymeric
materials, the type of these polymers and temperature of the samples.
Biofilm samples were collected by scraping biofilms from all wetted surfaces of
an untreated annular reactor after seven days of biofilm growth in continuous flow mode.
These samples were stored at 3 ~ 5 0C until use. A cone/plate (CP40) type viscometer
(LVDV-11++CP, Brookfield Engineering Laboratories, Inc.) was used for biofilm
viscosity measurement. It consists of a spinning cone and a stationary plate in which
samples are held. This type of viscometer only requires 0.5 ml of sample that is very
suitable for a biofilm study since generating of large amount of biomass is very difficult.
A computer was connected to the viscometer using a serial port. Data recording and cone
rotation speed could be automatically controlled using a software application called
WinGather (Brookfield Engineering Laboratories, Inc). The temperature inside the
measuring chamber of viscometer was controlled by a circulating water bath (RTE-221,
NESLAB). The cone’s revolution speed was fixed at 0.6 rpm to avoid shear-thinning and
also to simulate the shear stress (about 4 dynes/cm2) experienced in the annular reactor.
First 0.5 mL of biofilm sample was transferred into the plate of cone/plate viscometer.
Viscosity of this biofilm sample was measured after assembling the plate to the
viscometer. Then the plate was disassembled and 0.1 mL of desired chemical solution
19
was added into the plate. The viscosity of the treated biofilm sample was then measured.
The detailed procedure to perform viscosity measurement is described below.
Viscosity measurement protocol
1) Turn on the water bath (RTE-221, NESLAB) and stabilize the temperature at 25 ± 0.1
0C.
2) Turn on the viscometer, screw in the rotating cone (type cp40) after autozeroing
according to instructions in the viscometer’s manual.
3) Drop the viscometer temperature probe into the water tank of the water bath, and
make sure the temperature readings from the two instruments are the same.
4) Connect the data communication cable from the viscometer to the serial port of a
computer.
5) Turn on the computer and open up the WinGather application.
6) Press the print button on the front panel of the viscometer for about 20 seconds to
enable data transfer between the viscometer and computer.
7) If there is no data showing on the WiriGather’s window, select a different port, either
COMl or COM2, by clicking on the communication port button on WinGather’s
menu.
8) Transfer 0.5 mL of biofilm sample into the plate (cup shape) of viscometer.
9) Assemble the plate to the viscometer.
10) Set the rotation speed to 0.6 rpm using the speed selection button on the viscometer
front panel.
20
11) Turn on the cone rotation by pressing the on/off button on the viscometer front panel.
12) Wait two or more revolutions of the cone to let the viscosity reading stabilize.
13) Record viscosity data in a time stop fashion by putting 30 in the data field and 00:10
in the time interval field. This will record data for 5 minutes with total of 30 data
points.
14) Turn off the cone rotation and disassemble the plate. Add 0.1 ml of desired chemical
solution into the plate, then reassemble.
15) Switch on the cone and repeat steps 12 and 13.
Source of Reagents
All general chemicals, such as various salts and glucose, were bought from Fisher.
Surfactants were from Sigma. Dequest 2006 (Aminotri(methylene-phosphonic acid),
pentasodium salt) was bought from Solutia. Enzymes, including amylase (10-30 units per
mg solid for a-Amylase, from porcine pancreas), cellulase (5,000 units, from aspergillus
niger), lysozyme (50,000 units per mg protein, from chicken egg white, 3X crystallized,
dialyzed and lyophilized), nuclease (from penicillium citrinum lyophilized powder) and
protease (type 1,10 units/mg, from bovine pancreas), were all bought from Sigma.
RESULTS
This section reports the results of the two types of experiments performed,
viscometry of biofilm suspensions and detachment from an operating reactor. In both
types of experiment, data were collected before and after a variety of chemical treatments.
Data Analysis and Repeatability
Viscometry
Viscosity results were obtained before and after addition of treatment chemicals,
but special care had to be used to obtain satisfactory results. Two practical complications
were encountered in performing viscometry, both of which were thought to reflect the
natural heterogeneity of a scraped biofilm suspension. Viscometry of untreated biofilm
samples always resulted in an oscillatory response with a period corresponding to one full
rotation of the viscometer cone (Fig. I). Although oscillation was observed, the mean
value of the apparent viscosity prior to treatment was relatively stable. After the addition
of various salts, viscosities tended to become stable arid nonoscillatory. This behavior
could be attributed to the fact that the large aggregates of biofilm initially presented in
suspension were disassociated into many small particles after the addition of salts.
2 2
MgCI2 added
10
11
Time (min)
Figure I. Viscosity of biofilm sample before and after addition of 0.22M MgCl2. Results
from control experiment are represented by filled circles (•).
A second problem encountered in viscosity measurements was the lack of ability
to control the initial viscosity of each biofilm sample. This was also attributed to the
inherent heterogeneous nature of the biofilm suspension. To facilitate the comparison of
viscosity changes measured from different starting points, several experiments were
23
performed over a range of initial viscosities. In each experiment viscosity data were
averaged for the last two revolution periods prior to the addition of a chemical. This
average constituted the initial viscosity. Initial viscosities typically ranged from 20cP to
400 cP. A chemical was added and the post-treatment viscosity was measured. The initial
and final viscosities were correlated (Figure 2). The correlation of viscosities of biofilm
samples after addition of each chemical at a specific concentration with viscosities of the
original biofilm samples before the addition of chemicals was established by using linear
regression. Linear regression parameters were used to predict the post-treatment viscosity
corresponding to an initial viscosity of 150 cP. The standard errors of the prediction
(Using MiniTab version 12, MiniTab Inc.) were used to construct the error bars.
Interpolated post-treatment viscosities corresponding to an initial viscosity of 150 cP
were used to compare the effectiveness of viscosity changes by additions of various
chemicals. The changes of viscosity brought by the treatment chemicals themselves in the
absence of biofilm only contributed to about ±6 cP or ±4% of the initial viscosity of 150
cP. An exception was protease, which itself increased the viscosity about 24 cP or 16%
(see Appendix A).
Experiments with enzymes required a different approach for data analysis. Simply
blending biofilm samples with enzyme solutions resulted in instantaneous changes of
viscosity. These viscosity changes should not be attributed to enzymatic lysis. Viscosity
data were collected over time at 20-40 minute intervals over a two-hour period. The
viscosity measured at time zero (immediately after mixing of biofilm and enzyme
solutions) was used to normalize subsequent measurements (Fig. 3). Decreases of the
24
normalized viscosity from 1.0 represented decreases of viscosity in biofilm sample.
Linear regressions were used as described above to estimate the solution viscosity ratio at
a fixed time (80min) after enzyme addition.
Y=0.08X+14.0
error of Slope=O.02
r :=0.855
P=0.02
100
150
200
250
300
350
400
450
Initial viscosity of biofilm sample (cP)
Figure 2. Correlation of viscosities of biofilm samples after addition of MgCl2to the
initial viscosities. The line was fit using least squares regression. Dashed line indicates
interpolation to predict post-treatment viscosity corresponding to an initial viscosity of
150 cP.
Lysozyme
1 .0
0.8
CO
8co
"> 0.6
T3
CD
N
ro
E
o 0.4
Z
0.2
Y=-0.0056X+1.14
error of slope=0.0012
r2=0.49
P=0.0003
0.0
j ______ i_________ i_____ i________ i_________ i________I_______
0
20
40
60
80
100
120
140
Time (min)
Figure 3. Viscosity changes of biofilm samples with time after addition of lysozyme.
Different symbols represent different trials. The line was fit using least squares
regression.
26
To test the repeatability of viscometry data, sixteen individual control experiments
were performed. They are graphically presented in Figure 4. A linear relationship was
found with r2of 0.94.
Y = 1 .1 4 X -1 4 .2
e rro r of Slope = O.075
r2= 0.94
P = 0 .0001
B iofilm in itia l v is c o s ity (cP )
Figure 4. Viscosity of untreated control biofilm samples. The line was fit using least
squares regression.
Annular reactor step change experiments
Three different measurements were used to track the total amount of biomass
detached from a continuous flow biofilm reactor following an environmental step change:
27
a) effluent turbidity, b) total protein in effluent and from biofilm sample slides, and c)
effluent cell count. The percentage of the biofilm that was detached was calculated two
ways. The first was the cumulative amount of total protein discharged in the effluent over
a 75 minute period following the treatment divided by the initial total protein inside the
biofilm reactor.
Effluent volume (ml) x V Effluent total protein (//g/ml)
% detachment, effluent =-----------------------------——-------------------------;------------ r - (I)
Total wetted area (mm ) x Initial slide total protein (/zg/mm )
where effluent volume =15 (min) x 31 (ml/min) = 465 (ml)
Total wetted area = 1600.4 (mm2)
The percentage of biofilm detached was also calculated based on biofilm slide sample. It
was the difference of total protein from a biofilm slide coupon before and after treatment
divided by the initial total protein inside the biofilm reactor.
% detachment, slide=
A slide total protein W m rn ^ )
Initial slide total protein (jug/mm )
(2)
The correlation of biofilm detachment represented by the total protein measurement from
effluent and biofilm slide coupon is shown graphically in Figure 5. Although the
relationship is noisy (r2 =0.61), the slope of the regressed line is not statistically
significantly different from one (m=0.95; p=0.89) indicating that these two measures are
consistent. Integrated effluent protein concentration and integrated effluent optical
density changes were also positively correlated (r2=0.85) (see Fig. 6).
2 8
Y=O. 95X
error of Slope=O.065
P=O.0002
Detachment (% based on effluent)
Figure 5. Correlation of percentage detachment determined by scraping bio film from
sample slides and by integrating biomass appearing in the effluent. The line was fit using
least squares regression.
29
Y=0.332X+4.15
error of slope=0.048
^=0.83
P=0.0003
S (Aprotein) (mg)
Figure 6. Correlation of the total amount of biomass detached in effluent by integrating
total protein and by integrating optical density changes. The line was fit using least
squares regression.
30
Electrostatic Interaction
Influence of different salt additions
Viscosity changes by addition of various salts are compared in Figure 7 at an
ionic strength of 0.3 for each chemical. The addition of cations other than iron decreased
biofilm viscosity by an average of 73%. Treatments with Fe2"1"and Fe3+increased biofilm
viscosity about 50%, but they were not statistically significant as the P value was greater
than 0.05 (0.095 and 0.1248, respectively) (see Table 3). The method of 2-sample T test,
where variances are not necessary equal, was used in this and following statistical
analyses for all viscometry experiments. The probability of each individual experiment
differing from the control was presented as P value. A 95% confidence level was used to
reject the hypothesis that there are no statistically significant viscosity changes by
addition of various chemicals when the P values are greater than 0.05. A non-ionic
treatment with 0.47 M sucrose (osmotically equivalent to 0.3M NaCl) Caused a viscosity
reduction of 7% (not shown in Fig. 7).
Table 3. Statistical analysis of the changes of biofilm viscosity at ionic strength of 0.3
when compared to the control.
P
LiCl
NaCl
KCl
CaCl2
MgCl2
FeCl2
Fe(NOg)^
Sucrose
0.0002
0.0001
0.0718
0.0014
0.0002
0.095
0.1248
0.0314
31
250
1.
2.
3.
4.
5.
200
Control
LiCI
NaCI
KCI
CaCI2
6. MgCI2
CL
7. FeCI2
T
8. Fe(NO3)
3/3
S- 150
Z'
"55
8Crt
> 100
T
T
T
1
2
3
X
4
5
6
7
8
Salt
Figure 7. Viscosity measurement of biofilm samples with addition of various salts, all at
ionic strength of 0.3. Error bars represent the standard errors.
Effect of ionic strength
The viscosity of biofilm was shown to be influenced by the ionic strength of
various salt treatments. The change of biofilm viscosity upon addition of salts was small
when the ionic strength of treatment was less than about 0.1. Under these conditions, the
32
final viscosity ranged from 60% to 105% of the initial viscosity (80%~93% for NaCl,
97%~105% for CaCl2, 61%~80% for FeCl2 and 73%~103% for Fe(NO3)3). These changes
of viscosity were considered insignificant (P > 0.05, Table 4) based on statistical analysis.
However changes from addition OfFeCl2 resulted in P values ranging from 0.0001 to
0.0018 (Table 4) at low ionic strength. Biofilm viscosity was dramatically changed when
the ionic strength was great than 0.1. It dropped significantly to about 20% of the initial
value with addition of sodium (Fig. 8) and calcium ions (Fig. 9), but increased to about
150% with addition of Fe2+Or Fe3"1"ions (Fig. 10 and 11, respectively), which was not
statistically significant. Since addition OfFeCl2 or Fe(NO3)3 also caused the pH to
decrease from 6.5 to 2.1, other experiments were performed to investigate the effect of
pH change alone by acidifying biofilm samples with 6N HCL Biofilm viscosities
decreased about 40% from the initial value of 150 cP when the pH of biofilm samples
changed from 6.5 to 2.0, which indicates that the increases of biofilm viscosity by
addition of Fe2"1"and Fe3"1"might even be higher if the pH of the biofilm sample were held
steady.
33
O
80
0.001
Ionic Strength
Figure 8. The influence o f ionic strength on biofilm viscosity when treated with NaCl.
The initial viscosity was 150 cP. Error bars represent the standard errors.
34
& 100
>
80
0.001
Ionic Strength
Figure 9. The influence o f ionic strength on biofilm viscosity when treated with CaCl2.
The initial viscosity was 150 cP. Error bars represent the standard errors.
35
S
150
0.001
Ionic Strength
Figure 10. The influence o f ionic strength on biofilm viscosity when treated with FeCl2.
The initial viscosity was 150 cP. Error bars represent the standard errors.
36
250 -
200
-
o. 150
‘5)
8
.$2
> 100
-
0.01
0.1
Ionic Strength
Figure 11. The influence o f ionic strength on biofilm viscosity when treated with
Fe(NO3)3. The initial viscosity was 150 cP. Error bars represent the standard errors.
37
Table 4. Statistical analysis of the changes of biofilm viscosity at different ionic strength
when compared to the control.
P
1=0.002
0.0766
1=0.01
0.3002
NaCl
1=0.1
0.002
1=0.3
0.0001
___________________________________ CaCh_________________________
1=0.002
1=0.01
1=0.03
1=0.3
1=0.663
P
0.6908
0.2262
0.9796
0.0044
0.0001
___________________________________ FeCl2_____________
1=0.002
1=0.01
1=0.03
1=0.3
P
0.0018
0.0001
0.0002
0.1796
Fe(NO3)3
P
1=0.002
0.05
1=0.01
0.8604
1=0.03
0.252
1=0.3
0.1616
1=0.6
0.5928
Step change experiments were conducted in an annular reactor to further test the
effect of ionic strength on actual biofilm detachment. Step additions of various salts in the
influent of a continuous flow biofilm reactor resulted in removal of approximately 5 to 75
percent of the biofilm within a few hours. A typical result is plotted in Figure 12 for a
step change addition of 0.3M NaCl. Biofilm detachment was reflected in the transient
increase in the concentration of biomass present in the reactor effluent as measured
optically (Figure 12A), as total protein (Figure 12B), and by viable plate counts (Figure
12C). A control experiment in which the same volume of buffer lacking NaCl was added
did not cause detachment as indicated by stable levels of biomass in the effluent (Figure
13 ).
38
S
20
CL 15
1e+9
1 e +8
S
1e+7
1 e+6
T im e (m in)
Figure 12. The measurements of biomass concentration versus time in the effluent of a
biofilm reactor following biofilm treatment with 0.3M NaCl. Treatment was initiated at
t=0 and terminated at t=60 minutes. Biomass was determined by turbidimetry (A), total
protein (B), and by colony formation on agar plates (C). In panel C, (•) denotes P.
aeruginosa and (■) denotes K. pneumoniae.
39
ro 10
1 e+9
Z-
1 e+8
o
1 e+7
1 e+6
30
4£
T im e (m in)
Figure 13. The measurements of biomass concentration versus time in the effluent of a
biofilm reactor in an untreated control. Biomass was determined by turbidimetry (A),
total protein (B), and by colony formation on agar plates (C). In panel C, (•) denotes P.
aeruginosa and (■) denotes K. pneumoniae.
40
Biofilm detachment was also demonstrated by scraping and assaying biofilm from
slides samples before and after the treatment. The percentages of biofilm removal as
determined by total protein assay of biofilm samples and by integration of the increased
protein released in the effluent are tabulated in Table 5. Net detachment caused by
various salts was 48 percent by 0.3M sodium chloride, 48 percent by 0.22M calcium
chloride, and 23 percent by 0.21M magnesium chloride. A pH downshift resulted in
average of 15% of biofilm detachment. A pH upshift caused more than double that
amount averaging 46%. The net detachment caused by sucrose was only approximately
7.5%. In another sodium chloride treatment experiment, No.6, chloramphenicol (200
mg/L), a protein synthesis inhibitor, was added to block any biological responses that
could cause biofilm detachment. This generated more biofilm detachment (69%) than
using sodium chloride alone (48%), indicating, that de novo protein synthesis was not
involved in mediating biofilm detachment.
Table 5. Summary of biofilm detachment caused by environmental step changes of
various salts and related compounds.
No #
Treatment
% detachment,
% detachment
Average
reactor effluent
biofilm slides
%
I
Control
0
4
2
2
NaCl (0.3M)
60
55
58
3
NaCl (0.3M)
52
29
40
4
NaCl (0.3M)
51
42
47
5
NaCl (0.3M), 4.5 hrs after No.4
6
8
7
6
NaCl (0.3M), chloramphenicol
63
74
69
(200 mg/L) added 3 minutes before
7
CaCl2 2H20 (0.221M)
48
48
48
8
MgCl26H20 (0.206M)
24
22
23
9
pH (6.4 -> 2.9)
20
11
16
10
pH (6.6 —> 11.2)
48
45
47
11
Sucrose (0.47M)
5
10
8
Species compositions of the biofilm before and after treatment were compared to
test whether biofilm treatment caused preferential detachment of one of the two microbial
species (Figure 14). One would expect that a treatment that, for example, increased the
fraction of Pseudomonas in the effluent should concomitantly reduce the fraction of
Pseudomonas in the biofilm. Neither this relationship nor any other pattern could be
discerned by the analysis presented in Figure 14, suggesting that both species are
similarly detached by the various treatments.
42
10
11
Experim ent No.
Figure 14. Comparison of biofilm species composition before and after treatment. The yaxis is the ratio of the percentage of P. aeruginosa in the sample after treatment to the
percentage of P. aeruginosa in the sample before treatment. Symbols indicate effluent
(O) and biofilm ( • ) samples.
Cation bridging
Crosslinking by divalent cations has been considered to play a major role in the
structure of biofilm. Two chemicals treatments were tested to explore this idea: EDTA
(ethylene diamine tetraacetic acid), a well-known chelating agent, and Dequest 2006, a
phosphonate used in water treatment and reported to be superior to such chelants as
EDTA and EGTA in terms of binding with calcium. Viscometry experiments showed that
43
Dequest 2006 reduced biofilm viscosity by 50% whereas EDTA reduced it only 20%
(Fig. 15). From statistical analysis these viscosity reductions were significant in
comparison to the control. P value were 0.0001 and 0.002 for EDTA and Dequest 2006
(Table 6), respectively.
In situ step changes using EDTA and Dequest 2006 were also performed. The
results presented in Table 7 shown an average of 26% detachment for EDTA and 27%
detachment for Dequest 2006.
Table 6. Statistical analysis of the changes of biofilm viscosity treated with EDTA or
Dequest 2006 when compared to the control.
■ EDTA
P
Dequest 2006
0.0001
0.002
Table 7. Summary of biofilm detachment caused by environmental step changes of
chelants.
Treatment
N o#
% detachment,
% detachment
Average
reactor effluent
biofilm slides
%
I
Control
0
4
2
12
EDTA (0.01M, 2.92g/L)
13
40
26
13
Dequest 2006 (Ig/L)
28
26
27
160
1. Control
2. ED TA
3. D e q ue st 2006
Figure 15. Viscosity measurement of biofilm samples treated with EDTA or Dequest
2006. Error bars represent the standard errors.
The elemental composition of selected metals in biofilm samples was also
measured and is reported in Table 8. The most dominant elements, such as potassium,
sodium and magnesium, are components of the medium. Calcium and magnesium,
however, were considered to be selectively accumulated in the biofilm, because the ratios
of these metal elements’ concentrations in biofilm samples over their respective
concentrations in medium were high, about 75 and 43, respectively. In comparison the
ratios for iron, potassium and sodium were only about 2, 0.4, and 0.4, respectively. There
were no detectable metal elements in the water supply.
Table 8. Metal elements in biofilm samples (normalization of raw data was performed
based on dry weight).
Dry
Total
Ca
Fe
Mg
K
Na
weight
protein
mg/L
mg/L
mg/L
mg/L
mg/L
mg/mL
mg/mL
No. I
8.8
1.91
11.7
0.7
16.6
434
220
No. I,
normalized
I
0.22
1.33
0.08
1.89
49.32
25.00
No.2
25.4
6.84
17.1
3.8
39.4
1149
709
No.2,
normalized
I
0.27
0.67
0.15
1.55
45.24
27.91
Hydrophobic Interaction
Various types of surfactants were tested to investigate the influence of these on
the structural integrity of biofilm. These included SDS (sodium dodecyl sulfate), an
anionic surfactant; hexadecyltrimethylammonium bromide, which is cationic; and Tween
20 and Triton X-100, which are both non-ionic surfactants. With addition of SDS and
Tween 20, biofilm samples appeared to be more transparent and swollen than the original
ones. These two agents caused the smallest changes in viscosities, a 12% increase and 9%
decrease, respectively (Fig. 16). Triton X-100 and hexadecyltrimethylammonium
bromide reduced viscosities of biofilm samples about 43% and 38%, respectively (Fig.
16). Lignosulfate, regarded as a dispersant, caused the biofilm viscosity to decrease about
23%. However, only the viscosity reductions from Triton X-100 and
46
hexadecyltrimethylammonium bromide were statistically significant with P values of
0.0001 and 0.001 (Table 9), respectively. Biofilm viscosity changes by treatments of
SDS, Tween 20, and lignosulfate may not be considered different from the control
statistically (P > 0.05, Table 9).
In situ step change experiments provided direct information about biofilm
detachment by some of these agents. Net detachment was approximately 71 percent after
addition of 1000 mg/L sodium dodecyl sulfate (SDS), 26 percent by 1000 mg/L Tween
20, and 48% by 1000 mg/L Triton X-100 (see Table 5, the control is included for
comparison). Biofilm detachment could be induced even when the viscosity change was
small or positive, as with SDS. A protein synthesis inhibitor, chloramphenicol
(lOOmg/L), was used again in a SDS duplicate experiment (No. 16). The protein
synthesis inhibitor increased biofilm detachment by 20%. This indicates that biologically
mediated responses were not a mechanism that caused biofilm detachment by addition of
this surfactant.
Table 9. Statistical analysis of the changes of biofilm viscosity treated with surfactants
when compared to the control.
SDS
Triton X-100
Tween 20
Hexadecyltrimethyl-
Lignosulfate
ammonium bromide
P
0.3986
0.0001
0.131
0.001
0-1902
47
(1). Control
(2). SDS
(3). Triton X-100 (4). Tween-20
(5)
. Hexadecyltrimethylammonium
Bromide
(6)
. Lignosulfate
CL
o
Zr
(Z)
8
5
Surfactants
Figure 16. Viscosity measurement of biofilm samples with additions of various
surfactants. Error bars represent the standard errors.
48
Table 10. Summary of biofilm detachment caused by environmental step changes of
surfactants.
N o#
Treatment
% detachment,
% detachment
average
reactor effluent
biofilm slides
%
I
Control
0
4
2
14
SDS (1000 mg/L)
38
88
63
15
SDS (1000 mg/L)
90
68
79
16
SDS (1000 mg/L), chloramphenicol
94
88
91
(100 mg/L) added 3 minutes before
17
Tween 20 (1000 mg/L)
34
20
27
18
Triton X-100 (1000 mg/L)
53
43
48
Depolymerization
Two types of agents that could degrade the biofilm matrix were tested: oxidants
and enzymes. With addition of hypochlorite (50 mg/L) biofilm viscosity decreased about
40% (see Fig. 17). Treatment with monochloramine only caused a 10% decrease of
biofilm viscosity (Fig. 17). These viscosity reductions were all statistically significant
based on the P values of 0.0026 and 0.0012 for hypochlorite and monochloramine,
respectively (Table 11).
1: Control
2: Hypochlorite (50mg/L)
3: Monochloramine (50mg/L)
Q, 100
Chlorines
Figure 17. Viscosity measurement of biofilm samples treated with chlorine (pH 7.7) and
monochloramine (pH 7.3). The concentration refers to total chlorine. Error bars represent
the standard errors.
Enzymatic lyases are well known to cleave specific polymer bonds. Their
influence on biofilm structure was tested by viscometry. Lysozyme and protease were the
most effective ones in reducing biofilm viscosities (Fig. 18). On average, the enzymes
reduced biofilm viscosity by 35% compared to the control, which increased 27% during
the same 80 minute period (27% for amylase, 28% for cellulase, 46% for lysozyme, 34%
for nuclease and 40% for protease). However, only the viscosity reductions from
50
lysozyme (P = 0.01) and protease (P = 0.04) were statistically significant. Amylase,
cellulase, and nuclease all reduced biofilm viscosity in a insignificant amount from
statistical analysis (P > 0.05, Table 11). Viscosity reduction with addition of these lyases
was far less than that from treatment with salts (average of 73%).
1.
2.
3.
4.
5.
6.
E
o
CO
Control
Amylase
Cellulase
Lysozyme
Nuclease
Protease
CD
0)
O
)
C
ro
JZ
O
I
O
CO
Dd
Enzymes
Figure 18. Viscosity measurement of biofilm samples with additions of enzymatic lyases.
Error bars represent the standard errors.
51
Table 11. Statistical analysis of the changes of biofilm viscosity treated with degradation
agents when compared to the control.
P
OCl'
NH2Cl
Amylase
Cellulase
0.0026
0.0012
0.10
0.13
■
Lysozyme
Nuclease
Protease
0.01
0.07.
0.04
Biofilm detachment caused by these depolymerization agents was also measured
in situ in the biofilm reactor by a step change addition. Results are listed in Table 6.
Removal by chlorination was about 50% higher at pH=10.9 (average of 65%) than
pH=6.4 (average of 42%). This is consistent with Characklis’ report (Characklis, 1980)
and with data on depolymerization of polysaccharides (starch) by chlorine, for which the
optimum pH is greater than 7 (Whister and Schweiger, 1957). There was no notable
difference in the amount of detachment caused by hypochlorite and monochloramine.
52
Table 12. Summary of biofilm detachment caused by environmental step changes of
chlorine, monochloramine and lysozyme.
No#
Treatment
% detachment,
% detachment
average
reactor effluent
biofilm slides
%
I
Control
0
4
2
10
pH (6.6->11.2)
48
45
46
19
Chlorine (15 mg/L, pH=6.4)
33
62
47
20
Chlorine (15mg/L, pH=6.4)
52
22
37
21
Chlorine (15mg/L, pH=10.9)
60
70
65
22
NH2Cl (lOOmg/L)
68
64
66
23
NH2Cl (25mg/L)
32
18
25
24
NH2Cl (25mg/L)
72
68
70
25
NH2Cl (lOmg/L)
27
17
22
26
NH2Cl (7.5mg/L)
33.0
42.3
38
27
NH2Cl (5mg/L)
52
60
56
28
NH2Cl (25mg/L), NaCl (0.3M)
64
45
54
36
45
40
added 60min later
29
Lysozyme (0.5g/L)
Relationship between Biofilm Detachment and Viscosity Change
When all treatments were grouped together, there was no apparent relationship
between the reduction in biofilm viscosity caused by a particular agent and the amount of
biofilm detachment induced by that agent (Figure 19, Table 13). The correlation
53
coefficient (r2) for all treatments was 0.01, indicating that there is no general relationship
between viscosity reduction and biofilm detachment. Considering the salt group alone
(salts, chelants, sucrose), however, a noisy, but positive, correlation could be discerned
(rW).57, slope=0.392). The values of the slopes from an analogous linear regression for
surfactants and depolymerization agents were -0.068 and 0.676, respectively. This
indicates that these three groups of chemicals exhibited different mechanism(s) in the
changes of biofilm structure that caused detachment. Because of the high variability and
uncertainty of each individual data point in Fig. 19, which invalidates the basic
assumption of equal variability of each data, a multivariable variance statistical analysis
could not be performed. But, if one assumed the variance of each point is equal, the salt
group would be statistically different from the other two groups, which also can be
visually discerned by drawing a line that separates salts group from the other two.
Table 13. Summary of linear regression results from correlation of the percentage of
biofilm detachment and the percentage of biofilm viscosity reduction.
Linear regression
R2
Y = 0.392X + 9.22
0.57
Surfactants
Y = -0.068X + 37.33
0.003
Depolymerization agents
(OCl", NH2Cl, lysozyme)
Y = 0.676X+ 17.59
0.46
All chemical
Y = 0.074X + 33.43
0.01
Chemicals
Salts, chelants, sucrose
54
80
70
—
A sds
5?
c
^NaCI
Iysozymer.
50
^H2CI
E
iTiton
®CaCI2
s:
o
B 40
<u
-
Chlorine
"O
Request
A
• EDTA
Tween
^vigCi2
#pH upshift
I
-10
^Sucrose
^Control
™ I
I
0
10
20
I
I
30
40
l
50
l
l
60
70
I
I
80
90
100
viscosity reduction %
Figure 19. Correlation of the percentage of biofilm viscosity reduction and the percentage
of biofilm detachment. Symbols indicate results from salt group (#), including pH and
sucrose, from surfactant group (A) and depolymerization group (O).
55
Time Scale of Biofilm Detachment
The relative rapidity of the detachment response following a chemical treatment
(e.g., immediate versus delayed) provides clues to the underlying mechanisms of
cohesion and detachment. In particular, it could be expected that challenges to the biofilm
by disruption of physico-chemical forces (e.g., electrostatic or hydrophobic interactions)
or rapid degradation reactions (e.g. with the chlorine) would result in detachment
occurring very rapidly, on a time scale of seconds or minutes. On the other hand,
challenges to the biofilm matrix that are based on relatively slow degradative action (e.g.,
added enzymes or induced release of lyases from the cell in response to a treatment)
would be expected to require more time to trigger detachment.
To address the question of the time scale of biofilm detachment, we defined two
measures of the detachment response time. The first of these was the time to the
maximum effluent optical density. The second was the integrated effluent optical density
over the first 18 minutes, which corresponds to the average residence time, divided by the
75 minutes treatment period. Both measures are presented graphically in Figure 20. This
analysis indicates that the “salt” group, including ionic (including salts, No.2-8), osmotic
(sucrose, No.11), and chelant treatments (No. 12 and 13) all induced detachment rapidly,
with peak times averaging 5 minutes. These treatments likewise yielded a large fraction
of the total detachment, averaging 45 percent, within the first 18 minutes. Treatment with
hypochlorite (No.21) also induced rapid detachment (peak time 8 minutes). By
56
comparison, treatment with SDS (No.14-16), Tween 20 (No. 17), pH shifts (No. 18) and
monochloramine (No.22-27) exhibited relatively delayed responses. The peak times were
averaged 20 minutes for SDS, 17 minutes for Tween 20, 35 minutes for pH shift, 32
minutes for monochloramine, and 29 minutes for lysozyme. Similarly, the fraction of the
total detachment occurring in the first 18 minutes was lower than for the ionic treatments;
it averaged 27 percent for SDS treatment, 24 percent for Tween 20, 8 percent for the pH
treatments, 21 percent for monochloramine, 12 percent for lysozyme. Results from this
data analysis suggested that salts caused biofilm detachment via a different mechanism(s)
than what causes detachment after exposure to surfactants, pH shift, monochloramine and
lysozyme.
57
nn .
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
Experiment No.
Experiment No.
Figure 20. Comparison of relative rapidity of biofilm detachment with various treatments.
Peak time (A) is defined as the time to the maximum effluent optical density. Relative
rate (B) is defined as the integrated effluent optical density over the first 18 minutes
divided by the 75 minute treatment period. The dashed line in panel B indicates the
relative rate that would be measured if the detachment rate were uniformly distributed
over the 75 minute measurement period.
DISCUSSION
Experiments measuring biofilm detachment and the apparent viscosity of biofilm
suspensions in response to diverse chemical treatments suggest that multiple types of
cohesive forces maintain biofilm structural integrity. These experimental results are
consistent with a model of biofilm cohesion in which two types of structural elements,
namely EPS and cells, are crosslinked by a combination of electrostatic and hydrophobic
interactions.
The evidence that microbial cells are structural elements is limited to the
observation that the enzyme lysozyme, which specifically degrades the bacterial cell wall,
reduces the viscosity of biofilm suspension (46%). Results from step change experiment
treated by lysozyme showed that cell viability from slide coupon decreased about an
order of magnitude after treatment, while it only removed about half of the biomass as
measured from the loss of total protein on the slide. This indicated that in addition to the
loss of biomass by detachment, there was a loss of cell viability showing that whole cells
were attacked by lysozyme. If cells were simply inclusions in the biofilm matrix that bore
no mechanical load, then disruption of the cell envelope would be expected to lead to
holes in the biofilm matrix, but not to detachment or aggregate dispersal. An alternative
explanation for the effects of lysozyme treatment is that peptidoglycan, the cell wall
structural material, is also a component of EPS.
59
Proteins play a role in biofilm cohesion based on the observation that protease
caused significant reduction in biofilm viscosity (40% compared to control). Proteins
may be either structural polymer in the EPS itself or may function as a bridge between
polymer strands or between cells and polymers. The potential importance of protein in
contributing to biofilm structural integrity is consistent with some recent reports that
proteins constitute a much larger fraction of EPS than previously recognized (Nielsen,
et.al., 1996).
Interpretation of Viscometry Study
The viscosity of polymer solution depends on the concentration of polymers and
can be represented mathematically by the expression of Tanford (1961):
^ = M + k M 2C
where p = viscosity of polymer solution
C = polymer concentration
[r|] = intrinsic viscosity
k = coefficient
The intrinsic viscosity can be determined by the Mark-Houwink (Tanford, 1961)
equation:
(3)
60
[%] = W
(4)
Where M= molepular weight
a = an exponent dependent upon the stiffness of the chain.
Biofilm samples are highly heterogeneous. The majority of the biofilm viscosity is
contributed by few big chunks of biofilm aggregates. In this heterogeneous environment,
the local concentrations of biopolymer in these .big chunks of biofilm may change very
little or not at all, despite a total volume increase of 16.7% with addition of treatment
chemicals. This was confirmed from the statistically insignificant change of biofilm
viscosity in the control viscosity experiment. The viscosity in control changed to 157+6
cP from the initial viscosity of 150 cP. The molecular weight of biofilm polymer during
chemical treatment could be changed as a result of degradation of polymer (for example,
chlorine and lyase), or damage of the structure of biopolymer by disrupting crosslihking
interactions that hold polymer together. Therefore the changes of viscosity by chemical
addition can be related to changes in molecular weight of the polymer or the stiffness of
the polymer chain. The polysaccharides, which are major components of biofilm
extracellular polymers, are usually random coils with a - 0.5 to 0:8 (Christensen and
Characklis, 1990). If the viscosity reduction by treatment of certain chemical were 50%,
this would translate to about 50% reduction of intrinsic viscosity from equation (3). From
equation (4) with the assumption of a = 0.5, a 50% of reduction in intrinsic viscosity
61
would correspond to a 75% reduction in molecular weight of biofilm polymer. This
represents a huge change in biofilm polymer structure.
Electrostatic Interaction
Electrostatic interactions clearly play a role in biofilm cohesion. Mono and
divalent cation (Li, Na, K, Mg, Ca) chloride salts all caused large reductions in biofilm
viscosity as the ionic strength of the treatment exceeded about 0.1. Treatment of biofilm
reactors with salts (Na, Mg, Ca) caused biofilm detachment. The DLVO theory explains
very well why there were no significant viscosity changes in the range of ionic strength
1=0.001 to 0.1 in our viscometry experiments. It could not explain the sudden drastic
changes of viscosity at high ionic strength (I>0.1). The observation that the mechanism
for bacterial cohesion differs from the prediction of DLVO theory when the electrolyte
concentration is increased over 1=0.1 has been reported previously in studies of floe
stability (Zita and Hermansson, 1994) and bacterial adhesion to solid surfaces (Gordon
and Millero, 1984). One of the possible explanations for this could be that the binding
capacity of the cations that crosslink the exopolymers changes with change of ionic
strength. It was reported (Svensson et ah, 1991; Schiewer and Volesky, 1997) that the
binding constants of divalent cations, such as calcium, could decrease by roughly 2 orders
of magnitude at high ionic strength (I=I) as compared to low ionic strength (1=0.002).
Iron, in the form of Fe2+, Fe3"1"or both, probably crosslinks more strongly with
negatively charged biofilm structural elements. FeCl2 and Fe(NO3)3were the only salts
62
that when added to biofilm suspensions in high concentrations caused significant
viscosity increases (52% and 45% at 1=0.3, respectively). All other salts caused
significant viscosity reduction (avg. 73%) when added at the same ionic strength. This
iron phenomenon was also found by Sutherland (1980). It was found that
polysaccharides, separated from several marine and freshwater bacteria, precipitated only
with an addition of Fe2"1"or Fe3+and there was no effect when Na+, Ca2"1", Cu2+, Mg2"1", Zn2"1",
or Al3"1"was added. Caccavo et al. also reported that Fe3"1"was a better crosslinking ion in
activated sludge (Caccavo et al., 1996). This may be explained from the Schulze-Hardy
rule (Gregory, 1989): aggregation should be ion dependent and effectiveness of ions
should increase with valency. Trivalent ions (e.g. Fe34") should be more effective than
divalent ions (e.g. Ca24", Mg2+). In conflict with previous reports and hypotheses (Turakhia
and Characklis, 1989, Applegate and Dryers, 1991, Huang and Finder, 1995) we found no
direct evidence that divalent calcium played an important role in biofilm crosslinking.
Calcium addition caused biofilm detachment and reduction in biofilm viscosity that were
comparable to those measured for sodium. Two chelating agents, EDTA and Dequest
2006, both caused reductions in biofilm viscosity and induced biofilm detachment, which
further supports, a role for crosslinking multivalent cations.
The above-listed effects caused by salts should not be considered osmotic effects.
Treatment of a biofilm reactor with 0.47 M sucrose (osmotically equivalent to the 0.3 M
NaCl treatment) caused only negligible biofilm detachment (8% versus 48% for NaCl).
Treatment of biofilm suspension with sucrose reduced the viscosity negligibly (7% versus
83% for the osmotically equivalent NaCl treatment).
63
Hydrophobic Interaction
Surfactants are widely used in biological research, for example to solubilize
hydrophobic components of various tissues and cellular structures. All surfactants consist
of a hydrophobic residue terminating in a hydrophilic head group and can be divided into
anionic, cationic, or non-ionic depending on whether the head group is negatively,
positively or not charged, respectively. Protein, which is a very important component of
biofilm consisting of about 25% of dry weight in our system (Table 8), is probably a
target of surfactants. For anionic surfactants initial binding occurs to the cationic sites on
the protein surface, while for non-ionic surfactants the binding sites will be hydrophobic
patches on the protein surface and no further binding occurs after these are saturated.
Anionic surfactants may, however, induce protein unfolding to expose many more
hydrophobic binding sites previously buried in the core of the tertiary structure.
Therefore, with addition of surfactants the initial conformations of proteins might be
altered. This structural disturbance of proteinaceous matrix Components may be one of
the mechanisms by which surfactants disrupt biofilm structural integrity. Surfactant might
also disrupt the cell membrane and thereby weaken or release EPS molecules that are
putatively anchored to the cell via a membrane interaction. Membrane disruption could
also release stockpiled lyase enzymes that then degrade EPS. Another possibility is that
surfactants interfere with crosslinking interaction between hydrophobic moieties on
carbohydrate or proteinaceous components of the EPS.
Depolymerization
It has been found that hypochlorite ion attacks polysaccharides with extensive
oxidation at the C2 and C3positions of D-glucose units, which results in cleavage of the
C2-C3 bond (Hullinger, 1963; Whistler et al, 1953). Depolymerization can result from the
inductive effects of the glucosidic bond, or from degradation of the intermediate carbonyl
compound.
A small viscosity reduction by monochloramine in comparison with chlorine
(11.5% versus 47.7%, repsectively) was found, suggesting that monochloramine is a
much weaker depolymerization agent than chlorine. In its ability to induce biofilm
detachment, however, monochloramine was very comparable with chlorine, 46.3% versus
49.7%, respectively. This indicates that depolymerization itself was not the only
mechanism that caused biofilm detachment when treated with monochloramine. This
leads to a speculation that, when challenged with certain environmental stresses, biofilms
might respond with a variety of physiological changes, including release of lyase
enzymes that degrade EPS. The high variation in the percentage of biofilm detachment
(ranging from 21.9% to 70.4%, which shown no apparent relation to the concentration of
monochloramine added) may suggest the existence of a biologically mediated response
that may not be induced each time.
Enzymatic lyases specifically attack certain component of biofilms. The amylase
targets the alpha-linkage between glucose molecules. The cellulase attacks carbohydrate
65
molecules; the protease, in the other hand, only cleaves extracellular protein molecules.
Nuclease certainly attacks various forms of nucleic acid. Although all of enzymes tested
may be very effective for their particular use, their influence on biofilm detachment is
generally small, with about 40% biomass detached from biofilm reactor for the most
effective viscosity reduction enzyme—lysozyme, which breaks down the cell wall
membranes. Nevertheless enzymatic lyases could cause biofilm detachment by
destruction of particular biofilm structural constituents.
66
CONCLUSIONS
Experimental investigation of biofilm detachment from binary population biofilm
of P. aeruginosa and K. pneumoniae showed that:
1) Biofilm detachment can be induced by addition of diverse chemicals, including NaCl
(51 percent based on total protein), CaCl2 (48 percent), MgCl2 (23 percent), Sucrose
(8 percent), pH upshift (16 percent), EDTA (26 percent), Dequest 2006 (27 percent),
sodium dodecyl sulfate (71 percent), Triton X-100 (48 percent), Tween 20 (27
percent), chlorine (50 percent), monochloramine (46 percent), and lysozyme (40
percent).
2) Cells and EPS are structural components of biofilms. Destruction of biofilm
components, including EPS or cells, can cause biofilm detachment.
3) Electrostatic interactions (in particular cation bridging) and hydrophobic interactions
are (wo major forces that maintain the integrity of biofilm structure. The biofilm
viscosity reduction, which was used to reflect the changes of biofilm structure when
challenged with various of chemicals, averaged of 73 percent when treated by salt, 7
percent by sucrose, 20 percent by EDTA, 49 percent by Dequest 2006, 42 percent by
Triton X-100, 9 percent by Tween 20,42 percent by chlorine, 12 percent by
monochloramine, and 46 percent by lysozyme. Disruption of these forces can cause
biofilm detachment.
67
RECOMMENDATIONS FOR FUTURE WORK
After this preliminary investigation of chemically induced biofilm detachment, I
find that additional work is necessary to further distinguish and quantify the contributions
of crosslinking interactions to biofilm structural integrity. For example, hydrogen
bonding may be another short-range force working in conjunction with electrostatic and
hydrophobic interactions that hold biofilms together. Distinguishing hydrogen bonding
from other crosslinking interactions merits further investigation. Since hydrogen bonding
is very sensitive to temperature, a measurement of viscosity change with temperature may
help understand its role in maintaining biofilm structure.
A better understanding of the interior structure of biofilms is also a key to
successful control of biofilm by artificially inducing biofilm detachment. What exactly
are the basic structural elements of biofilm? There may be individually separated cells
and EPS or a combination of these two.
68
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in the application of flocculation theory. Environ. Tecbnol. Lett. 3:363-374.
49. Whister, R. L. and Schweiger, R. 1957. Oxidation of amylopectin with hypochlorite
at different hydrogen ion concentrations. J. Am. Chem. Soc. 79:6460-6464.
72
50. Whister, R. L. Linke, E.G. and Kazenniac, S. 1956. Action of alkaline hypochlorite
on com starch amylase and methyl 4-O-Methyl-D-glucopyranosides. J. Am. Chem.
Soc. 78:4704-4709.
51. Wiatr, C. L. 1991. Enzyme blend containing cellulase to control industrial slime. .US
Patent. No. 4994390.
52. Zita, A. and Hermansson, M. 1994. Effects of ionic strength on bacterial adhesion and
stability of floes in a wastewater activated sludge system. AppL Environ. Microbiol.
60:3041-3048.
73
APPENDICES
74
APPENDIX A
RAW DATA FROM VISCOMETRY EXPERIMENTS
75
I . Control
No.
Before
After
(cP)
(cP)
I
122.5
136.6
2
140.8
177.1
3
95.2
67.8
4
188.1
152.8
5
128.7
150.7
6
57.7
49.6
7
102.5
99.9
8
159.7
143.6
9
83.1
47.8
10
81.5
91.5
11
110.6
134.5
12
57.1
64.8
13
270.9
286.0
14
321.1
366.7
15
132.2
150.4
16
264.9
296.3
2. LiCl (1=0.3)
No.
Before
After
(CP)
(CF)
I
174.4
57.1
2
324.2
67.2
3
175.8
73.9
4
131.8
28.1
5
129.4
17.9
6
469.5
66.5
3. NaCl (1=0.002)
Before
After
(cP)
(cP)
I
161.6
110.6
2
238.7
187.4
3
91.8
96.4
4
157.3
128.0
No.
4. NaCl (1=0.01)
Before
After
(cP)
(cP)
I
264.5
198.6
2
30.7
37.0
3
140.2
178.3
4
79.6
70.2
No.
5. NaCl (1=0.1)
No.
Before
After
(CP)
(CP)
I
135.2
108.5
2
121.4
87.9
3
81.4
54.7
4
65.4
60.8
6. NaCl (1=0.3)
Before
After
(cP)
(cP)
I
103.1
17.6
2
163.2
30.1
3
406.1
77.8
4
62.4
14.8
5
356.1
41.8
No.
7. KCl (1=0.3)
Before
After
(cP)
(cP)
I
249.4
115.6
2
105.5
26.9
3
91.1
16.3
4
322.5
42.4
5
322.5
220.3
No.
8. MgCl2 (1=0.3)
No.
Before
After
(cP)
(cP)
I
395.4
68.7
2
124.1
15.2
3
82.5
14.1
4
416.9
30.3
5
124.9
17.1
9. MgCl2 (1=0.6)
Before
After
(cP)
(cP)
I
430.8
516
2
267.5
29.0
3
331.9
40.0
4
90.3
216
5
189.6
32.0
No.
10. CaCl2 (1=0.002)
Before
After
(cP)
(cP)
I
197.4
264.6
2
303.8
219.9
3
180.1
144.2
4
152.6
126.5
5
188.4
159.1
6
101.5
104.3
No.
11. CaCl2 (1=0.01)
Before
After
(cP)
(cP)
I
202.3
172.7
2
334.4
258.7
3
79.8
107.7
4
92.3
102.3
5
195.3
189.2
No.
12. CaCl2 (1=0.03)
Before
After
(cP)
(cP)
I
418.1
280.2
2
324.1
299.0
3
136.5
203.8
4
87.6
119.6
5
278.1
218.4
6
95.6
74.9
No.
13. CaCl2 (1=0.3)
I
Before
(cP)
259.9
After
(cP)
61.9
2
287.1
67.1
3
227.6
100.2
4
83.3
45.9
5
121.7
44.0
No.
14. CaCl2 (1-0.663)
No.
Before
After
(cP)
(cP)
I
162.9
24.9
2
301.4
45.9
3
143.3
37.4
4
166.9
46.5
5
230.5
39.4
15. FeCl2 (1=0.002)
Before
After
(cP)
(cP)
I
211.6
154.9
2
45.3
79.1
3
70.6
85.4
4
164.9
117.8
No.
16. FeCl2 (1=0.01)
Before
After
(cP)
(cP)
I
162.3
92.8
2
86.9
63.9
3
30.5
45.7
4
182.6
108.6
No.
17. FeCl2 (1=0.03)
Before
After
(cP)
(cP)
I
211.5
105.4
2
107.0
83.1
3
38.5
81.4
4
162.3
110.4
No.
18. FeCl2 (1=0.3)
Before
After
(cP)
(cP)
I
122.1
210.4
2
42.7
518
3
123.5
2318
4
215.9
286.0
No.
19. Fe(NO3)3 (1=0.002)
Before
After
(cP)
(cP)
I
98.1
119.5
2
238.9
170.1
3
93.9
76.2
4
185.1
96.7
5
2219
142.1
No.
20. Fe(NO3)3 (1=0.01)
No.
Before
After
(CP)
(CP)
I
207.4
246.0
2
176.2
181.5
3
105.5
121.4
4
100.2
89.6
5
261.2
212.5
21. Fe(NO3)3(1=0.03)
Before
After
(cP)
(cP)
I
202.0
130.0
2
100.3
90.3
3
237.1
160.9
4
321.5
322.5
5
295.8
201.3
No.
22. Fe(NO3)3(1=0.3)
Before
After
(cP)
(cP)
I
219.7
217.4
2
130.9
170.2
3
86.8
160.0
4
277.9
353.3
5
169.8
307.9
No.
23. Fe(NO3)3 (1=0.6)
No.
Before
After
(CF)
(CP)
I
181.6
125.4
2
177.9
195.7
3
320.3
279.9
4
185.9
201.6
5
87.0
151.7
6
255.5
232.3
24. Sucrose (0.47M)
Before
After
(cP)
(cP)
I
81.6
88.5
2
235.5
209.4
3
167.4
158.8
4
113.9
114.6
5
144.3
120.3
No.
84
25. EDTA (0.01M)
No.
Before
After
(CF)
(CP)
I
122.8
105.7
2
58.1
48.8
3
76.2
61.7
4
22.3
26.0
5
178.9
140.4
26. Dequest-2006 (Ig/L)
Before
After
(cP)
(cP)
I
28.4
33.8
2
170.8
69.7
3
170.3
103.8
4
101.9
48.2
5
12.9
15.4
No.
27. pH downshift (pH 6.8 -> 2.0)
Before
After
(cP)
(cP)
I
30.6
38.4
2
130.4
80.9
3
76.3
62.2
4
170.4
102.3
No.
28. SDS(IgZL)
No.
Before
After
(cP)
(cP)
I
167.2
191.9
2
43.8
96.3
3
76.6
145.0
4
176.8
168.4
5
41.7
94.4
29. Triton X-IOO(IgZL)
Before
After
(cP)
(cP)
I
150.7
85.2
2
113.2
69 8
3
60.5
27.7
4
6.0
9.3
5
99.5
63 9
No.
30. Tween-20 (IgZL)
Before
After
(cP)
(cP)
I
25.6
20.3
2
36.0
22.9
3
61.8
76.5
4
42.4
36.1
5
210.6
189.2
No.
8 6
31. Hexadecyltrimethylammonium bromide (Ig/L)
No.
Before
After
(CP)
(cP)
I
94.9
65.2
2
10.6
13.1
3
102.6
45.7
4
24.4
10.2
5
204.4
133.6
32. Lignosulfonic acid, sodium salt (Ig/L)
Before
After
(cP)
(cP)
I
35.5
41.8
2
96.4
81.0
3
102.3
64.9
4
111.2
106.9
No.
33. NaClO (50mg/L)
Before
After
(cP)
(cP)
I
64.8
32.7
2
177.9
102.4
3
34.7
24.5
4
669
51.6
No.
87
34. NH2Cl (50mg/L)
No.
Before
After
(CP)
(CP)
I
44.4
43.8
2
36.2
38.1
3
74.7
67.5
4
187.3
164.6
35. Amylase (Ig/L)
Time after mix
Run I
Run 2
Run 3
(min)
(CP)
(cP)
(cP)
Initial
150.6
201.6
106.5
2
165.9
125.4
100.3
32
86.4
102.2
111.4
62
190.4
96.1
116.3
92
130.0
84.0
110.3
122
190.1
80.7
108.2
36. Cellulase (Ig/L)
Time after mix
(min)
Initial
Run I
(cP)
132.7
Run 2
(cP)
129.6
Run 3
(cP)
99.6
2
119.0
154.8
133.0
32
64.0
197.8
143.4
62
121.6
53.6
134.6
92
76.5
178.8
132.0
122
71.4
174.5
143.3
88
37. Lysozyme (Ig/L)
Time after mix
Run I
Time after mix
(min)
Run 2
(min)
(cP)
Initial
235.3
Initial
2
185.0
46
Time after mix
(min)
Run 3
205.0
Initial
215.7
2
144.7
2
97.8
144.4
33
112.2
35
53.6
72
130.1
64
94.1
62
57.0
93
110.4
93
87.8
88
54.9
121
106.0
124
81.1
115
57.3
(cP)
38. Nuclease (Ig/L)
Time after mix
Run I
Run 2
Run 3
(min)
(cP)
(cP)
(cP)
Initial
70.0
167.9
90.3
2
89.5
124.6
130.6
32
98.0
76.7
111.3
62
125.7
818
121.0
92
86.6
94.6
814
122
818
78.9
78.9
(cP)
89
39. Protease (Ig/L)
Time after mix
Run I
Time after mix
(min)
Run 2
(min)
(CP)
2
143.7
Initial
18
123.2
51
Time after mix
(min)
Run 3
179.5
Initial
167.7
2
128.9
2
138.1
116.5
33
126.0
32
146.4
81
89.7
62
106.3
58
60.5
111
79.7
93
116.0
90
55.3
142
92.6
127
114.3
115
46.5
170
77.9
(cP)
40. Control (viscosity change with time)
Time
Run I
Run 2
Run 3
(min)
(cP)
(cP)
(cP)
0
173.6
110.4
205.1
30
200.5
124.6
210.7
60
222.4
132.4
239.6
90
235.0
148.1
258.9
120
249.5
154.8
272.6
(cP)
90
41. Control (water viscosity changes with addition o f chemicals)
Chemical
Water viscosity, before
Water viscosity, after
(cP)
(cP)
LiCl (0.3M)
5.4
9.8
NaCl (0.3M)
5.0
10.8
KCl (0.3M)
3.2
6.2
MgCl2 (0.2M)
5.3
10.7
CaCl2 (0.2M)
4.3
10.7
FeCl2 (0.1M)
5.2
10.6
Fe(NO3)3 (0.1M)
5.8
11.4
Sucrose (0.47M)
4.7
9.2
EDTA (0.01M)
3.4
5.1
Bequest 2006 (Ig/L)
4.1
3.5
pH downshift
4.8
5.0
SDS(IgZL)
4.1
3.9
Triton X-100 (Ig/L)
4.4
2.0
Tween-20 (Ig/L)
1.9
2.9
Hexadecyltrimethyl
3.9
1.0
Lignosulfate (lg/L)
3.7
2.5
NaClO (SOmgZL)
4.5
5.2
NH2Cl (SOmgZL)
5.1
6.2
Amylase (IgZL)
4.6
9.1
Cellulase (IgZL)
3.6
5.7
Lysozyme (IgZL)
4.1
6.8
Nuclease (IgZL)
5.6
6.9
Protease (IgZL)
3.8
27.7
Ammonium bromide
(lg/L)
91
APPENDIX B
RAW DATA FROM ENVIRONMENTAL STEP CHANGE EXPERIMENTS
92
Raw data from optical density measurements
I . Optical density measurement results from experiment No. I to No. 9
T im e
E xp
E xp
E xp
E xp
E xp
E xp
E xp
E xp
( m in )
N o. I
N o .2
N o .3
N o .5
N o .6
N o .7
N o .8
N o .9
4 .1 5 e - 3
-2 5
1 .1 7 e - 3
- 9 .3 3 e - 3
-2 4
0 .0 1
- 6 .1 9 e - 3
2 .0 8 e - 3
-2 3
3 .5 0 e - 3
- 0 .0 1
4 .1 5 e - 3
-2 2
4 .2 7 e - 3
- 4 .6 2 e - 3
0 .0 1
-2 1
- 3 .5 4 e - 3
- 3 .8 6 e - 3
2 .0 8 e - 3
-2 0
3 .5 0 e - 3
- 3 .0 6 e - 3
1 .0 3 e - 3
0 .0 1
-1 9
3 .5 0 e - 3
- 0 .0 1
2 .0 8 e - 3
4 .8 3 e - 3
6 .6 2 e - 3
-1 8
3 .5 0 e - 3
- 5 .3 9 e - 3
- 4 .1 l e - 3
-1 7
8 .1 8 e - 3
- 4 .6 2 e - 3
2 .0 8 e - 3
0 .0 4
0 .0 2
-1 6
0 .0 3
- 6 .9 6 e - 3
- 8 .1 9 e - 3
0 .0 3
0 .0 2
-1 5
0 .0 2
3 .1 8 e - 3
-9 .8 9 e - 4
0 .0 3
-1 4
2 .7 0 e - 3
- 3 .8 6 e - 3
-4 .1 l e - 3
4 .7 2 e - 3
0 .0 2
-1 3
5 .0 7 e - 3
- 3 .0 6 e - 3
-9 .8 9 e -4
6 .8 5 e - 3
0 .0 1
-1 2
0 .0 1
- 6 .9 6 e - 3
- 6 .1 3 e - 3
8 .9 0 e - 3
7 .6 3 e - 3
0 .0 2
- 7 .7 6 e - 3
8 .7 9 e - 3
- 3 .0 5 e - 3
- 3 .5 6 e - 3
-1 0
-3 .3 0 e -3
4 .2 7 e - 3
- 7 .7 6 e - 3
0 .0 1
- 5 .1 2 e - 3
6 .2 4 e - 4
0 .0 2
-9
-5 .4 5 e -3
0 .0 2
- 7 .7 6 e - 3
8 .7 9 e - 3
1 .0 3 e - 3
2 .6 7 e - 3
- 9 .1 3 e - 3
3 .0 5 e - 3
-8
-3 .2 6 e - 3
0 .0 1
- 2 .2 9 e - 3
- 1 .9 3 e - 3
- 3 .0 5 e - 3
6 .8 5 e - 3
0 .0 1
9 .2 8 e - 3
-7
-5 .4 5 e - 3
0 .0 2
8 .4 8 e - 4
0 .0 1
2 .0 8 e - 3
2 .6 7 e - 3
5 .9 1 e - 3
8 .4 l e - 3
-6
0 .0 2
0 .0 2
- 6 . 1 9 e -3
4 .5 2 e - 3
2 .0 8 e - 3
4 .7 2 e - 3
0 .0 2
6 .6 2 e - 3
-5
7 .6 3 e - 3
0 .0 5
- 5 .3 9 e - 3
2 .3 4 e - 3
6 .1 7 e - 3
- 0 .0 1
3 .8 0 e - 3
6 .6 1 e - 3
1 .0 3 e - 3
- 5 .6 9 e - 3
5 .9 1 e - 3
8 .4 1 e - 3
- 5 .6 9 e - 3
- 5 .1 0 e - 4
4 .8 3 e - 3
-1 1
-4
- 0 .0 2
0 .0 5
- 3 .8 6 e - 3
9 .2 8 e - 3
0 .0 1
- 0 .0 2
-3
5 .4 5 e - 3
0 .0 4
- 5 .3 9 e - 3
- 1 .9 3 e - 3
3 .0 9 e - 3
-2
-3 .2 6 e - 3
0 .0 3
- 5 .3 9 e - 3
0 .0 1
8 .2 3 e - 3
4 .7 2 e - 3
- 0 .0 1
6 .2 4 e - 4
3 .8 0 e - 3
0 .0 1
0 .0 1
0 .0 1
5 .7 4 e - 3
0 .0 6
-I
- 0 .0 1
0 .0 3
- 0 .0 1
2 .3 4 e - 3
0 .0 2
0
-1 .1 2 e - 3
0 .0 3
- 7 .2 0 e - 4
8 .7 9 e - 3
0 .0 2
8 .9 0 e - 3
0 .0 2
0 .0 2
0 .2 7
0 .3 1
0 .4 8
0 .0 4
I
-1 .1 2 e - 3
0 .4 0
0 .8 2
0 .0 6
2
9 .8 2 e - 3
0 .6 1
0 .8 7
0 .0 8
0 .2 7
0 .2 5
0 .5 5
0 .0 3
0 .1 2
0 .2 7
0 .2 2
0 .6 2
0 .0 3
3
-5 .4 5 e - 3
0 .6 0
0 .8 9
4
- 0 .0 1
0 .6 0
0 .8 8
0 .1 1
0 .2 6
0 .2 8
0 .6 5
0 .0 2
0 .1 2
0 .2 4
0 .2 5
0 .7 1
0 .0 2
5
4 .3 7 e -3
0 .6 5
0 .8 5
6
0 .0 0
0 .6 1
0 .8 4
0 .1 3
0 .2 5
0 .2 8
0 .6 6
0 .0 2
0 .8 1
0 .1 2
0 .2 3
0 .3 2
0 .6 6
0 .0 2
0 .5 1
0 .7 8
0 .1 2
0 .2 4
0 .2 8
0 .6 5
0 .0 3
0 .1 2
0 .2 0
0 .4 0
0 .6 3
0 .0 2
0 .1 0
0 .2 1
0 .3 9
0 .6 1
0 .0 5
7
8
-2 .1 9 e - 3
-2 .1 9 e - 3
0 .5 7
9
0 .0 0
0 .4 6
0 .7 6
10
1 .0 7 e - 3
0 .5 1
0 .7 3
93
I. Optical density measurement results from experiment No. I to No. 9 (continued)
T im e
E xp
E xp
E xp
E xp
E xp
E xp
E xp
E xp
( m in )
N o. I
N o .2
N o .3
N o .5
N o .6
N o .7
N o .8
N o .9
11
5 .4 5 e - 3
0 .4 7
0 .7 0
0 .1 0
0 .1 9
0 .3 9
0 .5 9
0 .0 3
12
2 .1 9 e -3
0 .4 4
0 .6 7
0 .0 9
0 .1 9
0 .2 0
0 .5 6
0 .0 3
13
7 .6 3 e - 3
0 .3 9
0 .6 4
0 .0 9
0 .1 8
0 .3 9
0 .5 5
0 .0 3
14
3 .2 6 e - 3
0 .3 6
0 .6 1
0 .0 9
0 .1 7
0 .3 4
0 .5 2
0 .0 3
15
-4 .3 7 e - 3
0 .3 5
0 .5 5
0 .0 9
0 .1 7
0 .1 3
0 .4 8
0 .0 2
16
-3 .2 6 e - 3
0 .3 4
0 .5 9
0 .0 8
0 .1 7
0 .5 3
0 .4 6
0 .0 4
17
4 .3 7 e -3
0 .3 1
0 .5 7
0 .0 9
0 .1 6
0 .5 0
0 .4 5
0 .0 4
18
5 .4 5 e - 3
0 .2 9
0 .5 5
0 .0 9
0 .1 6
0 .1 2
0 .4 3
0 .0 4
19
1 .0 7 e - 3
0 .3 0
0 .5 3
0 .0 9
0 .1 7
0 .1 4
0 .4 0
0 .0 3
20
-1 .0 7 e -3
0 .3 0
0 .5 4
0 .0 7
0 .1 8
0 .1 5
0 .3 8
0 .0 4
21
7 .6 3 e -3
0 .3 8
0 .5 1
0 .0 7
0 .1 8
0 .2 0
0 .3 6
0 .0 3
22
6 .5 2 e - 3
0 .3 5
0 .5 0
0 .0 6
0 .1 8
0 .0 5
0 .3 4
0 .0 6
23
-1 .0 7 e -3
0 .3 1
0 .4 9
0 .0 7
0 .1 8
0 .1 5
0 .3 3
0 .0 3
24
8 .7 5 e - 3
0 .3 1
0 .4 7
0 .0 4
0 .1 9
0 .4 9
0 .3 3
0 .0 3
25
-7 .6 3 e - 3
0 .3 4
0 .4 6
0 .0 5
0 .2 0
0 .1 7
0 .3 2
0 .0 3
0 .0 5
0 .2 1
0 .1 1
0 .2 9
0 .0 3
0 .0 5
0 .2 8
0 .0 2
26
27
28
8 .7 0 e - 3
4 .3 3 e -3
- 0 .0 1
0 .3 3
0 .4 5
0 .3 2
0 .4 4
0 .0 5
0 .2 2
0 .3 7
0 .4 0
0 .0 5
0 .2 1
0 .1 2
0 .2 8
0 .0 4
0 .4 2
0 .0 6
0 .1 9
0 .1 3
0 .2 6
0 .0 4
0 .0 6
29
3 .3 0 e - 3
0 .3 3
30
7 .6 3 e - 3
0 .3 3
0 .4 3
0 .0 5
0 .1 9
6 .2 4 e - 4
0 .2 3
0 .4 3
0 .0 5
0 .2 0
-0 .0 1
0 .2 3
0 .0 3
31
-6 .5 6 e - 3
0 .3 1
32
-9 .8 2 e -3
0 .3 0
0 .4 0
0 .0 4
0 .2 0
0 .0 4
0 .2 4
0 .0 3
33
-2 .1 9 e -3
0 .3 1
0 .4 0
0 .0 5
0 .1 8
- 9 .8 7 e - 3
0 .2 2
0 .0 3
34
7 .6 3 e - 3
0 .2 9
0 .3 9
0 .0 4
0 .1 9
- 0 .0 3
0 .2 2
0 .0 8
35
0 .0 1
0 .3 0
0 .3 8
0 .0 3
0 .2 0
0 .0 5
0 .2 1
0 .1 3
36
- 0 .0 1
0 .2 8
0 .3 5
0 .0 3
0 .1 8
0 .0 6
0 .1 9
0 .1 6
37
- 0 .0 1
0 .2 7
0 .3 5
0 .0 4
0 .1 7
- 0 .0 2
0 .1 9
0 .1 5
38
3 .2 6 e - 3
0 .2 6
0 .3 3
0 .0 3
0 .1 5
- 9 .8 7 e - 3
0 .1 9
0 .1 6
39
0 .0 2
0 .2 5
0 .3 2
0 .0 4
0 .1 8
0 .0 1
0 .1 9
0 .1 5
40
0 .0 0
0 .2 4
0 .3 2
0 .0 3
0 .1 5
- 0 .0 4
0 .1 8
0 .1 3
41
- 0 .0 1
0 .2 2
0 .3 0
0 .0 3
0 .1 4
- 0 .0 5
0 .2 0
0 .1 2
0 .1 1
42
5 .4 5 e - 3
0 .2 2
0 .2 9
0 .0 4
0 .1 5
- 0 .0 2
0 .1 8
43
0 .0 1
0 .2 1
0 .2 8
0 .0 3
0 .1 3
- 0 .0 3
0 .2 0
0 .1 1
44
-4 .3 7 e - 3
0 .2 3
0 .2 7
0 .0 3
0 .1 3
0 .1 9
0 .2 1
0 .1 1
45
-3 .2 6 e - 3
0 .2 1
0 .2 6
0 .0 3
0 .1 2
0 .0 7
0 .2 1
0 .1 1
46
6 .5 6 e - 3
0 .2 0
0 .2 5
0 .0 3
0 .1 2
- 0 .0 6
0 .1 9
0 .0 9
47
-5 .4 5 e - 3
0 .1 9
0 .2 4
0 .0 4
0 .1 1
- 0 .0 6
0 .1 9
0 .0 1 0
48
-1 .0 7 e -3
0 .1 9
0 .2 3
0 .0 3
0 .1 0
- 0 .0 5
0 .1 8
0 .0 8
49
5 .4 5 e - 3
0 .1 7
0 .2 3
0 .0 3
0 .1 1
- 0 .0 6
0 .1 9
0 .0 9
50
9 .7 8 e - 3
0 .1 6
0 .2 1
0 .0 2
0 .1 1
- 0 .0 5
0 .2 0
0 .0 7
51
-7 .6 3 e -3
0 .1 6
0 .2 0
0 .0 3
0 .0 9
- 0 .0 6
0 .1 8
0 .0 8
52
-5 .4 5 e - 3
0 .1 5
0 .2 0
0 .0 3
0 .0 8
- 0 .0 5
0 .1 8
0 .0 8
53
5 .4 5 e - 3
0 .1 4
0 .2 0
0 .0 2
0 .0 9
- 0 .0 4
0 .1 7
0 .0 7
54
0 .0 0
0 .1 4
0 .1 9
0 .0 3
0 .0 8
- 0 .0 4
0 .1 7
0 .0 7
55
0 .0 0
0 .1 4
0 .1 7
0 .0 2
0 .0 8
- 0 .0 4
0 .1 8
0 .0 6
94
I. Optical density measurement results from experiment No. I to No. 9 (continued)
T im e
E xp
E xp
E xp
E xp
E xp
E xp
E xp
E xp
( m in )
N o. I
N o .2
N o .3
N o .5
N o .6
N o .7
N o .8
N o .9
56
-8 .7 0 e - 3
0 .1 5
0 .1 7
0 .0 2
0 .0 8
- 0 .0 6
0 .1 7
0 .0 6
57
3 .2 6 e - 3
0 .1 6
0 .1 6
8 .7 9 e - 3
0 .0 7
- 0 .0 4
0 .1 7
0 .0 6
58
4 .3 7 e - 3
0 .1 9
0 .1 6
8 .7 9 e - 3
0 .0 7
- 0 .0 5
0 .1 4
0 .0 5
59
0 .0 0
0 .1 8
0 .1 6
0 .0 2
0 .0 6
- 0 .0 7
0 .1 6
0 .0 5
60
4 .3 3 e - 3
0 .1 7
0 .1 5
0 .0 1
0 .0 8
- 0 .0 4
0 .1 6
0 .0 4
61
0 .0 0
0 .1 7
0 .1 4
0 .0 1
0 .0 6
- 0 .0 1
0 .1 7
0 .0 4
62
-3 .2 6 e - 3
0 .1 7
0 .1 4
0 .0 3
0 .0 5
- 0 .0 5
0 .1 6
0 .0 4
63
6 .5 6 e - 3
0 .1 6
0 .1 3
0 .0 4
0 .0 4
- 0 .0 5
0 .1 6
0 .0 5
0 .0 4
64
-2 .1 9 e -3
0 .1 6
0 .1 3
0 .0 3
0 .0 4
- 0 .0 5
0 .1 6
65
-4 .3 7 e -3
0 .1 6
0 .1 2
0 .0 2
0 .0 4
- 0 .0 4
0 .1 6
0 .0 4
66
-3 .2 6 e -3
0 .1 5
O il
0 .0 2
0 .0 7
- 0 .0 3
0 .1 6
0 .0 3
67
0 .0 0
0 .1 5
0 .1 1
0 .0 3
0 .0 3
- 0 .0 4
0 .1 6
0 .0 3
68
0 .0 2
0 .1 4
0 .1 1
0 .0 2
0 .0 3
- 0 .0 3
0 .1 6
0 .0 4
69
0 .0 0
0 .1 4
0 .1 0
0 .0 2
0 .0 3
- 0 .0 3
0 .1 8
0 .0 3
70
3 .2 6 e - 3
0 .1 3
0 .0 1 0
0 .0 3
0 .0 2
- 0 .0 5
0 .1 2
0 .0 2
71
2 .1 9 e -3
0 .1 2
0 .0 9
0 .0 3
0 .0 4
0 .3 1
0 .1 1
0 .0 2
72
0 .0 0
0 .1 1
0 .0 1 0
0 .0 2
0 .0 3
0 .1 7
0 .1 2
0 .0 2
73
1 .0 7 e - 3
0 .1 0
0 .0 9
0 .0 3
0 .0 3
0 .2 0
0 .1 1
0 .0 1
74
-9 .7 8 e -3
0 .0 1 0
0 .1 3
0 .0 2
0 .0 2
0 .1 6
0 .1 0
0 .0 2
75
-3 .2 6 e -3
0 .1 0
0 .0 2
0 .1 2
0 .0 9
0 .0 2
0 .0 4
0 .1 3
76
0 .1 1
0 .0 8
0 .0 3
0 .0 2
0 .1 0
0 .0 1
77
0 .1 1
0 .0 7
0 .0 2
0 .1 4
0 .0 1
78
0 .1 0
0 .0 8
0 .0 2
0 .0 3
0 .0 2
0 .0 8
0 .0 4
79
0 .1 1
0 .0 6
0 .0 1
80
0 .1 4
0 .0 6
6 .1 7 e - 3
0 .0 7
0 .0 1
0 .0 3
0 .0 3
81
0 .1 3
0 .0 6
6 .1 7 e - 3
82
0 .1 2
0 .0 6
6 .1 7 e - 3
0 .0 2
0 .0 2
0 .0 2
0 .0 1
0 .0 2
83
0 .1 0
0 .0 6
7 .2 2 e - 3
84
0 .1 2
0 .0 5
3 .0 9 e - 3
0 .0 1
0 .0 5
0 .0 1
- 5 .6 9 e - 3
0 .0 2
0 .0 5
7 .2 2 e - 3
- 0 .0 2
0 .0 1
0 .0 3
85
0 .1 1
86
0 .1 0
87
0 .0 1 0
0 .0 5
8 .2 3 e - 3
- 0 .0 2
88
0 .1 0
0 .0 5
7 .2 2 e - 3
- 9 .8 7 e - 3
89
0 .1 2
0 .0 5
6 .1 7 e - 3
- 0 .0 4
5 .7 4 e - 3
0 .0 1
90
0 .0 9
0 .0 6
0 .0 1
0 .0 1
91
0 .0 8
0 .1 0
8 .2 3 e - 3
0 .0 1
92
0 .0 7
0 .0 5
8 .4 1 e - 3
93
0 .0 6
0 .0 5
0 .0 1
94
0 .0 6
0 .0 4
0 .0 1
0 .0 4
0 .0 1
0 .0 1
95
0 .0 5
96
0 .0 4
0 .0 3
97
0 .0 4
0 .0 4
5 .7 4 e - 3
98
0 .0 3
0 .0 3
9 .2 8 e - 3
99
0 .0 3
0 .0 3
0 .0 1
100
0 .0 2
0 .0 3
0 .0 2
101
0 .0 3
0 .0 3
102
0 .0 3
0 .0 2
103
0 .0 3
0 .0 2
95
2. Optical density measurement results from experiment No. 10 to No. 21
T im e
E xp
E xp
E xp
E xp
E xp
E xp
E xp
E xp
( m in )
N o . 10
N o . 11
N o . 12
N o . 14
N o . 15
N o . 16
N o . 17
N o .2 1
-2 5
0 .0 4
-2 4
0 .0 3
0 .0 3
-2 3
0 .0 3
2 .4 2 e -3
0 .0 5
9 .1 0 e -3
0 .0 2
0 .0 5
4 .0 7 e -3
0 .0 2
-2 2
-2 1
-2 0
0 .0 1
0 .0 2
0 .0 4
-1 9
5 .7 4 e - 3
0 .0 2
0 .0 5
5 .7 2 e - 3
-1 8
7 .8 9 e - 3
7 .1 4 e - 3
0 .0 4
7 .4 5 e - 3
0 .0 2
-1 7
7 .8 9 e - 3
0 .0 1
0 .0 5
-1 6
- 6 .8 1 e - 3
0 .0 1
0 .0 4
0 .0 3
-1 5
- 8 .8 7 e - 3
0 .0 1
0 .0 4
0 .0 2
-1 4
0 .0 2
0 .0 1
0 .0 2
0 .0 1
-1 3
0 .0 1
0 .0 1
0 .0 4
0 .0 1
-1 2
0 .0 2
8 .1 2 e - 3
0 .0 5
0 .0 1
-1 1
7 .8 9 e - 3
- 9 .7 8 6 - 3
0 .0 7
0 .0 2
-1 0
- 5 .3 3 e - 4
0 .0 1
0 .0 1
0 .0 7
0 .0 1
0 .0 3
-9
0 .0 3
5 .8 3 e - 3
0 .0 2
0 .0 4
0 .0 1
3 .0 3 e - 3
-8
0 .0 2
5 .8 3 e - 3
5 .1 4 e - 3
0 .0 7
0 .0 1
0 .0 3
3 .0 3 e - 3
-7
0 .0 4
5 .8 3 e - 3
7 .1 4 e - 3
0 .0 6
0 .0 3
4 .0 7 e -3
0 .0 2
0 .0 1
0 .0 3
- 1 .9 8 e - 3
5 .1 4 e - 3
0 .0 6
3 .0 4 e - 3
0 .0 3
-5
1 .2 9 e - 3
0 .0 4
- 1 .9 8 e - 3
2 .1 7 e - 3
0 .0 4
6 .1 9 e - 3
0 .0 1
- 0 .0 1
-4
0 .0 3
0 .0 1
- 3 .9 9 e - 3
0 .0 1
0 .0 5
0 .0 2
9 .1 0 e - 3
- 1 .3 9 e - 3
7 .7 2 e -4
-6
-3
7 .7 8 e - 3
0 .0 2
- 3 .9 9 e - 3
8 .1 2 e - 3
0 .0 3
- 6 . 4 1 e -3
0 .0 1
-2
0 .0 2
0 .0 3
- 1 .9 8 e - 3
7 .1 4 e - 3
0 .0 5
4 .5 8 e - 3
7 .0 1 e -4
-0 .0 1
-I
3 .4 2 e - 3
0 .0 3
- 5 .9 3 e - 3
0 .0 4
0 .0 4
1 .4 3 e - 3
7 .4 5 e - 3
9 . 6 1e - 3
0
-3 .0 7 e - 3
0 .0 2
5 .8 3 e - 3
6 .1 6 e - 3
0 .0 5
0 .0 3
5 .7 2 e - 3
- 1 .3 9 e - 3
I
1 .2 9 e - 3
0 .0 3
0 .0 8
8 .1 2 e - 3
0 .1 5
0 .0 2
0 .0 8
3 .0 3 e - 3
2
- 9 .3 3 e - 4
0 .0 5
0 .1 3
0 .0 1
0 .3 8
0 .1 7
0 .0 2
5 .1 9 e - 3
3
7 .7 8 e - 3
0 .1 3
0 .1 4
1 .7 1 e - 4
0 .5 8
0 .3 0
0 .0 4
0 .1 1
4
0 .0 1
0 .1 3
0 .1 4
0 .0 1
0 .7 2
0 .1 1
0 .0 5
0 .2 9
5
-5 .2 0 e - 3
0 .1 2
0 .1 5
3 .1 5 e - 3
0 .7 1
0 .5 2
0 .0 4
0 .4 2
6
1 .2 9 e - 3
0 .1 3
0 .1 4
0 .0 3
0 .7 3
0 .4 4
0 .0 6
0 .4 9
7
0 .0 1
0 .1 0
0 .1 2
0 .0 3
0 .7 3
0 .6 4
0 .0 5
0 .5 0
8
0 .0 4
0 .0 1 0
0 .1 4
0 .0 2
0 .7 7
0 .7 7
0 .0 5
0 .5 0
9
0 .0 8
0 .1 0
0 .1 2
0 .0 2
0 .7 9
0 .8 0
0 .0 5
0 .4 8
10
0 .0 9
0 .0 8
0 .1 1
0 .0 5
0 .7 6
0 .8 1
0 .0 5
0 .4 8
11
0 .1 2
0 .0 8
0 .0 1 0
0 .1 1
0 .7 8
0 .7 8
0 .0 6
0 .4 7
12
0 .1 0
0 .0 9
0 .1 0
0 .1 3
0 .7 5
0 .7 8
0 .0 1 0
0 .4 4
13
0 .1 0
0 .0 9
0 .0 9
0 .1 6
0 .8 0
0 .7 9
0 .1 0
0 .4 6
14
0 .1 5
0 .0 8
0 .0 9
0 .1 6
0 .7 7
0 .7 9
0 .0 1 0
0 .4 7
15
0 .1 5
0 .0 8
0 .0 9
0 .1 7
0 .6 4
0 .7 9
0 .1 1
0 .4 4
16
0 .1 0
0 .0 7
0 .0 8
0 .1 8
0 .8 7
0 .8 1
0 .1 2
0 .4 6
17
0 .0 9
0 .0 7
0 .0 9
0 .1 7
0 .8 3
0 .8 0
0 .1 2
0 .4 6
18
0 .1 0
0 .0 6
0 .0 9
0 .1 8
0 .7 8
0 .8 1
0 .1 1
0 .4 7
19
0 .0 8
0 .0 5
0 .1 1
0 .1 7
0 .7 1
0 .8 0
0 .1 0
0 .4 4
20
0 .0 8
0 .0 6
0 .1 2
0 .1 7
0 .8 4
0 .7 7
0 .0 8
0 .4 3
21
0 .1 2
0 .0 4
0 .0 1 0
0 .1 5
0 .7 3
0 .7 7
0 .1 0
0 .4 0
96
2. Optical density measurement results from experiment No. 10 to No. 21 (continued)
T im e
E xp
E xp
E xp
E xp
E xp
E xp
E xp
E xp
( m in )
N o . 10
N o . 11
N o . 12
N o .1 4
N o . 15
N o . 16
N o . 17
N o .2 1
22
0 .1 6
0 .0 6
0 .0 8
0 .1 6
0 .7 5
0 .7 6
0 .0 9
0 .3 9
23
0 .2 7
0 .0 4
0 .1 1
0 .1 5
0 .7 8
0 .7 5
0 .0 8
0 .3 8
24
0 .2 0
0 .0 5
0 .0 9
0 .1 8
0 .7 8
0 .7 5
0 .0 7
0 .3 4
25
0 .2 6
0 .0 4
0 .0 9
0 .2 6
0 .7 7
0 .7 3
0 .0 8
0 .3 4
0 .3 0
26
0 .0 7
0 .0 3
0 .1 0
0 .2 5
0 .7 7
0 .7 3
0 .0 7
27
0 .2 3
0 .0 5
0 .0 9
0 .2 5
0 .8 5
0 .7 2
0 .0 6
0 .2 9
28
0 .0 7
0 .0 5
0 .0 9
0 .2 5
0 .8 3
0 .7 0
0 .0 6
0 .3 1
29
0 .0 9
0 .0 4
0 .0 8
0 .2 5
0 .7 4
0 .6 9
0 .0 6
0 .2 5
30
0 .0 7
0 .0 1
0 .0 9
0 .2 4
0 .7 7
0 .6 8
0 .0 5
0 .2 6
31
0 .3 5
0 .0 4
0 .0 8
0 .2 4
0 .8 3
0 .6 7
0 .0 5
0 .2 6
32
0 .1 1
0 .0 3
0 .1 1
0 .2 5
0 .6 3
0 .6 3
0 .0 4
0 .2 4
0 .2 5
33
0 .1 0
0 .0 4
0 .0 8
0 .2 4
0 .6 9
0 .6 3
7 .0 1 e -4
34
0 .2 2
0 .0 2
0 .1 0
0 .2 2
0 .7 0
0 .6 2
0 .0 1
0 .2 5
35
0 .1 5
0 .0 2
0 .1 1
0 .2 3
0 .7 4
0 .6 2
- 2 .6 7 e - 3
0 .2 3
36
0 .2 4
0 .0 2
0 .0 9
0 .2 2
0 .7 3
0 .6 0
0 .0 2
0 .2 0
37
0 .2 1
7 .8 9 e -3
0 .0 9
0 .2 0
0 .7 2
0 .5 9
0 .0 2
0 .1 8
38
0 .2 6
-5 .3 3 e - 4
0 .0 8
0 .2 0
0 .7 3
0 .5 7
2 .4 2 e -3
0 .1 9
39
0 .3 2
0 .0 1
0 .0 9
0 .1 9
0 .5 8
0 .5 5
0 .0 2
0 .1 8
40
0 .2 6
0 .0 1
0 .0 8
0 .1 8
0 .7 3
0 .5 3
4 .0 7 e -3
0 .2 4
41
0 .2 7
1 .6 2 e - 3
0 .0 9
0 .2 0
0 .6 6
0 .5 3
-9 .5 1 e - 4
0 .1 6
42
0 .2 2
5 .7 4 e - 3
0 .0 7
0 .1 9
0 .7 0
0 .5 2
7 .4 5 e - 3
0 .2 0
43
0 .2 2
5 .7 4 e - 3
0 .0 7
0 .1 9
0 .7 4
0 .4 9
7 .4 5 e -3
0 .1 8
44
0 .1 9
0 .0 1
0 .0 6
0 .1 7
0 .6 6
0 .4 8
0 .0 2
0 .2 0
45
0 .2 5
9 .9 5 e - 3
0 .0 7
0 .1 7
0 .6 5
0 .4 6
4 .0 7 e -3
0 .1 4
0 .1 1
46
0 .2 0
0 .0 2
0 .0 6
0 .1 9
0 .6 5
0 .4 6
0 .0 1
47
0 .1 9
- 6 .8 1 e - 3
0 .1 0
0 .1 6
0 .6 8
0 .4 6
0 .0 2
0 .2 0
0 .3 4
0 .4 6
48
0 .1 8
7 .8 9 e - 3
0 .0 6
0 .1 7
0 .5 8
0 .5 1
2 .4 2 e -3
49
0 .1 4
7 .8 9 e - 3
0 .0 5
0 .1 5
0 .5 7
0 .5 3
-9 .5 1 e - 4
9 .1 0 e - 3
0 .4 3
-9 .5 1 e - 4
0 .4 5
50
0 .1 7
- 0 .0 1
0 .0 8
0 .1 4
0 .7 3
0 .5 5
0 .6 7
0 .5 7
51
0 .1 7
- 4 .7 5 e - 3
0 .0 8
0 .1 4
52
0 .1 3
-5 .3 3 e - 4
0 .0 7
0 .1 3
0 .7 5
0 .5 5
0 .0 3
0 .3 8
0 .5 5
0 .0 3
0 .3 7
53
0 .1 6
5 .7 4 e - 3
0 .0 7
0 .1 3
0 .6 3
54
0 .2 2
- 4 .7 5 e - 3
0 .0 5
0 .1 0
0 .5 9
0 .5 2
0 .0 6
0 .3 2
0 .1 1
0 .5 7
0 .5 0
5 .7 2 e - 3
0 .3 3
55
0 .1 8
- 5 .3 3 e - 4
0 .0 7
56
0 .1 4
- 8 .8 7 e - 3
0 .0 8
0 .0 9
0 .6 4
0 .5 0
0 .0 1
0 .3 0
0 .6 3
0 .4 7
0 .0 1
0 .3 6
57
0 .1 2
- 0 .0 1
0 .0 3
0 .0 9
58
0 .1 2
3 .6 8 e - 3
0 .0 8
0 .0 8
0 .6 6
0 .4 7
2 .4 2 e -3
0 .2 4
59
0 .1 2
- 6 .8 1 e -3
0 .0 6
0 .0 7
0 .7 6
0 .5 5
-9 .5 1 e - 4
0 .3 3
60
0 .1 6
9 .9 5 e -3
0 .0 6
0 .0 7
0 .5 3
0 .4 3
9 .1 0 e -3
0 .2 6
0 .0 6
0 .0 6
0 .5 9
0 .4 1
5 .7 2 e -3
0 .2 6
61
0 .1 1
- 2 .6 0 e - 3
62
0 .1 0
1 .6 2 e - 3
0 .0 6
0 .0 6
0 .5 0
0 .4 2
0 .0 1
0 .1 8
63
0 .1 4
1 .6 2 e - 3
0 .0 5
0 .0 6
0 .5 1
0 .3 9
0 .0 6
0 .1 9
64
0 .0 1 0
-5 .3 3 e - 4
0 .0 4
0 .0 6
0 .5 2
0 .3 7
0 .1 7
0 .2 3
65
0 .0 1 0
3 .6 8 e - 3
0 .0 4
0 .0 5
0 .4 7
0 .3 7
-9 .5 1 e - 4
0 .2 2
66
0 .3 6
3 .6 8 e - 3
0 .0 5
0 .0 5
0 .1 9
0 .3 4
0 .0 1
0 .2 6
67
0 .0 1 0
1 .6 2 e - 3
0 .0 4
0 .0 4
0 .1 2
0 .3 2
-2 .6 7 e -3
0 .1 3
68
0 .0 7
1 .6 2 e - 3
0 .0 5
0 .0 4
0 .1 1
0 .3 1
-9 .5 1 e - 4
0 .1 9
97
2. Optical density measurement results from experiment N o.10 to No. 21 (continued)
T im e
E xp
E xp
E xp
E xp
E xp
E xp
E xp
E xp
( m in )
N o . 10
N o . 11
N o . 12
N o . 14
N o . 15
N o .1 6
N o . 17
N o .2 1
69
0 .0 8
- 4 .7 5 e - 3
0 .0 5
0 .0 4
0 .4 3
0 .3 1
0 .0 1
70
0 .0 8
- 0 .0 1
0 .0 4
0 .0 5
0 .6 3
0 .3 0
7 .4 5 e - 3
0 .1 5
71
0 .0 7
- 0 .0 1
0 .0 6
0 .0 3
0 .7 8
0 .2 8
0 .0 3
0 .2 0
0 .1 6
72
0 .0 6
- 0 .0 1
0 .0 5
0 .0 2
0 .6 4
0 .2 7
0 .0 5
0 .1 7
73
0 .0 8
- 5 .3 3 e - 4
0 .0 6
0 .0 3
0 .6 5
0 .2 7
0 .0 5
0 .1 4
74
0 .0 7
- 6 . 8 1e - 3
0 .0 3
0 .0 3
0 .8 0
0 .3 1
0 .0 5
0 .1 7
75
0 .0 6
- 8 .8 7 e - 3
0 .0 4
3 .1 5 e - 3
0 .7 3
0 .3 8
0 .0 6
0 .0 7
76
0 .0 5
- 0 .0 2
0 .0 3
0 .0 3
0 .6 3
0 .2 5
0 .0 5
0 .0 8
77
0 .0 6
- 5 .3 3 e - 4
0 .0 3
0 .0 4
0 .8 1
0 .2 4
78
- 2 .6 0 e - 3
0 .0 4
0 .0 2
0 .7 2
0 .2 6
0 .1 0
0 .0 1 0
79
7 .8 9 e - 3
0 .0 4
0 .0 2
0 .7 8
0 .2 4
0 .1 2
80
1 .6 2 e - 3
0 .0 3
0 .0 3
0 .8 1
0 .2 3
0 .0 6
81
0 .0 2
0 .0 3
0 .0 1
0 .6 3
0 .2 3
0 .1 1
82
9 .9 5 e - 3
0 .0 5
4 .1 7 e - 3
0 .7 5
0 .2 1
0 .0 5
83
7 .8 9 e - 3
0 .0 6
4 .1 7 e - 3
0 .7 4
0 .2 0
0 .0 4
0 .0 8
84
0 .0 2
0 .0 6
- 8 .8 0 e - 3
0 .7 1
0 .2 0
85
0 .0 2
0 .0 5
7 .1 4 e - 3
0 .7 6
0 .2 0
0 .0 5
86
0 .0 2
0 .0 6
0 .0 1
0 .5 6
0 .2 0
0 .0 6
87
0 .0 3
0 .0 5
0 .0 8
0 .6 3
0 .1 9
88
0 .0 6
0 .5 4
0 .1 9
0 .0 6
89
0 .0 7
0 .5 0
0 .1 7
0 .0 6
90
0 .6 1
0 .1 8
0 .0 5
91
0 .6 8
0 .1 6
0 .0 2
92
0 .6 3
0 .1 7
0 .1 4
93
0 .7 7
0 .1 5
0 .0 3
94
0 .7 1
0 .1 7
0 .0 4
95
0 .4 1
0 .1 4
- 3 .6 5 e - 3
0 .2 2
- 3 .6 5 e - 3
96
0 .2 0
97
0 .6 4
98
0 .6 0
0 .0 1
98
3. Optical density measurement results from experiment No.22 to No. 29
T im e
E xp
E xp
E xp
E xp
E xp
E xp
( m in )
N o. 22
N o . 23
N o. 24
N o. 26
N o. 27
N o. 29
-2 0
0 .0 1
-1 9
0 .0 1
-1 8
0 .0 1
-1 7
8 .5 6 e - 3
-1 6
5 .5 0 e - 3
-1 5
0 .0 1
-1 4
8 .5 6 e - 3
-1 3
4 .0 0 e - 3
-1 2
7 .0 6 e - 3
0 .0 1
-1 1
-1 0
0 .1 5
- 3 .6 5 e - 3
2 .0 1 e - 3
0 .0 2
4 .5 3 e - 3
0 .0 2
-9
0 .0 2
3 .0 3 e - 3
4 .3 5 e -4
9 .0 8 e - 3
- 2 .2 0 e - 3
0 .0 2
-8
3 .9 5 e - 3
7 .7 2 e -4
8 .7 8 e - 3
5 .3 7 e - 3
-0 .0 1
0 .0 5
1 .0 8 e - 3
1 .4 4 e - 5
0 .0 7
-7
7 .9 7 e - 3
- 1 .3 9 e - 3
6 .5 6 e - 3
-6
-4 .0 9 e - 3
7 .3 5 e - 3
4 .3 3 e - 3
3 .2 3 e - 3
0 .0 3
0 .0 6
-5
7 .9 7 e - 3
9 .6 1 e - 3
0 .0 5
3 .2 3 e - 3
0 .0 2
0 .0 2
-4
-4 .0 9 e - 3
0 .0 2
0 .0 2
- 3 .3 1 e - 3
0 .0 1
0 .0 5
-3
3 .9 5 e - 3
7 .3 5 e - 3
0 .0 2
1 .0 8 e - 3
0 .0 3
0 .0 5
-2
0 .0 1
0 .0 2
0 .0 6
3 .2 3 e - 3
0 .0 4
0 .0 6
-I
5 .9 2 e - 3
7 .3 5 e - 3
8 .7 8 e - 3
0 .0 1
0 .0 2
0 .0 4
0
7 .9 7 e - 3
7 .7 2 e -4
-2 .1 4 e -4
- 1 .1 6 e - 3
0 .0 6
0 .0 5
I
3 .9 5 e - 3
7 .3 5 e - 3
0 .0 2
0 .0 3
0 .1 2
0 .0 2
2
0 .0 3
9 .6 1 e - 3
0 .0 5
0 .1 3
6 .7 4 e - 3
0 .0 2
3
3 .9 5 e - 3
0 .0 2
0 .1 3
0 .1 8
- 0 .0 2
0 .0 3
4
0 .0 3
0 .0 3
0 .1 8
0 .1 8
0 .0 7
2 .4 4 e - 3
5
0 .0 3
0 .0 4
0 .2 2
0 .2 1
0 .0 5
0 .0 2
6
0 .0 4
0 .0 7
0 .3 2
0 .2 2
0 .0 7
0 .0 3
7
0 .0 6
0 .0 7
0 .2 8
0 .2 4
0 .2 0
0 .0 3
8
0 .0 4
0 .0 9
0 .2 9
0 .2 2
0 .1 5
0 .0 6
9
0 .0 7
0 .0 1 0
0 .2 6
0 .2 5
0 .1 4
0 .0 2
10
0 .0 9
0 .1 0
0 .2 9
0 .2 0
0 .2 3
0 .1 9
11
0 .1 2
0 .1 1
0 .2 6
0 .2 0
0 .2 0
0 .2 4
12
0 .1 4
0 .2 0
0 .2 2
0 .2 1
0 .2 1
0 .1 6
13
0 .1 4
0 .2 0
0 .2 6
0 .1 9
0 .2 5
0 .2 3
14
0 .1 6
0 .2 1
0 .2 7
0 .1 8
0 .2 2
0 .1 6
15
0 .1 8
0 .2 3
0 .2 5
0 .1 6
0 .2 0
0 .1 9
16
0 .1 9
0 .2 4
0 .2 6
0 .1 9
0 .1 8
0 .2 4
17
0 .1 6
0 .3 4
0 .2 4
0 .2 0
0 .1 3
0 .1 9
18
0 .1 9
0 .2 7
0 .2 1
0 .2 8
0 .1 0
0 .3 6
19
0 .2 0
0 .2 3
0 .2 0
0 .1 9
0 .1 3
0 .4 1
20
0 .2 2
0 .2 8
0 .2 0
0 .1 6
0 .1 4
0 .4 5
99
3. Optical density measurement results from experiment No.22 to No. 29 (continued)
T im e
E xp
E xp
E xp
E xp
E xp
E xp
( m in )
N o. 22
N o . 23
N o. 24
N o. 26
N o. 27
N o. 29
21
0 .2 5
0 .3 0
0 .1 9
0 .1 5
0 .1 5
0 .4 7
22
0 .2 3
0 .3 4
0 .1 8
0 .1 7
0 .2 4
0 .4 7
23
0 .2 5
0 .2 3
0 .1 6
0 .1 6
0 .1 6
0 .4 5
24
0 .2 5
0 .2 2
0 .1 8
0 .1 7
0 .2 7
0 .4 6
25
0 .2 6
0 .2 6
0 .1 8
0 .1 5
0 .2 5
0 .4 3
26
0 .2 7
0 .2 4
0 .1 7
0 .1 6
0 .2 7
0 .4 4
27
0 .2 7
0 .2 4
0 .1 6
0 .1 6
0 .2 9
0 .4 2
28
0 .2 8
0 .2 4
0 .1 5
0 .1 4
0 .2 7
0 .4 3
0 .4 5
29
0 .2 8
0 .2 3
0 .1 4
0 .1 2
0 .2 7
30
0 .3 4
0 .2 4
0 .1 6
0 .1 2
0 .2 9
0 .4 7
31
0 .4 0
0 .2 5
0 .1 6
0 .1 3
0 .3 0
0 .4 5
32
0 .4 6
0 .2 9
0 .1 5
0 .1 2
0 .2 5
0 .4 4
33
0 .4 6
0 .2 6
0 .1 7
0 .1 1
0 .1 8
0 .4 5
34
0 .4 3
0 .2 6
0 .1 5
0 .1 3
0 .2 3
0 .4 6
35
0 .4 3
0 .2 6
0 .1 6
0 .1 1
0 .2 2
0 .4 8
36
0 .4 1
0 .2 6
0 .1 4
0 .1 3
0 .3 2
0 .4 6
37
0 .3 9
0 .2 5
0 .1 2
0 .1 3
0 .3 4
0 .4 5
38
0 .3 5
0 .2 5
0 .1 2
0 .1 1
0 .2 9
0 .4 4
39
0 .3 4
0 .2 7
0 .1 1
0 .1 1
0 .4 4
0 .4 3
40
0 .3 3
0 .2 5
0 .1 9
0 .1 2
0 .2 5
0 .4 2
41
0 .3 0
0 .2 5
0 .5 1
0 .0 1 0
0 .2 9
0 .4 2
42
0 .3 0
0 .2 3
0 .5 3
0 .0 8
0 .2 2
0 .3 6
43
0 .2 8
0 .2 3
0 .4 4
0 .0 9
0 .2 9
0 .3 6
44
0 .2 9
0 .2 2
0 .6 1
0 .0 8
0 .3 3
0 .3 6
45
0 .2 5
0 .2 1
0 .5 8
0 .0 7
0 .2 5
0 .3 4
46
0 .2 6
0 .2 1
0 .5 0
0 .0 6
0 .2 3
0 .3 4
0 .3 4
47
0 .2 5
0 .2 2
0 .5 3
0 .0 5
0 .2 2
48
0 .2 3
0 .2 1
0 .5 0
0 .0 7
0 .2 3
0 .3 3
0 .3 3
0 .2 8
49
0 .2 2
0 .2 0
0 .5 3
0 .0 8
50
0 .2 4
0 .1 7
0 .5 3
0 .0 8
0 .2 2
0 .2 7
0 .4 8
0 .0 7
0 .2 5
0 .2 7
51
0 .2 1
0 .1 8
52
0 .2 3
0 .1 7
0 .5 0
0 .0 7
0 .2 3
0 .2 5
0 .0 6
0 .2 0
0 .2 5
53
0 .2 2
0 .1 8
0 .4 7
54
0 .2 3
0 .1 7
0 .4 5
0 .0 6
0 .2 2
0 .2 5
0 .0 6
0 .2 3
0 .2 4
55
0 .2 2
0 .1 7
0 .4 6
56
0 .2 0
0 .1 6
0 .3 8
0 .0 7
0 .2 4
0 .2 0
57
0 .2 0
0 .1 4
0 .4 0
0 .0 5
0 .2 8
0 .1 7
58
0 .2 1
0 .1 7
0 .3 9
0 .0 5
0 .2 7
0 .1 8
0 .1 8
0 .1 3
0 .3 8
0 .0 4
0 .2 7
0 .1 8
60
0 .1 8
0 .1 5
0 .3 3
0 .0 9
0 .2 4
0 .1 8
61
0 .1 6
0 .1 4
0 .3 2
0 .0 4
0 .3 4
0 .1 5
0 .1 8
0 .0 1 0
0 .3 0
0 .0 9
0 .3 0
0 .1 5
0 .3 1
0 .0 8
0 .2 0
0 .1 5
59
62
63
0 .1 8
0 .2 1
64
0 .1 7
0 .2 6
0 .3 4
0 .0 6
0 .2 3
0 .1 3
65
0 .1 7
0 .2 3
0 .3 1
0 .0 6
0 .1 9
0 .1 3
100
3. Optical density measurement results from experiment No.22 to No. 29 (continued)
T im e
E xp
E xp
E xp
E xp
E xp
E xp
( m in )
N o. 22
N o. 23
N o. 24
N o. 26
N o. 27
N o. 29
66
0 .1 6
0 .2 5
0 .3 0
0 .0 6
0 .2 2
0 .1 2
0 .2 5
0 .2 8
0 .0 6
0 .2 1
0 .0 9
67
0 .2 1
68
0 .1 8
0 .2 5
0 .2 7
0 .0 6
0 .2 6
0 .0 9
0 .2 4
0 .2 5
0 .0 4
0 .2 4
0 .0 9
69
0 .1 6
70
0 .1 8
0 .3 0
0 .2 4
0 .0 3
0 .2 3
0 .1 0
71
0 .1 6
0 .2 3
0 .2 2
0 .0 4
0 .2 7
0 .0 8
72
0 .1 7
0 .2 4
0 .2 2
0 .0 4
0 .2 0
0 .1 8
73
0 .1 8
0 .2 1
0 .2 2
0 .0 4
0 .2 2
0 .0 8
74
0 .1 6
0 .1 9
0 .2 3
0 .0 3
0 .2 2
0 .0 5
75
0 .1 5
0 .2 0
0 .2 1
0 .0 3
0 .2 1
76
0 .1 5
0 .2 0
0 .0 2
0 .0 5
0 .0 3
77
0 .1 5
0 .2 2
0 .0 3
78
0 .1 9
0 .1 9
0 .0 3
0 .0 3
79
0 .1 6
0 .1 9
80
0 .1 7
0 .2 1
0 .0 3
81
0 .1 7
0 .1 7
0 .0 5
82
0 .1 8
0 .1 6
0 .0 3
83
0 .1 7
0 .1 9
0 .0 3
84
0 .1 8
0 .2 0
0 .0 3
85
0 .1 8
0 .1 5
86
0 .1 7
0 .1 4
87
0 .1 7
0 .1 9
88
0 .1 2
0 .1 2
89
0 .1 4
90
0 .1 4
101
Raw data from total protein and cell enumeration measurements
E x p e r im e n t N o . I — C o n tr o l
E f f lu e n t
T im e
K. pneum oniae
P. aeruginosa
T o ta l p r o te in
s a m p le s
( m in )
( c f u /m L )
( c f u /m L )
(p g /m L )
A
0
5 .2 0 e + 7
7 .0 0 e + 6
1 .0 4
B
15
7 .0 0 e + 7
5 .0 0 e + 6
1 .1 9
1 .2 3
C
30
7 .7 0 e + 7
3 .0 0 e + 6
D
45
6 .8 0 e + 7
4 .0 0 e + 6
1 .0 3
E
60
5 .3 0 e + 7
1 .6 0 e + 6
0 .9 8
F
75
4 .9 0 e + 7
1 .4 0 e + 6
0 .8 9
S li d e
T im e
K. pneum oniae
P. aeruginosa
T o ta l p r o te in
s a m p le s
( m in )
( c f u / m m 2)
( c f u /m m 2)
( p g / m m 2)
SO
0
3 .1 2 e + 8
2 .7 9 e + 7
1 6 .8
S30
30
2 .9 9 e + 8
1 .8 1 e + 7
1 6 .3
S75
75
3 .4 5 e + 8
1 .4 8 e + 7
1 6 .2
E x p e r im e n t N o . 2 — N a C l
E f f lu e n t
T im e
K. pneum oniae
P. aeruginosa
T o ta l p r o te in
s a m p le s
( m in )
( c f u /m L )
( c f u /m L )
(p g /m L )
A
-1 5
1 .3 0 e + 7
1 .3 0 e + 6
0 .5 7
B
0
2 .9 0 e + 7
1 .7 0 e + 6
0 .7 5
C
15
2 .5 0 e + 8
1 .3 0 e + 7
1 4 .7 4
D
30
2 .1 0 e + 8
1 .0 0 e + 7
1 1 .1 3
E
45
1 .5 2 e + 8
1 .1 0 e + 7
5 .1 9
F
60
9 .8 0 e + 7
5 .1 0 e + 6
3 .2 9
G
75
7 .7 0 e + 7
5 .7 0 e + 6
2 .9 3
H
90
4 .8 0 e + 7
3 .7 0 e + 6
1 .9 2
S lid e
T im e
K. pneum oniae
P. aeruginosa
T o ta l p r o te in
s a m p le s
( m in )
( c f u / m m 2)
( c f u / m m 2)
( p g / m m 2)
SO
0
3 .2 9 e + 8
2 .3 0 e + 7
1 6 .4 6
S75
75
1 .6 9 e + 8
8 .2 2 e + 6
7 .3 9
102
Experiment N o.3—NaCl
E fflu e n t
T im e
K. pneum oniae
P. aeruginosa
T o t a l p r o te in
s a m p le s
( m in )
( c f u /m L )
( c f t i/m L )
(p g /m L )
A
-1 5
2 .1 0 e + 7
1 .2 0 e + 6
0 .8 7
B
0
3 .2 0 e + 7
2 .3 0 e + 6
0 .7 0
C
15
8 .4 0 e + 8
5 .0 0 e + 6
1 5 .3 6
D
30
6 .1 0 e + 8
6 .0 0 e + 6
1 3 .7 2
E
45
2 .7 0 e + 8
3 .7 0 e + 6
9 .9 0
60
1 .4 0 e + 8
2 .9 0 e + 6
6 .7 4
F
G
75
1 .0 2 e + 8
2 .2 0 e + 6
3 .7 0
H
90
7 .9 0 e + 7
1 .5 0 e + 6
2 .6 3
S li d e
T im e
K. pneum oniae
P. aeruginosa
T o ta l p r o te in
s a m p le s
( m in )
( c f u / m m 2)
( c f u / m m 2)
( p g / m m 2)
SO
0
6 .9 0 e + 8
1 .4 8 e + 7
2 5 .3 6
S30
30
2 .8 4 e + 9
6 .4 1 e + 7
2 0 .8 2
S75
75
2 .6 3 e + 8
1 .3 0 e + 7
1 8 .1 1
E x p N o . 4 —- N a C l
E fflu e n t
T im e
K. pneum oniae
P. aeruginosa
T o ta l p r o te in
s a m p le s
( m in )
( c f u /m L )
( c f u /m L )
(p g /m L )
A
0
5 .1 0 e + 7
6 .2 0 e + 6
1 .2 0
B
15
6 .0 0 e + 8
5 .0 0 e + 7
1 8 .4 7
C
30
3 .0 0 e + 8
3 .5 0 e + 7
9 .8 8
D
45
1 .3 0 e + 8
1 .9 0 e + 7
7 .0 9
E
60
1 .0 5 e + 8
1 .1 0 e + 7
5 .7 8
F
75
7 .8 0 e + 7
7 .2 0 e + 6
3 .6 5
S lid e
T im e
K. pneum oniae
P. aeruginosa
T o t a l p r o te in
s a m p le s
( m in )
( c f u /m m 2)
( c f u /m m 2)
( p g / m m 2)
SO
0
3 .9 4 e + 8
6 .9 0 e + 7
2 2 .2 7
S30
30
1 .5 4 e + 8
6 .2 4 e + 7
1 3 .3 6
S75
75
1 .4 8 e + 8
4 .2 7 e + 7
1 2 .8 0
103
Experiment N o.5—NaCl
E f f lu e n t
T im e
K. pneum oniae
P. aeruginosa
T o ta l p r o te in
s a m p le s
( m in )
( c f u /m L )
( c f u /m L )
(p g /m L )
A
0
2 .5 0 e + 7
3 .9 0 e + 6
1 .3 9
B
15
8 .0 0 e + 7
7 .3 0 e + 6
3 .3 0
C
30
6 .7 0 e + 7
6 .0 0 e + 6
3 .1 4
D
45
3 .1 0 e + 7
5 .0 0 e + 6
2 .6 3
E
60
2 .9 0 e + 7
4 .4 0 e + 6
1 .7 9
75
2 .6 0 e + 7
4 . 1 O e+ 6
1 .3 4
F
S li d e
T im e
K. pneum oniae
P. aeruginosa
T o t a l p r o te in
s a m p le s
( m in )
( c f u / m m 2)
( c f u /m m 2)
( p g / m m 2)
sO
0
2 .5 3 e + 8
9 .2 0 e + 7
2 3 .2 7
s60
30
2 .4 3 e + 8
5 .2 6 e + 7
2 1 .7 0
s75
75
1 .8 4 e + 8
5 .9 2 e + 7
2 1 .5 1
E x p e r im e n t N o . 6 — N a C l
E f f lu e n t
T im e
K. pneum oniae
P. aeruginosa
T o ta l p r o te in
s a m p le s
( m in )
( c f u /m L )
( c f u /m L )
(p g /m L )
A
0
3 .5 0 e + 7
7 .7 0 e + 6
0 .6 1
B
15
5 .4 0 e + 8
3 .8 0 e + 7
1 6 .0 1
1 1 .1 8
C
30
3 .9 0 e + 8
3 .0 0 e + 7
D
45
4 .1 0 e + 8
3 .6 0 e + 7
8 .8 5
E
60
2 .0 0 e + 8
2 .2 0 e + 7
5 .9 1
F
75
1 .3 0 e + 8
9 .0 0 e + 6
5 .7 3
S li d e
s a m p le s
K. pneum oniae
P aeruginosa
T o t a l p r o te in
( m in )
( c f u / m m 2)
( c f u /m m 2)
( p g / m m 2)
T im e
SO
0
2 .7 6 e + 8
1 .7 4 e + 7
2 0 .7 0
S30
30
6 .9 0 e + 7
1 .2 2 e + 7
6 .9 0
S75
75
3 .7 8 e + 7
3 .9 4 e + 6
5 .2 7
S90
90
3 .5 8 e + 7
4 .9 6 e + 6
4 .4 6
104
Experiment No.7— CaCl2
E f f lu e n t
T im e
K. pneum oniae
P. aeruginosa
T o ta l p r o te in
s a m p le s
( m in )
( c f u /m L )
( c f u /m L )
(p g /m L )
A
0
1 .1 0 e + 7
2 .4 0 e + 6
1 .1 5
B
15
5 .6 0 e + 7
1 .8 0 e + 7
1 6 .7 2
1 2 .7 4
C
30
2 .6 0 e + 7
1 .8 0 e + 7
D
45
2 .1 0 e + 7
1 .0 0 e + 7
6 .6 4
E
60
1 .5 0 e + 7
2 .3 0 e + 6
4 .3 3
F
75
8 .2 0 e + 7
1 .1 0 e + 7
1 0 .5 6
G
90
7 .5 0 e + 7
4 .0 0 e + 6
6 .7 5
S li d e
T im e
K. pneum oniae
P. aeruginosa
T o ta l p r o te in
s a m p le s
( m in )
( c f u / m m 2)
( c f u /m m 2)
( p g / m m 2)
SO
0
4 .2 7 e + 8
2 .3 0 e + 7
2 7 .2 8
S30
30
3 .2 5 e + 8
6 .5 7 e + 6
1 1 .6 7
S75
75
3 .0 2 e + 8
9 .8 6 e + 6
1 4 .1 1
S90
90
2 .2 3 e + 8
3 .2 9 e + 7
1 6 .0 0
E x p e r im e n t N o . S - M g C l 2
E f f lu e n t
T im e
K. pneum oniae
P. aeruginosa
T o ta l p r o te in
s a m p le s
( m in )
( c f u /m L )
( c f u /m L )
(p g /m L )
A
0
6 .7 0 e + 7
6 .2 0 e + 6
1 .4 7
B
15
5 .3 0 e + 8
2 .7 0 e + 7
1 5 .0 1
1 1 .2 3
C
30
4 .9 0 e + 8
3 .0 0 e + 7
D
45
1 .6 0 e + 8
1 .7 0 e + 7
7 .4 6
E
60
8 .6 0 e + 7
1 .4 0 e + 7
2 .6 6
F
75
1 .0 0 e + 8
6 .4 0 e + 6
1 .4 9
S lid e
T im e
K. pneum oniae
P. aeruginosa
T o ta l p r o te in
s a m p le s
( m in )
( c f u / m m 2)
( c f u /m m 2)
( p g / m m 2)
SO
0
7 .2 3 e + 8
5 .5 9 e + 8
3 7 .3 2
S60
60
3 .2 9 e + 8
7 .2 3 e + 7
2 9 .7 9
S75
75
8 .8 7 e + 8
8 .2 2 e + 7
2 9 .2 3
105
Experiment N o.9— pH
E f f lu e n t
T im e
K. pneum oniae
P. aeruginosa
T o ta l p r o te in
s a m p le s
( m in )
( c f u /m L )
( c f u /m L )
(p g /m L )
A
0
6 .4 0 e + 6
2 .2 0 e + 6
0 .3 1
B
15
6 .9 0 e + 6
1.4 0 e + 6
0 .3 4
C
30
6 .8 0 e + 6
5 .0 0 e + 5
0 .3 3
D
45
1 .0 3 e + 8
5 .3 0 e + 6
2 .4 2
E
60
5 .0 0 e + 7
1 .4 0 e + 6
2 .2 2
F
75
2 .3 0 e + 7
5 .0 0 e + 5
1 .4 2
S lid e
T im e
K. pneum oniae
P. aeruginosa
T o ta l p r o te in
s a m p le s
( m in )
( c f u / m m 2)
( c f u /m m 2)
( p g / m m 2)
SI
0
1 .1 0 e + 8
1 .1 5 e + 8
7 .3 5
S2
15
9 .5 3 e + 7
6 .5 7 e + 7
5 .8 2
S3
30
9 .6 9 e + 7
4 .9 3 e + 7
8 .7 3
S4
45
7 .8 9 e + 7
2 .1 4 e + 7
6 .4 0
S5
60
1 .2 5 e + 8
4 .9 3 e + 7
7 .3 8
S6
75
8 .5 4 e + 7
4 .9 3 e + 7
6 .5 2
E xp N o . 10— pH
E f f lu e n t
T im e
T o ta l p r o te in
s a m p le s
( m in )
(p g /m L )
A
0
0 .7 2
B
15
4 .6 9
C
30
4 .8 9
D
45
6 .6 1
E
60
5 .2 5
F
75
3 .4 8
S li d e
T im e
K. pneum oniae
P. aeruginosa
T o ta l p r o te in
s a m p le s
( m in )
( c f u / m m 2)
( c f u /m m 2)
( p g / m m 2)
SO
0
1 .9 7 e + 8
5 .9 2 e + 7
1 2 .8 6
S lO
10
1 .6 4 e + 6
4 .9 3 e + 5
1 0 .3 5
S20
20
8 .2 2 e + 5
2 .7 9 e + 5
1 1 .4 2
S30
30
1 . 15 e + 6
3 .6 1 e + 5
8 .4 1
S45
45
6 .9 0 e + 5
2 .3 3 e + 5
6 .8 4
S60
60
7 .7 2 e + 5
1 .3 1 e + 5
7 .5 3
S75
75
4 .4 4 e + 5
9 .8 6 e + 4
6 .9 6
106
Experiment No. 11 — Sucrose
E f f lu e n t
T im e
K. pneum oniae
P. aeruginosa
T o ta l p r o te in
s a m p le s
( m in )
( c f u /m L )
( c f u /m L )
(p g /m L )
A
0
2 .7 0 e + 7
7 .0 0 e + 6
0 .8 2
B
15
1 .1 8 e + 8
1 .1 0 e + 7
1 .8 0
C
30
1 .1 5 e + 8
8 .0 0 e + 6
2 .0 8
D
45
6 .9 0 e + 7
8 .0 0 e + 6
1 .9 5
E
60
4 .8 0 e + 7
3 .6 0 e + 6
1 .3 2
F
75
2 .6 0 e + 7
5 .0 0 e + 6
1 .0 5
S li d e
T im e
K. pneum oniae
P. aeruginosa
T o ta l p r o te in
s a m p le s
( m in )
( c f u / m m 2)
( c f u /m m 2)
( p g / m m 2)
0
2 .4 6 e + 8
1 .3 1 e + 7
2 3 .9 6
S30
30
4 .1 l e + 8
1 .3 1 e + 7
2 2 .8 3
S75
75
3 .1 2 e + 8
2 .1 4 e + 7
2 1 .4 5
SO
E x p N o . 1 2 - -E D T A
E f f lu e n t
T im e
K. pneum oniae
P. aeruginosa
T o ta l p r o te in
s a m p le s
( m in )
( c f u /m L )
( c f ii/ m L )
(p g /m L )
A
0
2 .1 0 e + 7
1 .2 0 e + 6
0 .8 7
B
15
1 .3 4 e + 8
4 .4 0 e + 6
4 .2 0
C
30
1.1 l e + 8
6 .5 0 e + 6
2 .4 2
D
45
6 .7 0 e + 7
6 .3 0 e + 6
1 .8 9
E
60
6 .9 0 e + 7
3 .6 0 e + 6
1 .3 5
F
75
3 .5 0 e + 7
4 .8 0 e + 6
0 .7 3
G
90
2 .4 0 e + 7
2 .3 0 e + 6
0 .7 6
S li d e
T im e
K. pneum oniae
P. aeruginosa
T o ta l p r o te in
s a m p le s
( m in )
( c f u / m m 2)
( c f u /m m 2)
( p g / m m 2)
SO
0
2 .1 4 e + 8
8 .0 5 e + 6
1 3 .9 8
S30
30
2 .7 9 e + 8
1 .3 1 e + 6
9 .0 4
S75
75
3 .2 9 e + 8
1. 15 e + 6
8 .4 4
107
Experiment No. 13— Dequest 2006
E f f lu e n t
T im e
T o t a l p r o te in
s a m p le s
( m in )
(p g /m L )
A
0
1 .1 7
B
15
9 .7 6
C
30
3 .7 5
D
45
3 .9 9
E
60
2 .0 1
F
75
1 .3 0
S li d e
T im e
T o ta l p r o te in
s a m p le s
( m in )
( p g / m m 2)
SO
0
1 8 .6 4
S75
75
1 3 .7 1
E x p e r im e n t N o . 1 4 — S D S
E f f lu e n t
T im e
K. pneum oniae
P. aeruginosa
T o ta l c e ll
T o t a l p r o te in
s a m p le s
( m in )
( c f u /m L )
( c f u /m L )
( c f u /m L )
(p g /m L )
A
0
2 .4 0 e + 7
9 .0 0 e + 5
3 .3 4 e + 7
0 .3 2
B
15
2 .0 0 e + 7
1 .5 0 e + 6
3 .4 1 e + 7
4 .9 6
C
30
1 .6 0 e + 7
2 .4 0 e + 6
3 .2 9 e + 7
6 .8 1
D
45
1 .4 0 e + 7
2 .1 0 e + 6
3 .1 6 e + 7
4 .4 8
3 .8 1
E
60
1 .6 0 e + 7
1 .7 0 e + 6
3 .1 8 e + 7
F
75
1 .5 0 e + 7
2 .0 0 e + 6
3 .1 2 e + 7
5 .7 9
G
90
1 .3 0 e + 7
1 .6 0 e + 6
3 .0 9 e + 7
3 .6 1
S li d e
T im e
K. pneum oniae
P. aeruginosa
T o ta l c e ll
T o ta l p r o te in
s a m p le s
( m in )
( c f u / m m 2)
( c f u /m m 2)
( c f u /m m 2)
( p g / m m 2)
SI
0
3 .1 2 e + 8
1 .0 7 e + 7
5 .2 8 e + 8
1 8 .7 9
S2
15
3 .2 0 e + 8
3 .2 9 e + 6
5 .1 9 e + 8
1 3 .2 5
S3
30
3 .0 4 e + 8
9 .0 4 e + 6
4 .9 6 e + 8
9 .1 0
S4
45
2 .7 1 e + 8
6 .5 7 e + 6
5 .0 4 e + 8
7 .0 6
S5
60
2 .7 9 e + 8
7 .3 9 e + 6
4 .8 2 e + 8
4 .8 9
S6
75
2 .1 4 e + 8
4 .9 3 e + 6
4 .3 5 e + 8
2 .1 8
108
Experiment No. 15— SDS
E fflu e n t
T im e
T o ta l p r o te in
s a m p le s
( m in )
(p g /m L )
A
0
1 .1 7
B
15
1.11
C
30
1 4 .0 6
D
45
2 1 .9 8
E
60
1 7 .7 4
F
75
1 0 .6 2
G
90
4 .0 3
S li d e
T im e
K. pneum oniae
P. aeruginosa
T o ta l p r o te in
s a m p le s
( m in )
( c f u / m m 2)
( c f u /m m 2)
( p g / m m 2)
SI
-I
2 .7 9 e + 8
8 .2 2 e + 6
2 2 .6 4
S2
0
1 .9 7 e + 8
4 .9 3 e + 6
1 9 .3 0
S3
I
3 .6 1 e + 7
5 .4 2 e + 5
6 .0 7
S4
3
2 .8 4 e + 7
3 .9 4 e + 5
5 .8 4
S5
4
1 .0 7 e + 6
2 .6 3 e + 4
5 .6 5
S6
10
9 .0 4 e + 5
1 .2 2 e + 4
4 .0 2
S7
2 2 .5
4 .6 0 e + 6
2 .6 3 e + 4
4 .4 6
S8
3 1 .5
7 .7 2 e + 6
2 .9 6 e + 5
5 .1 5
S9
4 6 .5
2 .1 0 e + 7
3 .9 4 e + 6
7 .8 4
S lO
52
3 .9 4 e + 7
7 .3 9 e + 6
1 3 .8 0
S ll
71
1 .5 6 e + 8
2 .4 6 e + 7
1 8 .6 3
S12
95
3 .7 8 e + 8
4 .1 l e + 7
2 4 .7 7
E x p e r im e n t N o . 16 — S D S
E f f lu e n t
T im e
K. pneum oniae
P. aeruginosa
T o ta l p r o te in
s a m p le s
( m in )
( c f u /m L )
( c f u /m L )
(p g /m L )
A
0
7 .4 0 e + 7
1 .1 0 e + 6
1 .9 4
B
15
7 .6 0 e + 8
1 .2 0 e + 7
2 4 .2 4
C
30
6 .5 0 e + 8
1 .3 0 e + 7
1 9 .9 4
D
45
2 .1 0 e + 8
1 .4 0 e + 7
1 2 .9 3
E
60
2 .2 0 e + 8
8 .0 0 e + 6
1 0 .2 1
F
75
1 .4 0 e + 8
3 .3 0 e + 6
5 .4 0
T im e
K. pneum oniae
P. aeruginosa
T o t a l p r o te in
( m in )
( c f u /m m 2)
( c f u /m m 2)
( p g / m m 2)
SO
0
4 .4 4 e + 8
7 .7 2 e + 6
1 9 .3 7
S75
75
1 .3 l e + 7
1 . 15 e + 6
2 .4 1
S lid e
s a m p le s
109
Exp No. 11 — Tween 20
E fflu e n t
T im e
K. pneum oniae
P. aeruginosa
T o t a l p r o te in
s a m p le s
( m in )
( c f ii/ m L )
( c f u /m L )
(p g /m L )
A
0
4 .7 0 e + 7
7 .0 0 e + 5
0 .4 9
B
15
4 .8 0 e + 7
3 .0 0 e + 5
6 .7 2
C
30
4 .4 0 e + 7
5 .0 0 e + 5
4 .0 5
D
45
3 .8 0 e + 7
2 .0 0 e + 5
6 .2 8
E
60
3 .3 0 e + 7
4 .0 0 e + 5
4 .6 4
F
75
6 .5 0 e + 7
5 .0 0 e + 5
3 .5 9
S lid e
T im e
K. pneum oniae
P. aeruginosa
T o t a l p r o te in
s a m p le s
( m in )
( c f u / m m 2)
( c f u /m m 2)
( p g / m m 2)
0
4 .9 3 e + 8
3 .2 9 e + 6
1 9 .6 6
75
3 .3 7 e + 8
3 .2 9 e + 6
1 5 .7 3
SO
S75
E x p N o . 1 8 — T r ito n X - 1 0 0
E f f lu e n t
T im e
T o ta l p r o te in
s a m p le s
( m in )
(p g /m L )
A
0
2 .5
B
15
1 2 .4
C
30
1 5 .1
D
45
1 3 .3
E
60
5 .1
F
75
3 .3
S li d e
T im e
T o ta l p r o te in
s a m p le s
( m in )
( p g / m m 2)
SO
0
2 0 .1 3
S75
75
1 1 .4 3
HO
Experiment No. 19— Chlorine
E f f lu e n t
T im e
K. pneum oniae
P. aeruginosa
T o ta l p r o te in
s a m p le s
( m in )
( c f u /m L )
( c f u /m L )
(p g /m L )
A
-1 5
2 .3 0 e + 7
1 .5 0 e + 6
0 .6 9
B
0
2 .4 0 e + 7
1.6 0 e + 6
0 .7 3
C
15
1 .0 9 e + 7
4 .3 0 e + 5
8 .6 9
D
30
4 .7 0 e + 6
3 .7 0 e + 5
5 .3 6
E
45
1 .1 7 e + 7
1 .2 7 e + 6
4 .8 2
F
60
8 .8 0 e + 6
1 .2 0 e + 6
2 .9 4
G
75
2 .1 8
S li d e
T im e
K. pneum oniae
P. aeruginosa
T o ta l p r o te in
s a m p le s
( m in )
( c f u / m m 2)
( c f u /m m 2)
( p g / m m 2)
SO
-2
1 .6 4 e + 8
7 .5 6 e + 6
1 7 .2 5
6 .9 0 e + 6
1 8 .8 8
SI
0
1 .6 6 e + 8
S2
I
1 .2 2 e + 7
1 .3 6 e + 6
6 .9 6
S3
6
5 .2 6 e + 6
1 .1 7 e + 6
4 .8 3
S4
20
2 .7 3 e + 7
5 .4 2 e + 6
7 .7 8
S5
25
7 .0 7 e + 7
1 .4 8 e + 7
9 .9 7
S6
44
9 .6 9 e + 7
1 .4 0 e + 7
1 1 .7 9
SI
52
7 .5 6 e + 7
1 .1 5 e + 7
1 2 .3 0
S8
68
1 .0 0 e + 8
1 .5 4 e + 7
1 5 .7 4
S9
78
8 .3 8 e + 7
1 .8 1 e + 7
1 6 .4 3
E x p e r im e n t N o . 2 0 —- C h lo r in e
E f f lu e n t
T im e
T o ta l p r o te in
s a m p le s
( m in )
(p g /m L )
A
-1 5
0 .8 0
B
0
0 .7 4
C
15
8 .4 1
D
30
6 .7 4
E
45
2 .9 0
F
60
1 1 .2 4
G
75
7 .5 9
H
90
2 .8 6
S li d e
T im e
K. pneum oniae
P. aeruginosa
T o ta l p r o te in
s a m p le s
( m in )
( c f u / m m 2)
( c f u /m m 2)
( p g / m m 2)
SO
-2
2 .9 6 e + 8
4 .1 l e + 7
1 8 .0 6
SI
0
2 .1 4 e + 8
3 .6 1 e + 7
1 9 .2 6
S2
I
1 .4 5 e + 7
1 .8 1 e + 6
1 4 .4 9
S3
6
2 .2 5 e + 7
5 .2 6 e + 6
1 3 .0 5
1 0 .0 4
S4
20
2 .6 6 e + 7
1.4 l e + 7
S5
26
4 .7 7 e + 7
2 .0 5 e + 7
1 0 .9 8
S6
44
1 .0 4 e + 8
7 .3 9 e + 7
1 5 .3 1
SI
4 8 .5
1 .1 2 e + 8
6 .2 4 e + 7
1 7 .0 6
SB
68
2 .3 0 e + 8
7 .2 3 e + 7
1 8 .7 5
I ll
Experiment No. 21— Chlorine (15mg/L, pH= 10.9)
E f f lu e n t
T im e
T o ta l p r o te in
s a m p le s
( m in )
(p g /m L )
A
0
1 .3 4
B
15
8 .2 5
C
30
1 4 .8 6
D
45
6 .7 3
E
60
1 0 .3 4
F
75
3 .6 7
G
90
2 .0 4
S lid e
T im e
K. pneum oniae
P. aeruginosa
T o t a l p r o te in
s a m p le s
( m in )
( c f u / m m 2)
( c f u /m m 2)
( p g / m m 2)
SO
0
1 .8 1 e + 8
2 .3 0 e + 7
1 7 .9 6
S lO
10
2 .9 6 e + 7
U 5e+6
6 .2 7
S20
20
1 .8 6 e + 8
3 .4 5 e + 6
8 .0 9
S30
30
1 .8 6 e + 7
2 .1 4 e + 6
6 .0 9
S45
45
6 .9 0 e + 5
1 .2 5 e + 6
6 .1 5
S60
60
3 .6 1 e + 5
2 .9 6 e + 5
7 .2 2
S75
75
4 .2 7 e + 5
3 .2 9 e + 5
5 .4 6
S90
90
3 .1 2 e + 5
2 .6 3 e + 5
6 .9 6
E x p e r im e n t N o . 2 2 - - N H 2C l (lO O m g /L )
E f f lu e n t
T im e
T o ta l p r o te in
s a m p le s
( m in )
(p g /m L )
A
0
1 .3 9
B
15
4 .5 6
C
30
9 .7 3
D
45
1 4 .7 5
E
60
9 .3 4
F
75
4 .9 1
G
90
3 .0 3
S li d e
T im e
K. pneum oniae
P. aeruginosa
T o ta l p r o te in
s a m p le s
( m in )
( c f u /m m 2)
( c f u /m m 2)
( p g / m m 2)
SO
0
1 .3 1 e + 8
4 .9 3 e + 6
1 5 .5 6
S lO
10
1 . 15 e + 7
6 .5 7 e + 5
1 1 .9 2
S20
20
6 .7 4 e + 5
4 .9 3 e + 4
9 .5 4
S30
30
4 .6 0 e + 4
3 .2 9 e + 4
8 .9 1
S40
40
5 .7 5 e + 5
9 .8 6 e + 3
6 .2 1
5 .1 5
S50
50
1 .9 7 e + 5
1 .4 8 e + 4
S60
60
7 .2 3 e + 5
1 .3 1 e + 5
5 .6 5
S75
75
3 .4 5 e + 5
6 .5 7 e + 4
5 .8 4
S90
90
2 .4 6 e + 4
9 .8 6 e + 3
5 .2 7
112
Experiment No. 23 —NH2Cl (25mg/L)
E f f lu e n t
T im e
T o ta l p r o te in
s a m p le s
( m in )
(p g /m L )
A
0
0 .6 9
B
15
5 .3 9
C
30
7 .7 9
D
45
6 .0 6
E
60
4 .7 1
F
75
5 .5 1
S li d e
T im e
K. pneum oniae
P. aeruginosa
T o ta l p r o te in
s a m p le s
( m in )
( c f u / m m 2)
( c f u / m m 2)
( p g / m m 2)
SO
0
2 .6 3 e + 8
1 .6 4 e + 7
2 3 .3 3
S lO
10
6 .2 4 e + 7
6 .5 7 e + 6
2 2 .3 9
S20
20
1 .6 8 e + 6
1 .6 8 e + 6
2 1 .8 3
S30
30
1 .4 5 e + 6
5 .9 2 e + 5
2 1 .3 9
S45
45
1 .1 2 e + 6
5 .9 2 e + 5
2 0 .7 6
S60
60
7 .8 9 e + 5
2 .9 6 e + 5
2 0 .4 5
S75
75
4 .9 3 e + 5
1 .3 8 e + 5
1 9 .0 7
E x p e r im e n t N o . 2 4 - - N H 2C l ( 2 5 m g / L )
E f f lu e n t
T im e
T o t a l p r o te in
s a m p le s
( m in )
(p g /m L )
A
0
1 .7 6
B
15
1 1 .8 4
C
30
1 0 .9 6
D
45
1 6 .1 5
E
60
2 3 .7 9
F
75
1 1 .9 4
G
90
5 .7 3
S li d e
s a m p le s
T im e
K. pneum oniae
P. aeruginosa
T o ta l p r o te in
( m in )
( c f u /m m 2)
( c f u /m m 2)
( p g / m m 2)
SO
0
4 .9 3 e + 8
8 .2 2 e + 6
2 6 .4 7
S15
15
1 .0 2 e + 7
4 .9 3 e + 5
2 3 .8 3
S30
30
4 .1 l e + 6
1 .3 1 e + 5
1 9 .8 8
2 .9 6 e + 6
6 .2 4 e + 4
9 .7 2
1 .6 4 e + 6
2 .3 0 e + 4
9 .8 5
8 .3 4
8 .8 5
S45
S60
45
60
S75
75
2 .7 9 e + 6
6 .5 7 e + 4
S90
90
6 .0 8 e + 5
4 .1 l e + 4
113
Experiment No.25—NH2Cl (lOmg/L)
E fflu e n t
T im e
T o ta l p r o te in
s a m p le s
( m in )
(p g /m L )
A
O
1 .1 0
B
15
5 .3 6
C
30
6 .1 5
D
45
5 .3 4
E
60
7 .1 3
F
75
6 .2 6
S li d e
T im e
K. pneum oniae
P. aeruginosa
T o t a l p r o te in
s a m p le s
( m in )
( c f u / m m 2)
( c f u /m m 2)
( p g / m m 2)
0
2 .7 9 e + 8
8 .2 2 e + 6
2 6 .9 1
2 .1 4 e + 7
1 .1 5 e + 6
2 5 .9 7
SO
S lO
10
S20
20
5 .7 5 e + 6
2 .1 4 e + 5
2 4 .9 6
S30
30
7 .3 9 e + 5
2 .1 4 e + 4
2 5 .2 8
S45
45
1 .4 8 e + 5
2 .1 4 e + 4
2 2 .5 8
S60
60
8 .2 2 e + 4
4 .9 3 e + 3
2 1 .3 9
S75
75
7 .2 3 e + 4
6 .5 7 e + 3
2 2 .3 2
E x p e r im e n t N o . 2 6 — N H 2C l
E f f lu e n t
T im e
T o t a l p r o te in
s a m p le s
( m in )
(p g /m L )
A
0
1.51
B
15
9 .8 5
C
30
7 .8 4
D
45
4 .7 7
E
60
3 .9 5
F
75
3 .0 2
S li d e
T im e
K. pneum oniae
P. aeruginosa
T o ta l p r o te in
s a m p le s
( m in )
( c f u / m m 2)
( c f u /m m 2)
( p g / m m 2)
SO
0
2 .3 0 e + 8
6 .4 1 e + 7
1 9 .2 5
S lO
10
2 .2 0 e + 8
2 .7 9 e + 7
1 6 .5 0
S20
20
1 .8 4 e + 8
1 .9 7 e + 7
1 4 .1 0
S30
30
7 .3 9 e + 7
1 .8 1 e + 7
1 1 .7 3
S45
45
6 .0 8 e + 7
1 .1 8 e + 7
1 3 .1 0
S60
60
4 .4 4 e + 7
1 .0 0 e + 7
1 1 .1 2
S75
75
1 .8 2 e + 7
4 .6 0 e + 6
1 1 .1 1
114
Experiment No. 27 — NH2Cl (5mg/L)
E f f lu e n t
T im e
T o ta l p r o te in
s a m p le s
( m in )
(p g /m L )
A
0
0 .5 0
B
15
4 .6 5
C
30
3 .4 2
D
45
1 1 .4 4
E
60
8 .6 7
F
75
7 .2 3
S lid e
T im e
K. pneum oniae
P. aeruginosa
T o ta l
s a m p le s
( m in )
( c f t i/ m m 2)
( c f t i/ m m 2)
p r o te in
SO
0
1 .8 1 e + 8
1 .3 1 e + 7
1 8 .1 9
S lO
10
1 .4 0 e + 8
9 .6 9 e + 6
1 7 .0 0
( p g / m m 2)
S20
20
7 .0 7 e + 7
7 .8 9 e + 6
1 8 .3 8
S30
30
3 .9 4 e + 7
2 .7 9 e + 6
1 3 .6 7
7 .7 8
S45
45
1 .4 0 e + 7
1 .9 7 e + 6
S60
60
1 .6 8 e + 7
2 .6 3 e + 6
8 .4 1
S75
75
6 .4 1 e + 6
8 .2 2 e + 5
5 .5 8
E x p e r im e n t N o . 2 8 — N H 2C l ( 2 5 m g / L ) , N a C l ( 0 .3 M ) a d d e d at 6 0 m in u te
E f f lu e n t
T im e
T o t a l p r o te in
s a m p le s
( m in )
(p g /m L )
A
0
1 .0 1
B
15
1 4 .1 0
C
30
1 5 .3 5
D
45
1 1 .0 4
E
60
7 .4 2
F
75
1 8 .2 0
G
90
9 .5 3
H
105
4 .8 4
I
120
2 .1 4
S li d e
s a m p le s
SO
T im e
K. pneum oniae
P. aeruginosa
T o ta l p r o te in
( m in )
( c f u / m m 2)
( c f u /m m 2)
( p g / m m 2)
4 .9 3 e + 8
2 .1 0 e + 8
2 7 .9 3
9 .8 6 e + 5
2 3 .8 7
0
S lO
10
7 .5 6 e + 6
S20
20
5 .9 2 e + 5
1 .3 1 e + 5
2 2 .4 9
2 0 .2 9
S30
30
2 .3 0 e + 5
1 .3 5 e + 5
S45
45
5 .2 6 e + 5
9 .8 6 e + 4
2 1 .3 6
60
3 .2 9 e + 4
5 .5 9 e + 4
1 4 .7 1
75
4 .6 0 e + 4
1 .1 2 e + 5
1 5 .4 5
S90
90
1 .1 5 e + 5
1 .2 5 e + 5
9 .1 9
S120
120
2 .6 3 e + 4
2 .9 6 e + 4
4 .7 4
S60
S75
115
E x p e r im e n t N o . 2 9 — L y s o z y m e
E fflu e n t
T im e
K. pneum oniae
P. aeruginosa
T o ta l p r o te in
s a m p le s
( m in )
( c f ii/ m L )
( c f u /m L )
(p g /m L )
A
0
2 .8 0 e + 7
3 .0 0 e + 6
1 .1 6
B
15
3 .7 0 e + 7
1 .3 0 e + 7
7 .4 8
1 2 .3 4
C
30
5 .0 0 e + 7
1 .7 0 e + 7
D
45
2 .1 0 e + 7
5 .0 0 e + 6
8 .0 7
E
60
1 .0 0 e + 6
2 .0 0 e + 6
3 .5 7
F
75
3 .0 0 e + 6
1 .1 0 e + 6
0 .3 8
S li d e
T im e
K. pneum oniae
P. aeruginosa
T o ta l p r o te in
s a m p le s
( m in )
( c f u / m m 2)
( c f u /m m 2)
( p g / m m 2)
SO
0
4 .1 l e + 8
2 .4 6 e + 7
2 0 .8 3
S30
30
1 .1 5 e + 8
1 .6 4 e + 7
1 6 .1 3
S75
75
4 .0 3 e + 7
1 .8 1 e + 7
1 1 .5 2
MONTANA